Toll-like Receptor 4 Mediates the Antitumor Host Response Induced

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Toll-like receptor 4 mediates the antitumor host response induced by Ganoderma atrum polysaccharide Qiang Yu, Shaoping Nie, Junqiao Wang, Danfei Huang, Wenjuan Li, and Ming-Yong Xie J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/jf5041096 • Publication Date (Web): 30 Dec 2014 Downloaded from http://pubs.acs.org on January 8, 2015

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Journal of Agricultural and Food Chemistry

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Toll-like receptor 4 mediates the antitumor host response

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induced by Ganoderma atrum polysaccharide

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Qiang Yu, Shao-Ping Nie*, Jun-Qiao Wang, Dan-Fei Huang, Wen-Juan Li,

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Ming-Yong Xie*

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

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Nanchang 330047, China.

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*Corresponding to: Professor Ming-Yong Xie, PhD; Professor Shao-Ping Nie, PhD

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State Key Laboratory of Food Science and Technology, Nanchang University, 235

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Nanjing East Road, Nanchang 330047, China

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Tel.&Fax: +86 791-83969009 (M. Y. XIE); +86 791-88304452 (S. P. NIE)

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E-mail: [email protected] (M. Y. XIE), [email protected] (S. P. NIE)

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ACS Paragon Plus Environment


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ABSTRACT

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The aim of this study is to investigate the role of Toll-like receptor (TLR) 4 in

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Ganoderma atrum polysaccharide (PSG-1) induced antitumor activity. In vitro, the

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apoptosis rate of S-180 cells was increased in PSG-1 induced peritoneal macrophage

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derived from C3H/HeN (wild type) mice, but not from C3H/HeJ (TLR4 deficiency)

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mice. In S-180 tumor model, phagocytosis, NO and ROS release, phosphorylation of

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MAPKs and Akt, as well as expression of NF-κB were increased by PSG-1 in

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peritoneal macrophage derived from C3H/HeN mice. Furthermore, PSG-1 elevated

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Th1 cytokines production and enhanced cytotoxic activity of CTL and NK cells in

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C3H/HeN mice. In addition, PSG-1 decreased the tumor weight, increased the

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apoptosis rate and caspase 3, 9 activities of tumor derived from the C3H/HeN mice.

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However, none of these activities were observed in C3H/HeJ mice. In summary, these

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findings demonstrated that the antitumor activity of PSG-1 is mediated by TLR4.

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KEYWORDS: Toll-like receptor 4; Ganoderma atrum polysaccharide; Antitumor;

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Innate immunity; Adaptive immunity.

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INTRODUCTION

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Great efforts have been made in therapeutic treatments, including surgery, radiation

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and chemotherapy, however, due to severe adverse effects cancer is still the leading

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cause of death1. Recently, cancer immunotherapy that recruits the host immune

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response to exert anti-tumor effect without harming the host is of great interest2.

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Toll-like receptors (TLRs) are mammalian homologues of Drosophila toll receptor3.

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By now, there are 10 and 12 functional TLRs identified in humans and mice,

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respectively4. Among them, TLR4 is expressed by various immune cells, including

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macrophages, neutrophils and dendritic cells. Binding of ligands to TLR4 initiates

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signals transduction via recruitment of the adaptor protein myeloid differentiation

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protein 88 (Myd88) and the interleukin receptor associated kinase (IRAK), which in

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turn activate tumor necrosis factor receptor–associated factor 6 (TRAF6). This is

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followed by the activation of downstream signals, such as mitogen-activate protein

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kinase (MAPK) and nuclear factor-kB (NF-κB), and lead to the activation of a wide

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spectrum of target genes5.

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Emerging studies have shown that TLR4 agonists can serve as immunomodulating

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agent and exhibit promising therapeutic potential for cancer immunotherapy6, 7. It is

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worth noting that natural polysaccharides, which have been safely used as ingredients

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of traditional medicines and functional food for a long time in oriental countries, are

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attracting more and more attention since they trigger the response of innate immunity

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and in turn induces the adaptive immunity via TLR4 signaling8, 9.

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Ganoderma atrum has been used as Chinese traditional medicine and functional

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food for thousands of years. In recent years, it becomes more and more popular in

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developed countries as a dietary supplement 10. The polysaccharides are regarded as

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the major bioactive substances in G. atrum. We recently isolated and purified a

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polysaccharide from G. atrum, named as PSG-1, with a purity of >99.8%, whose

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primary structural features and molecular weight were characterized11, 12. Our in vitro

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study has demonstrated that TLR4 is the major receptor involved in specific binding

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of PSG-1 to macrophages, and PSG-1 induces macrophage activation through the

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TLR4-signaling pathway13.

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Interestingly, my colleagues showed that oral administration of PSG-1 could inhibit

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S-180 tumor growth and enhance host immune system through the TLR4

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mediated-signaling pathway14. However, the research results were carried out just by

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measuring the protein expression of TLR4 in peritoneal macrophage from BALB/c

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mice, and then they suggested PSG-1 may exert its antitumor effect through TLR4.

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Strictly speaking, the role of TLR4 in PSG-induced antitumor host response is still not

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fully determined. Therefore, in this study, we investigated the antitumor activity of

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PSG-1 in C3H/HeN (wild type) and C3H/HeJ (TLR4 deficiency) mice, especially

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focused on the contribution of TLR4 in PSG-1-mediated innate and adaptive immune

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responses. To the best of our knowledge, it is the first time using transgenic mice to

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establish tumor model for exploring the effect of TLR4 on the antitumor activity

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induced by PSG-1 in vivo. The results presented in our study could provide powerful

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evidence to support the vital role of TLR4 played in PSG-1-induced antitumor

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activity.

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MATERIALS AND METHODS

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Materials and reagents

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3-(4,

5-dimethylthiazol-2-yl)-2,

5-diphenyltetrazolim

bromide

(MTT)

and

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fluorescein isothiocyanate (FITC)-dextran were purchased from Sigma (St. Louis,

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MO, USA). NO assay kit was obtained from Beyotime Biotech Inc. (Jiangsu, China),

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Carboxyl-DCFH (CM-DCFH) was from Molecular Probes (Eugene, OR, USA).

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Antibodies against phospho-ERK1/2 (p-ERK1/2), phospho-JNK1/2(p-JNK1/2),

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phospho-p38 (p-p38), phospho-Akt (p-Akt) and NF-κB were purchased from Cell

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Signaling (Beverly, MA, USA). ELISA kits were from R&D Systems (Minneapolis,

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MN, USA). Annexin V-FITC apoptosis detection kits were obtained from Becton

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Dickinson Biosciences (San Jose, CA, USA). Caspase-3 and -9 colorimetric assay kits

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were purchased from BioVision, Inc. (Palo Alto, CA, USA). Cell culture products

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were from Life Technologies (Paisley, Scotland, UK).

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Cells and animals

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Murine S-180 cell line and YAC-1 cells were purchased from Type Culture

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Collection of Chinese Academy of Sciences, Shanghai, China. Cells were cultured in

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RPMI-1640 medium containing 10% fetal bovine serum, and maintained in a

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humidified incubator with 37 °C in 5% CO2 atmosphere.

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Six- to eight-week-old female C3H/HeN and C3H/HeJ mice were obtained from

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the Shanghai Laboratory Animal Center, China. Mice were housed 8 per cage at 25

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°C with12/12 h light/dark cycle. All mice were cared for in accordance with the

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Guidelines for the Care and Use of Laboratory Animals published by the National

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Institutes of Health, Bethesda, MD (NIH Publication 85-23, 1996). All procedures

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were approved by the Animal Care Review Committee, Nanchang University, China.

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Isolation of mouse peritoneal macrophages

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Each mouse was intraperitoneally injected with 5 mL cold PBS, peritoneal

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macrophages were aseptically harvested from peritoneal exudates by peritoneal lavage.

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Cells were resuspended in RPMI-1640 containing 10% heat-inactivated FBS. After 24

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h incubation, cells were washed by PBS to remove non-adherent cells. The resultant

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cells were collected as peritoneal macrophages.

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Macrophage cytotoxicity assay

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After incubating with PSG-1 (20, 40, 80, 160 µg/mL) for 48 h, the mouse

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peritoneal macrophages from C3H/HeN or C3H/HeJ were washed with fresh medium

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and co-incubated with S-180 cells at a ratio of 25:1 for 48 h. MTT method was

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performed to measure the growth inhibition of S-180 cells. Cytotoxicity was

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calculated as follows: % Cytotoxicity = (ODT − (ODS − ODE))/ODT×100, where ODT,

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ODS or ODE is optical density value of target cells control, test sample or effector

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cells control, respectively.

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Establishment of S-180 model

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After 1 week of acclimatization, C3H/HeN and C3H/HeJ mice were randomly

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divided into 3 experimental groups (n=8 per group), respectively, (i) Normal group:

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healthy mice without tumor inoculation were gavaged with 0.2 mL saline. The S-180

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model was established as previously reported15. Briefly, the S-180 mouse sarcoma

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cells with ascites were harvested under sterile conditions. 2×106 cells suspended in 0.2

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mL sterilized saline were subcutaneously inoculated into mice in the left axillary

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region. After the day of inoculation, (ii) Control group: the mice were gavaged with

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0.2 mL saline for 10 consecutive days. (iii) PSG-1 group: the mice were gavaged with

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0.2 mL PSG-1 (100 mg/kg body weight) for 10 consecutive days. Twenty-four hours

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after the last treatment, all the mice were sacrificed by cervical dislocation. Tumors

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and spleens were extirpated and weighed in a germ-free condition.

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Neutral red uptake

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Phagocytosis of macrophages was determined by neutral red uptake method as

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described with minor modification16. Briefly, peritoneal macrophages isolated from

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mice were seeded at a density of 5×105 cells/ well in the 24-well microplates and

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cultured for 24 h. After washed by PBS twice, the cells were incubated with neutral

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red (50 mg/mL) for another 3 h, and washed twice by PBS. The plate was blotted dry,

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then 50% ethanol containing 1% glacial acetic acid were added in each well. The

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absorbance at 540 nm was measured by microplate reader.

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FITC-dextran internalization

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The FITC dextran internalization of peritoneal macrophages was determined by

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flow cytometry. Briefly, 1×106 cells/well peritoneal macrophages were seeded in the

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24-well plate, and 1 mg/mL FITC-dextran was added to each well. After incubating at

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37 °C for 1 h, cells were washed three times and resuspended in 500 µL PBS for flow

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cytometric analysis.

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Measurement of ROS generation

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Peritoneal macrophages were harvested and seeded at a density of 1×106 cells/well

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in the 24-well plate. After incubating with 10 mM CM-DCFH at 37 °C for 30 min in

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dark, cells were washed three times and resuspended in 500 µL PBS, then analyzed by

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flow cytometry.

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Measurement of NO production

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NO production was measured according to the manufacture’s instruction. Briefly,

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5×105 cells/well peritoneal macrophages were placed in a 96-well plate. After

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incubating for 48 h, supernatants were collected and incubated with an equal volume

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of Griess reagent for 10 min at 25 °C. Nitrite concentrations were calculated by a

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standard curve.

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Western blot analysis

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The whole-cell protein or nuclear protein was prepared from peritoneal

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macrophages using Cell lysis kit or Nuclear and cytoplasmic extraction kit (Beyotime,

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Shanghai, China) according to the manufacturer’s instructions. 40 µg denatured

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protein were loaded onto 10% SDS polyacrylamide gel by electrophoresis and

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transferred to nitrocellulose membrane. 5% nonfat milk (prepared in TBS containing

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0.1% Tween 20) was used to block non-speciﬁc binding for 1 h at 37 °C. Then the

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membrane was incubated with specific primary antibodies at 4 °C overnight. After

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washing three times with TBST, the membrane was incubated at room temperature for

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1 h with secondary antibody and the signal was analyzed by using BeyoECL Plus kit

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(Beyotime, Shanghai, China). Densitometry of band was analyzed by Quantity One.

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Preparation of splenocytes

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The extirpated spleens were minced into small pieces in a germ-free condition.

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Single cell suspension was prepared by filtering the suspension through a sterile sieve

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mesh. The cells were treated with lysis buffer (0.15 M NH4Cl, 0.01 M KHCO3, and

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0.1 mM Na2EDTA, pH 7.4) to get rid of red blood cells, followed by washing twice

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with cold PBS. Then the cells were adjusted to the concentration of 5×106 cells/mL in

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in RPMI 1640 medium supplemented with 10% fetal calf serum, and incubated for 3 h

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in Petri dishes. The suspended cell populations were collected.

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Cytotoxicity assays of natural killer (NK) cell activity

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Splenocytes were used as the effector cells for splenic NK cell activity assay as

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described17. Briefly, 5×105 cells/well effector cells were co-cultured with YAC-1 cells

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in 96-well microplates at a ratio of 50:1 at 37 °C for 20 h. 20 µL MTT (5 mg/ml) was

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added to each well, and then MTT assay was employed after 4 h incubation. NK cell

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activity was calculated as the following equation: % Cytotoxicity = (ODT − (ODS −

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ODE))/ODT×100%, where ODT, ODS or ODE is optical density value of target cells

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control, test samples or effector cells control, respectively.

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Cytotoxicity assays of cytotoxic T lymphocyte (CTL) activity

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The CTL activity was measured using similar method as described above except

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that S-180 cells and splenocytes were used as target cells and effector cells,

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respectively, at a ratio of 50:1. CTL activity was calculated as the same equation as

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described above: % Cytotoxicity = (ODT − (ODS − ODE))/ODT × 100.

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Cytokine measurement by ELISA

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Levels of IL-2, IFN-γ and IL-12 were determined by the ELISA kits according to the manufacturer’s instructions.

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Analysis of apoptotic cells

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The tumors harvested from S-180-bearing mice were minced into small pieces in a

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germ-free condition. Single cell suspension was prepared by filtering the suspension

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through a sterile sieve mesh. The suspension was washed twice with cold PBS and

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adjusted to the concentration of 1×106 cells/mL. Then the cells were stained with

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Annexin V-FITC and PI for 15 min in dark, and analyzed by flow cytometry (FACS

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Calibur, Becton Dickinson, USA).

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Measurement of caspase activities

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Caspase-3 and -9 colorimetric assay kits were used to measure the activities of

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caspase-3 and -9 in S-180 cells. Briefly, 1×105 cells/mL were treated with cell lysis

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buffer for 10 min on ice and centrifuged at 10,000 g for 8 min. Then the supernatants

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were incubated with caspase substrate LEHD-AFC for caspase-9, and DEVD-AFC for

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caspase-3 at 37 °C for 1.5 h. Chromophore ρ-nitroanilide was measured by microplate

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reader at 405 nm.

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Statistical analysis

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Values are expressed as means ± SEM. One-way analysis of variance followed by

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the Student–Newman–Keuls test was used to determine the statistical signiﬁcance

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between various groups. A value of p

Toll-like Receptor 4 Mediates the Antitumor Host Response Induced

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