A Polysaccharide from Ganoderma atrum Inhibits Tumor Growth by

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A polysaccharide from Ganoderma atrum inhibits tumor growth by induction of apoptosis and activation of immune response in CT26-bearing mice Shenshen Zhang, Shaoping Nie, Danfei Huang, Jianqin Huang, Yanling Feng, and M. Y. Xie J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/jf503250d • Publication Date (Web): 01 Sep 2014 Downloaded from http://pubs.acs.org on September 3, 2014

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Journal of Agricultural and Food Chemistry

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A polysaccharide from Ganoderma atrum inhibits tumor growth by

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induction of apoptosis and activation of immune response in CT26-bearing

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mice

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Shenshen Zhang, Shaoping Nie,* Danfei Huang, Jianqin Huang, Yanling Feng,

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Mingyong Xie

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

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East Road, Nanchang, Jiangxi 330047, China

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* Corresponding authors: Professor Shao-Ping Nie, PhD, Nanchang University, 235 Nanjing

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East Rd., Nanchang 330047, China. Tel & Fax: +86-791-88304452 (S. P. NIE). E-mail

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address: [email protected].

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ACS Paragon Plus Environment


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ABSTRACT

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Ganoderma atrum is one species of edible and pharmaceutical mushroom with

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various biological activities. Recently, a novel polysaccharide PSG-1 was purified

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from G. atrum. The antitumor activity and its mechanism of action were studied. In

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vitro, PSG-1 has little effect on inhibiting proliferation of CT26 tumor cells. However,

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the tumor size was significantly decreased in PSG-1-treated mice. The results showed

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that PSG-1 induced apoptosis in CT26 cells. Moreover, the intracellular cyclic AMP

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(cAMP) level and protein kinase A (PKA) activity were markedly increased in

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PSG-1-treated mice. In contrast, the contents of cyclic GMP (cGMP) and DAG, and

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PKC activity were decreased. Similarly, the expression of PKA protein was

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upregulated, while PKC protein expression in PSG-1-treated group was lowered.

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Additionally, PSG-1 increased the immune organ index and serum biochemistry

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parameter. In general, PSG-1 enhances the antitumor immune response, induces

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apoptosis in CT26-bearing mice and could be a safe and effective adjuvant for tumor

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therapy or functional food.

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KEYWORDS: Antitumor; Apoptosis; Ganoderma atrum polysaccharide; PKA;

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PKC

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INTRODUCTION

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Colorectal carcinoma (CRC) is the most common leading cause of death from cancer

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of the gastrointestinal tract and the fourth most frequently diagnosed malignant

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neoplasm. Although early stage CRC can be cured with curative resection and

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neoadjuvant chemotherapy, advanced stage CRC is prone to recurrent, distant

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metastasis and become lethal, even though patients receive combination

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chemotherapy.1 Furthermore, many studies have demonstrated that lots of traditional

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chemotherapeutic drugs are toxic to cancer cells as well as normal cells and organs.2

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Therefore, there is urgent to discover and explore novel agents with hypotoxicity and

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effectiveness for the treatment of CRC. For decades, natural products have been

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considered as a significant source of safe drugs for various diseases. Hence, the

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natural drugs may be a promising new class of potent agents for patients with CRC.

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Apoptosis, well known as the regulated destruction of cell, is a controlled biological

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strategy to remove unwanted, injured, or virus-infected cells from a given tissue and

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may account for much of the spontaneous cell loss in many tumors. Apoptosis is

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triggered by many upstream signaling pathways and requires the coordinated

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activation and execution of multiple subprogrammes.3,

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cancer cells is their resistance to apoptosis induction.5 Therefore, most of the

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antineoplastic drugs kill cancer cells by inducing apoptotic death to block or suppress

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the growth of cancer cells. The process of apoptosis is controlled by a multiple

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biological process involving various signaling pathways, which may originate either

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extracellularly or intracellularly. Intracellular signaling mechanisms typically involve

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protein kinases as major regulators of cellular processes.6 cAMP and cGMP are

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important mediators that play pivotal roles in apoptosis.7,

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pathway conduces to regulate a wide range of biologically distinct cellular processes

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One critical hallmark of

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The cAMP signaling


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such as cell differentiation, metabolism, and apoptosis. cAMP could initiate apoptosis

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via increasing p53 and p21 expression and inhibite proliferation of human aortic

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vascular smooth muscle cells.9 Kizaki et al. indicated that cAMP causes apoptosis or

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potentiates its induction by other agents in thymocytes and T lymphocytes.10 The

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elevation of intracellular cAMP levels and the cAMP effector protein kinase A (PKA)

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have been proposed as targets to stimulate apoptosis, for example in the treatment of

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malignant lymphoid cells and other certain cancers.11 Moreover, protein kinases play

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key roles both in the upstream induction phase of apoptosis and in the downstream

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execution stage, as the direct targets for caspases.12 PKA can phosphorylate various

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proteins and regulate a cascade of transcription events implicated in the growth

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inhibition and differentiation of a broad spectrum of cancer cells.13 In addition,

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diacylglycerol (DAG), another intracellular second messenger, plays important roles

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in responding to the stimulation of extracellular signal. DAG can activate protein

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kinase C (PKC). Activation of PKC could lead to a cascade of phosphorylation of

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many targets, which regulates the proliferation, differentiation and other physiological

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functions of cells. The down-regulation of PKC activity by sorbitol rapidly induces

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apoptosis in gastric cancer cells.14 Taken together, the previous studies support the

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notion of an important role for cAMP-PKA and PKC pathways in the response to

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apoptosis of the tumor cells.

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Ganoderma (G.), which is also called ‘marvelous herb’, has been widely used as a

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tonic and medicinal food for longevity and health promotion in East Asia dates back

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to several centuries. Interestingly, many studies show that G. has various biological

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activities

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antiatherosclerotic and antimicrobial activities.15 G. atrum is a member of the most

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representative medical mushrooms of Ganodermataceae. The polysaccharide is the

and

therapeutic

effects

including

inhibiting

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the

tumor

growth,

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major source of its bioactive substance in G. atrum.16 For our unremitting pursuit of

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novel agents with antitumor activities, we reorganized and qualified the structure of

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PSG-1, which is a polysaccharide of G. atrum. The purity of PSG-1 is more than

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99.8%. The polysaccharide composed of glucose, mannose, galactose and

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galacturonic acid with molar ratio of 4.91: 1: 1.28: 0.71.17 Furthermore, PSG-1

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comprises a backbone of 1, 3-linked and 1, 6-linked β–Glcp residues substituted at

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O-3 and O-6 positions as the branch points.18, 19 Our previous studies suggested that

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PSG-1 had potent antioxidant and even stronger than that of G. Lucidium.17, 20 PSG-1

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has also shown to induce apoptosis in S180 xenografts in vivo.21 Recently, we verified

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that PSG-1 possessed potent antitumor activity by inducing apoptosis via

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mitochondrial pathways and enhanced the antitumor effect via improving immune

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system functions through TLR4 mediated-NF-κB and MAPK signaling pathway.22, 23

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Although PSG-1 is crowned as one of the most active antineoplastic agents in the past

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decades, the exact molecular mechanisms that integrate these events have remained

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largely unclear. Therefore, research and development of PSG-1 as a novel agent with

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anti-tumor activity is of great importance. This study was designed to evaluate and

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characterize the antitumor activity and potential molecular mechanism of PSG-1 in

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CT26-bearing mice. The data have verified the excellent antitumor effect of PSG-1 in

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CT26-bearing mice model, in which enhanced tumor growth inhibition accompanied

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by increased apoptosis as compared with control group was verified. We further

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demonstrated that such action may be associated with induction of apoptosis via the

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cAMP/PKA signaling pathway and down-regulation of PKC pathway, as well as

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improving the host immune system. These findings would pave the way for novel

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therapeutic approaches to treat CRC patients and open up new possibilities for the

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cancer control. 5



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MATERAL AND METHODS

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Materials

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PSG-1 was extracted and purified following our previously published method (18).

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RPMI 1640 and fetal bovine serum (FBS) were purchased from Hyclone (Logan, UT).

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Penicillin and streptomycin were purchased from Life Technologies, Inc.

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(Gaithersburg, MD). Antibodies for PKA and PKC were purchased from Abcam

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(Cambridge, MA). Anti-β-actin, goat anti-rabbit IgG-conjugated horseradish

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peroxidase (HRP) and goat anti-mouse IgG-conjugated HRP were purchased from

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ZSGB-Bio (Beijing, China). 5-fluorouracil was purchased from Sigma–Aldrich

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(Missour, USA). PKA, PKC, cAMP and cGMP assay kits were obtained from IBL

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(Hamburg, Germany). IL-1β and TNF-α ELISA kits were purchased from SenXiong

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Biotechnology (Shanghai, China). Cell culture products were obtained from Life

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Technologies (Paisley, Scotland).

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Cell Line and Animals

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CT26 WT mouse colon cell line was purchased from Type Culture Collection of

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Chinese Academy of Sciences, Shanghai, China. BALB/c mice (Female, 4-6 weeks

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old, weighted 20.0 ± 2.0 g) were purchased from Beijing HFK Biotechnology

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Company (Beijing, China; quality certificate number: SCXK (jing) 2009-0004). The

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animals were maintained in accordance with the Guide for the Care and Use of

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Laboratory Animals (NRC 2011), and all procedures were approved by Nanchang

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University Animal Ethnics Committee.

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Cell Viability Assay

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The effect of PSG-1 on the viability of CT26 cells were determined by MTT assay.

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CT26 cells were seeded in a flat-bottomed 96-well plate (2.0 × 104 cells/mL) and

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treated with PSG-1 (20 - 320 µg/mL). Controls were exposed to culture medium

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without PSG-1. After incubation for 48 h, 20 µL of MTT solution (5 mg/mL) were

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added to each well and the cells were incubated at 37 °C for 4 h. The formazan crystal

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was dissolved in 150 µL dimethyl suroxide (DMSO) and the absorbance at 490 nm

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was measured immediately on a microplate reader (Thermo, Shanghai, China).

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Animal Experiments

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The mice were injected hypodermically with 1×107 cells/mL of CT26 in 0.2 mL at the

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right hind groin. The xenograft tumor-bearing mice were divided randomly into 5

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groups (8 mice in each group). The mice were treated with PSG-1 via gastric tube at

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the doses of 50, 100 and 200 mg/kg body weight (BW) for 2 weeks. The negative

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control group was treated with 0.9% saline and the positive control received 5-Fu (20

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mg/kg BW). At the end of the experiments, mice were weighed and sacrificed by

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cervical dislocation, and then the solid tumors, thymus and spleen were harvested.

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Thymus and spleen indexes were expressed as the thymus and spleen weight relative

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to body weight. The rate of inhibition (IR) was calculated according to the following

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formula:

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Blood samples obtained from retrobulbar venous plexus of each group mice were

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centrifuged (12,000 rpm) for 10 min at 4°C to separate serum and blood cells. The

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serum was stored at −80°C for determination of cytokines (IL-2, IFN-γ and TNF-α).

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The serum cytokine levels were determined using ELISA kits (R&D, Minneapolis,

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USA). 7



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Preparation of Murine CT26 Cells in the Tumor of CT26-bearing

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Mice

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The tumor was isolated using sterile tissue dissection techniques from tumor-bearing

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mice and transferred to PBS. The tumor tissues were subsequently minced. The cell

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suspensions were filtered through a Steriflip unit. Then the cells were washed twice in

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PBS and further purified using gradient centrifugation. After centrifugation, the cells

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were resuspended in RPMI 1640 medium with 10% FBS (2 × 106/mL).

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Analysis of Cell Apoptosis

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To define the apoptosis of tumor cells, CT26 cells were strained with Annexin

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V-FITC/PI detection kit (KeyGen, Nanjing, China) according to the manufacturer’s

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instructions. Briefly, the CT26 cells from tumor-bearing mice were harvested and

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resuspended in 500 µL binding buffer containing the Annexin V-FITC and PI

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fluorescence dyes at 37 °C for 15 min in the dark. The strained cells were washed

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twice with PBS and resuspended. The fluorescence was detected by FACSCalibur

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(Becton Dickinson, USA) in the FL1-H and FL2-H channels.

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Determination of cAMP and cGMP Concentrations

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The intracellular cAMP and cGMP levels were determined by using the cAMP and

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cGMP ELISA kits (Enzo Life Sciences, PA). Firstly, the CT26 cells (2 × 105 cells/mL)

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were prepared as described above and then resuspended in lysis reagent provided by

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the kit, followed by centrifugation at 12,000 × g for 30 min at 4 °C. The supernatant

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was immediately collected and quantified by cAMP or cGMP kit. Each sample and

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standard value derived from triplicate measurements. 8


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Measurement of DAG and Adenylyl cyclase (AC) Contents

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The DAG and AC contents were determined by using the DAG and AC ELISA kits

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(Antibodies-online, Atlanta, USA). A total of 106 CT26 cells from tumor-bearing

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mice were washed with PBS and harvested by centrifugation. The cells were lysed by

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lysis reagent provided by the kit. The supernatant was collected and used for the

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determination of intracellular DAG and AC contents.

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PKA and PKC Activity Assays

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The CT26 cells from mice model were harvested as described above. The activities of

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intracellular PKC and PKA were determined by PKC and PKA kits (Enzo Life

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Sciences International, PA), respectively. In brief, CT26 cells were scraped into

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Ca2+-free PBS supplemented with 2.5 mM of EDTA or EGTA, and then pelleted by a

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brief centrifugation. Cells were lysed in an ice-cold homogenization buffer and then

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centrifuged at 15,000 × g for 30 min at 4 °C. The cell samples were added to the

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appropriate wells. The reaction was then stopped with acid-stop solution, and the

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absorbance was measured at 450 nm in a microplate reader (Thermo, Shanghai,

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China).

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Western Blot Analysis

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Total protein (15 µg) from CT26 cells was separated by SDS-PAGE and was

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transferred to nitrocellulose membranes. The membranes were blocked with

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Tris-buffered saline containing 5% fat-free dried milk for 2 h at room temperature,

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and further incubated 16 h at 4 °C with anti-PKA or anti-PKC antibodies (Cell

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Signaling Technology, Danvers, MA). After washed 3 times with TBS-0.05% Tween

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20, reactive bands were incubated with horseradish peroxidase-conjugated secondary

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antibody for 1 h at room temperature. The membranes were washed again, and the 9



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immunoreactive protein was visualized using enhanced chemiluminescent reagent

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(Beytin, Nanjing, China). The experiments were performed in triplicate, and

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representative results are shown.

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Neutral Red Phagocytosis Assay of Peritoneal Macrophages

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Macrophages were obtained from tumor-bearing mice peritoneal exudate cells (PECs).

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PECs were washed with PBS and resuspended in RPMI-1640 medium. Then PECs

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were seeded in a 96-well plate (2×105 cells per well) and incubated at 37 °C, 5% CO2

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for 4 h in a humidified atmosphere to allow peritoneal macrophages to adhere. The

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supernatants were discarded, and 100 µL 0.075% neutral red were added and

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incubated for another 1 h. Cells were washed with PBS for three times and incubated

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with cell lysis buffer (1 M acetic acid: ethanol = 1:1) overnight. The result was

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recorded with a micro-plate reader, using a test wavelength of 540 nm.

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Determination of Proliferation of Lymphocyte of CT26-bearing Mice

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CT26-bearing mice were killed and the spleens collected under aseptic condition were

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chopped into small pieces and filtered over a fine steel mesh. Lymphocytes were

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resuspended in lysis buffer to remove erythrocytes and then resuspended in 10% FBS

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RPMI-1640 (2×106 cells per well) medium. The cells were planted into 96-well plates

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with or without ConA 1 mg/L or LPS 5 mg/L, and incubated for 48 h. Cell

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proliferation was measured based on MTT method.

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Statistical Analysis

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All data were expressed as means ± S.E.M of at least three experiments. ANOVA was

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used to assess the statistical significance of the differences. Differences with P values

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A Polysaccharide from Ganoderma atrum Inhibits Tumor Growth by

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