Rhizopus nigricans polysaccharide activated macrophages and suppressed tumor growth in CT26 tumor-bearing mice

Article abstract of DOI:10.1016/j.carbpol.2018.06.076

In this study, a homogeneous polysaccharide (RPS-1) was extracted from liquid-cultured mycelia of Rhizopus nigricans. The weight-average molecular weight of RPS-1 was 1.617 × 10⁷ g/mol and structural characterization indicated that RPS-1 was a non-starch glucan which consisted of a backbone structure of (1→4)-linked α-D-glucopyranosyl residues substituted at the O-6 position with α-D-glucopyranosyl branches. RPS-1 stimulated the production of nitric oxide and tumor necrosis factor-α by triggering phosphorylation of mitogen-activated protein kinases and nuclear translocation of nuclear factor kappa B p65 in RAW 264.7 macrophage cells. Moreover, intragastric administration of RPS-1 improved the immune function of CT26 tumor-bearing mice and significantly inhibited the growth of transplanted tumor. In combination with 5-FU, RPS-1 enhanced antitumor activity of 5-FU and alleviated its toxicity on immune system. These findings suggested that RPS-1 has the potential for the development of functional foods and dietary supplements.

Full text of DOI:10.1016/j.carbpol.2018.06.076

CarbohydratePolymers198(2018)302–312
Contents lists available at ScienceDirect
Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol
Rhizopus nigricans polysaccharide activated macrophages and suppressed
tumor growth in CT26 tumor-bearing mice
⁎
Zhihong Weia,1, Guochuang Chenb,d, ,1, Pengying Zhangb, Lei Zhuc, Linan Zhange,
⁎
Kaoshan Chenb,c,
a
Gynecology Department, First Affiliated Hospital of Shenzhen University, Shenzhen Second People’s Hospital, Shenzhen, China
School of Life Science and National Glycoengineering Research Center, Shandong University, Jinan, China
Anhui Provincial Engineering Research Center for Polysaccharide Drugs, Anhui Province Key Laboratory of Active Biological Macro-molecules, School of Pharmacy,
b
c
Wannan Medical College, Wuhu, China
d
Institute of Biomedicine and Biotechnology, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen, China
Second Affiliated Hospital of China Medical University, Shenyang, China
e
A R T I C L E I N F O
A B S T R A C T
Keywords:
In this study, a homogeneous polysaccharide (RPS-1) was extracted from liquid-cultured mycelia of Rhizopus
nigricans. The weight-average molecular weight of RPS-1 was 1.617 × 107 g/mol and structural characterization
indicated that RPS-1 was a non-starch glucan which consisted of a backbone structure of (1→4)-linked α-D-
glucopyranosyl residues substituted at the O-6 position with α-D-glucopyranosyl branches. RPS-1 stimulated the
production of nitric oxide and tumor necrosis factor-α by triggering phosphorylation of mitogen-activated
protein kinases and nuclear translocation of nuclear factor kappa B p65 in RAW 264.7 macrophage cells.
Moreover, intragastric administration of RPS-1 improved the immune function of CT26 tumor-bearing mice and
significantly inhibited the growth of transplanted tumor. In combination with 5-FU, RPS-1 enhanced antitumor
activity of 5-FU and alleviated its toxicity on immune system. These findings suggested that RPS-1 has the
potential for the development of functional foods and dietary supplements.
Polysaccharides
Rhizopus nigricans
Macrophage
Antitumor
CT26 colon cancer
1. Introduction
through various signaling pathway (Brown & Gordon, 2001; Gantner,
Simmons, Canavera, Akira, & Underhill, 2003; Thornton, Vetvicka,
Fungal polysaccharides have long been appreciated for their biolo-
gical and pharmacological activities, including immunomodulatory,
antitumor, antioxidant, antiviral and anti-infection (Wasser, 2010; Yu,
Shen, Song, & Xie, 2018). A wide range of fungal glucans showed im-
munomodulatory and antitumor activities have been obtained from
fruit bodies, mycelia and liquid culture broth. These glucans present
diverse structures with (1→3)-, (1→4)- and/or (1→6)-linked linear or
branched α-glucans, β-glucans and mixed α/β-glucans, and molecular
weight ranged from thousands to millions Da (Synytsya & Novak,
2013). As the most studied fungal glucan, β-glucans are considered as
potent immunomodulators and responsible for the beneficial effects in
Pitman, Goldman, & Ross, 1996).
Despite the well documented β-glucans, little is known about the
immunoregulatory activities of fungus derived α-(1→4)-glucans and
their underlying mechanisms. Macrophages are one of the major types
of phagocytes and play vital roles in innate and adaptive immune
system. More important, macrophages orchestrate immune response
against cancer by phagocytosing aberrant cells, presenting tumor an-
tigens and secreting heterologous proinflammatory cytokines (Long &
Beatty, 2013). Recently research reported that maitake α-(1→4)-glu-
cans served as biological response modifiers with the capability to ac-
tivate macrophages and dendritic cells and inhibited tumor growth in
vivo (Masuda, Nakayama, Tanaka, Naito, & Konishi, 2017; Sun et al.,
2016; Wang et al., 2017). YCP, a homogeneous α-(1→6)-branched α-
(1→4)-glucan purified from the mycelium of marine fungus, was found
to be able to increase phagocytic activity in mice and stimulate NO
the outcome of malignant tumor (Ina, Kataoka,
& Ando, 2013;
Namikawa et al., 2015). Upon specific interactions with several cell
surface receptors, such as toll-like receptor, complement receptor 3 and
dectin-1, β-glucans can trigger a wide spectrum of immune responses
Abbreviations: MAPKs, mitogen-activated protein kinases; NF-κB, nuclear factor-κB; NO, nitric oxide; HPSEC-MALLS, high performance size-exclusion chromatography with multiangle
laser light scattering; PMB, polymyxin B; 5-FU, 5-fluorouracil
⁎
Corresponding authors. Postal address: School of Life Science and National Glycoengineering Research Center, Shandong University, No. 27 South Shanda Road, Jinan, China.
E-mail addresses: chenguochuang@126.com (G. Chen), ksc313@126.com (K. Chen).
These authors contributed equally to this work, thus share the first authorship.
1
https://doi.org/10.1016/j.carbpol.2018.06.076
Received 22 March 2018; Received in revised form 15 June 2018; Accepted 15 June 2018
Availableonline18June2018
0144-8617/©2018ElsevierLtd.Allrightsreserved.

Z. Wei et al.
CarbohydratePolymers198(2018)302–312
release in macrophages through p38 signaling axis (Chen et al., 2009;
Yang et al., 2005). Macrophage activation may partially underlie im-
munoregulatory and antitumor activities of α-(1→4)-glucans.
Rhizopus nigricans is a zygomycete filamentous fungus and widely
used in food and pharmaceutical industry. In this study, we obtained a
novel bioactive glucan from liquid-cultured mycelia of R. nigricans and
explored the underlying molecular mechanisms of macrophage activa-
tion. The antitumor effects of R. nigricans polysaccharide were further
investigated in CT26 colon cancer-bearing mice.
(HPSEC-MALLS). HPSEC-MALLS measurements were performed on a
Waters 2695 instrument (Waters, USA) equipped with OHpak SB-806M
(Shodex, Japan) at 25 °C. RPS-1 was dissolved in distilled water and
incubated for 3 h. All the solutions were purified by a 0.45 μm filter.
The elution was maintained at a flow rate of 0.5 ml/min and monitored
by Optilab T-rEX differential refractive index detector (Wyatt, USA) and
multiangle laser light scattering instrument equipped with DAWN
HELEOS II light scattering (Wyatt, USA) at angles of 35°, 43°, 52°, 60°,
69°, 80°, 90°, 100°, 111°, 121°, 132°, 142°, and 152°. The Astra software
was utilized for data acquisition and analysis.
2. Materials and methods
2.5. Monosaccharide composition analysis
2.1. Materials
Monosaccharide analysis was performed as described previously
(Chen et al., 2013).
DEAE sepharose Fast Flow and Sephacryl S-500 HR were obtained
from General Electric Healthcare Life Sciences (Pittsburgh, USA). Fetal
bovine serum, dulbecco's modified eagle medium (DMEM),
Lipofectamine® 2000, penicillin and streptomycin were provided by
Thermo Fisher Scientific (Waltham, USA). Monosaccharides, lipopoly-
saccharide (LPS), 4′,6-Diamidino-2-phenylindole dihydrochloride
(DAPI), polymyxin B (PMB) and 5-fluorouracil (5-FU) were purchased
from Sigma-Aldrich (St. Louis, USA). 1-Phenyl-3-methyl-5-pyrazolone
(PMP) was obtained from Acros Organics (Geel, Belgium). Antibodies
against IκB-α, NF-κB p65 (RelA), JNK1/2, phospho-JNK1/2, and β-actin
were purchased from Santa Cruz Biotechnology (Dallas, USA).
Antibodies against p38, phospho-p38, p44/42 (ERK1/2), phospho-p44/
42 (ERK1/2), and histone H2A were provided by Cell Signaling
Technology (Danvers, USA). The cell lysis buffer, nuclear and cyto-
plasmic protein extraction kit, NF-κB nuclear translocation assay kit
and horseradish peroxidase (HRP)-labeled second antibodies were
supplied by Beyotime Institute of Biotechnology (Shanghai, China). The
pNL3.2.NF-κB-RE and pRL-TK plasmids and dual-luciferase® reporter
assay system were obtained from Promega (Beijing, China). Mouse
TNF-α and interleukin-2 (IL-2) ELISA kits were purchased from
RayBiotech (Norcross, USA).
2.6. Methylation analysis
Methylation of RPS-1 was performed as previous described (Ciucanu
& Kerek, 1984). The resulting methylated product was hydrolyzed with
2 M TFA at 120 °C for 2 h followed by reduction with NaBD4 and
acetylation with acetic anhydride to yield partially methylated alditol
acetates which were further quantified by gas chromatograph-mass
spectrometer (GC–MS) using a HP-5 MS fused silica capillary column
(30 m × 0.25 mm i.d., 0.25 μm, Agillent, USA). The column tempera-
ture was set at 120 °C when injected, then increased to 200 °C at 4 °C/
min, then to 280 °C at 10 °C/min, and kept at 280 °C for 5 min. Helium
was used as the carrier gas.
2.7. Nuclear magnetic resonance analysis
A total of 10 mg of RPS-1 was dissolved in 0.5 ml of D2O. The 1H and
13C NMR spectra were recorded on a Bruker AVANCE-300 MHz NMR
spectrometer (Bruker, Germany) at 25 °C.
2.8. Cells and animals
2.2. Organism and growth conditions
Murine macrophage cell line RAW 264.7 and CT26 mouse colon cell
line were purchased from the Cell Bank of Type Culture Collection of
Chinese Academy of Sciences (Shanghai, China). Cells were maintained
in DMEM supplemented with 10% fetal bovine serum, 100 IU/ml pe-
nicillin and 100 μg/ml streptomycin. Cell cultures were incubated in
humidified atmosphere of 5% CO2 at 37 °C. Female BALB/c mice
weighing 18–22 g were purchased from Nanjing Qinglong mountain
farm (Nanjing, China) and acclimatized for 1 week before used. All the
animals were kept according to the National Institute of Health Guide
for the Care and Use of Laboratory Animals, and all experimental
protocols were in accordance with the institutional guidelines of the
Animal Care and Use Committee at Shandong University.
R. nigricans was preserved in Laboratory of Biomass Resources,
Shandong University (Jinan, China). The strain was cultured in shaking
flask containing potato dextrose broth under controlled conditions
(28 °C, 130 rpm) for 10 days.
2.3. Extraction and purification of the R. nigricans polysaccharides
Liquid-cultured mycelia of R. nigricans were extracted in boiling
water for 2 h and the extract was treated with 3 volumes of ethanol at
4 °C overnight. The precipitate was dissolved in distilled water and
deproteinated according to the method of Sevag (Sevag, 1938). The
resulted polysaccharide solution was decolorized with D301R resin,
followed by dialyzed and lyophilized to obtain the crude poly-
saccharides. The crude polysaccharides were loaded into DEAE Se-
pharose Fast Flow column (1.6 cm × 20 cm) and eluted with a linear
gradient of sodium chloride solution (0–0.5 M). The unabsorbed frac-
tion was collected and further purified using Sephacryl S-500 HR
column (1.6 cm × 60 cm) and the fraction of major peak was collected
and lyophilized to powder, named as RPS-1. The total sugar content of
RPS-1 was measured by phenol-sulfuric acid method and glucose was
used as standard. The endotoxin contamination was tested by the Li-
mulus amoebocyte lysate assay, showing negative value.
2.9. Measurement of NO and TNF-α
Logarithmically growing RAW 264.7 cells were placed in 96-well
plate and treated by a series of concentrations of RPS-1 for 24 h. LPS
(100 ng/ml) was used as positive control. After treatment, the levels of
NO and TNF-α were assayed by Griess reagent and enzyme-linked im-
munosorbent assay kit according to the manufacturer's protocols, re-
spectively.
2.10. Immunofluorescence staining
2.4. Homogeneity, molecular weight and root mean square radius
RAW 264.7 cells were plated in 6-well plate and exposed to RPS-1
(400 μg/ml) or LPS (100 ng/ml) for 30 min. After treatment, cells were
rinsed with cold phosphate buffer solution (PBS), fixed by paraf-
ormaldehyde and incubated with primary antibody against NF-κB p65
overnight at 4 °C overnight. And then cells were incubation with a Cy-3
The purity, molecular weight (Mw) and root mean square (RMS)
radius of RPS-1 were determined by using high performance size-ex-
clusion chromatography coupled with multiangle laser light scattering
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CarbohydratePolymers198(2018)302–312
Fig. 1. Separation and purification of R. nigricans polysaccharides. (A) The crude R. nigricans polysaccharides were extracted from liquid cultivated mycelium and
separated by (B) DEAE-Sepharose Fast Flow column and (C) Sephacryl S-500 HR. (D) The homogeneity of RPS-1 was analyzed by HPSEC-MALLS. LS: light scattering
detector, dRI: differential refractive index detector.
conjugated secondary antibody for 1 h at room temperature and stained
with DAPI for 5 min. The prepared samples were photographed by
using fluorescence microscopy (Olympus, Japan).
10–12% SDS–PAGE and then transferred onto polyvinylidene fluoride
member (Millipore, USA). The membrane was blocked with solution
containing 5% skim milk in Tris-buffered saline with 0.1% Tween-20
(TBST) for 2 h at room temperature, and incubated with primary anti-
bodies at 4 °C overnight. The blots were washed 3 times with TBST and
further probed with HRP-conjugated secondary antibodies for 1 h at
room temperature. The visualization of the immune complexes was
performed using an enhanced chemiluminescence detection system.
2.11. Western blot
RAW 264.7 cells were treated with RPS-1 or LPS for indicated time
and cell lysates were centrifuged at 12,000×g for 15 min at 4 °C to
remove cellular debris. The protein concentrations were determined
using BCA method. For the extraction of nuclear and cytoplasmic pro-
tein, a commercially available kit (Beyotime, China) was used ac-
cording to the manufacturer's protocols. The protein samples were
mixed with SDS-PAGE loading buffer and boiled at 100 °C for 5 min.
The samples containing equal amounts of protein were separated by
2.12. Dual-luciferase reporter assay
The plasmids used for transfection were purified by using an
E.Z.N.A.® Endo Free Plasmid Mini Kit (Omega, USA). Transfection was
performed by using the Lipofectamine® 2000 according to the
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Z. Wei et al.
CarbohydratePolymers198(2018)302–312
manufacturer’s recommendations. Briefly, the transfection mixture
consisted of 500 ng of reporter plasmid and 2 μl of transfection reagent
was added dropwise to each well and incubated for 6 h. The culture was
replaced with DMEM supplemented with 10% fetal bovine serum and
incubated for 12 h. After transfection, the cells were treated with RPS-1
or LPS for 12 h and the transcriptional activity of NF-κB was detected by
dual-luciferase reporter assay system. The results were recorded with a
Glomax® 96 microplate luminometer (Promega, USA).
2.13. In vivo model to assess anti-tumor effects of R. nigricans
polysaccharides
Model of subcutaneously transplanted tumor was established by
inoculating 0.2 ml of CT26 cell suspension (3 × 106 cells/ml) into the
right axillary region of mouse. After 24 h, the mice were randomly di-
vided into six groups (six mice in each group) and received intragastric
administration of RPS-1 or 5-FU for 10 days. The three groups of mice
received administration of RPS-1 at different dosages (50, 100 and
200 mg/kg/day), respectively. The positive control group was treated
with 5-FU (20 mg/kg/day), and the control group was administered
0.9% saline solution. In the combination group, RPS-1 (200 mg/kg/
day) was applied in combination with 5-FU (20 mg/kg/day). The ani-
mals were sacrificed when intervention caused mouse weight loss of
30% or more. Thymus and spleen indexes were calculated as the weight
of thymus and spleen relative to body weight: organ index (mg/g) =
Worgan/Wbody, where Worgan and Wbody were the weight of the organ
and body weight of mice, respectively. The tumor inhibitory rate was
Fig. 2. Monosaccharide composition analysis of RPS-1. RPS-1 was hydrolyzed
by 2.0 M trifluoroacetic acid (TFA) at 110 °C for 4 h and react with 0.5 M PMP.
PMP derivatives of RPS-1 (black line) and monosaccharide standard samples
(blue line) (1-Man, 2-GlcN, 3-Rha, 4-GlcA, 5-GalA, 6-GalN, 7-Glc, 8-Gal, 9-Xyl,
10-Ara and 11-Fuc) were analyzed by HPLC (UltiMate 3000) equipped with
Kromasil C18 HPLC column (4.6 mm × 150 mm, 5 μm) and ultraviolet detector
(For interpretation of the references to color in this figure legend, the reader is
referred to the web version of this article).
direct evidence of the chain conformation of RPS-1, molecular mor-
phology of RPS-1 in water was observed by atomic force microscope
imaging system (JPK, Germany). As shown in Supplementary Fig. 1,
individual spherical particles were imaged clearly. However, the slow
drying process of liquid samples prior to AFM analysis resulted in ag-
gregation of polysaccharide molecules.
calculated by following formula: tumor inhibitory rate (%) = (Wcontrol
-
Wtreated)/Wcontrol×100%, where Wtreated and Wcontrol were the average
tumor weight of the treated and control group, respectively.
2.14. Statistical analysis
RPS-1 showed negative reaction with iodine-iodide and had no
absorption at 280 nm and 260 nm, which indicated the absence of
starch-type polysaccharide, protein and nucleic acid. The total sugar
content of RPS-1 was 99.28%. According to monosaccharide composi-
tion analysis, only one peak was observed and identified as glucose
(Fig. 2). The IR spectrum of RPS-1 showed the characteristic peaks of
polysaccharide at 3310.41, 2928.66 and 1652.04 cm−1 (Fig. 3). Ac-
cording to data of methylation analysis, the linkage types of RPS-1
contained Glcp-(1→, (1→4)-linked glucopyranosyl units and (1→4, 6)-
linked glucopyranosyl units in the molar ratio of 6.9: 86.1: 6.9 (Sup-
plementary Fig. 2 and Supplementary Table 1). Signals of RPS-1 in 1D
NMR spectra were assigned on the basis of monosaccharide composi-
tion, linkage analysis, and chemical shift by comparing with previous
studies in combination with the peak intensity (Synytsya & Novak,
2013). Only signals of α-D-glucan appeared in the anomeric region of
both the 1H (5.35 and 4.92 ppm) and 13C NMR (99.86, 76.95, 69.43,
60.58 ppm) spectra, which indicated all of the residues are of α-con-
figuration (Fig. 4).
All data were analyzed using one-way ANOVA using SPSS13.0
software and presented as the mean standard deviation (SD). *P va-
lues of less than 0.05 were considered statistically significant.
3. Results
3.1. Isolation and purification of RPS-1
The crude polysaccharides from liquid-cultured mycelia of R. ni-
gricans were obtained by hot water extraction, ethanol precipitation,
deproteinization and decoloration (Fig. 1A). After separation by DEAE
Sepharose Fast Flow chromatography, the unabsorbed fraction, termed
as neutral polysaccharides (Fig. 1B), was further purified by Sephacryl
S-500 HR chromatography. The major peak, appeared as single and
symmetrical, was collected and lyophilized, named as RPS-1 (Fig. 1C).
The HPSEC profile of RPS-1 showed a single and symmetrical sharp
peak, indicating its homogeneity (Fig. 1D). The weight-average mole-
cular weight (Mw) of RPS-1 was 1.617 × 107 Da and its z-average root
mean square radius (RMS) was 27.5 nm (Table 1).
From the aforementioned results, RPS-1 was deduced to be a gly-
cogen-like polysaccharide consisting of a backbone structure of (1→4)-
linked α-D-glucopyranosyl residues substituted at the O-6 position with
α-D-glucopyranosyl branches on average 14.5 residues.
3.2. Physicochemical properties and structural feature of RPS-1
According to the data of MALLS, the slope of log(RMS radius)/log
(Mw) plot was 0.15, less than 0.33 (Table 1), which indicated that RPS-1
existed as compact spherical conformations (Wyatt, 1993). To provide
3.3. RPS-1 stimulated the production of NO and TNF-α in macrophages
Macrophages are key effector cells of the innate immune response to
Table 1
Experimental data of MALLS for RPS-1 in 0.2 mol/l sodium nitrate solution at 25 °C.
Molar mass (×107 Da)
Polydispersity index
RMS radius (nm)
Conformation plot slope
Mn
Mw
Mz
Mw/Mn
Mz/Mn
Rn
Rw
Rz
Log(radius)/log(Mw)
1.214
1.617
2.958
1.332
2.437
22.9
24.2
27.5
0.15
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CarbohydratePolymers198(2018)302–312
Fig. 3. Infrared spectrum analysis of RPS-1. The infrared spectrum of RPS-1 was recorded with a Nicolet Avatar 370 spectrometer (Nicolet, USA) in the range of
400–4000 cm−1
.
pathogen invasion and contribute to tumor destruction. They play im-
portant roles in the antigen presentation and clearance of pathogens
and cancerous cells. Several studied have reported the immuno-en-
hancing activity and antitumor effect of natural and enzymatically
synthesized (1→4)-α-glucan (Kakutani et al., 2012; Ryoyama, Kidachi,
Yamaguchi, Kajiura, & Takata, 2004; Takaya et al., 1998). To explore
the potential immunomodulatory activity of RPS-1, we studied the ef-
fects of RPS-1 on the activation of murine macrophages RAW 264.7
cells. Firstly, the cytotoxic effect on RAW 264.7 cells was analyzed by
MTT assay and RPS-1 failed to show any cytotoxicity up to 1 mg/ml in
vitro (data not shown). We detected the NO and TNF-α in RAW 264.7
cells challenged with RPS-1. As shown in Fig. 5, the release of TNF-α
was significantly stimulated by RPS-1 from 50 to 400 μg/ml, whereas
RPS-1 stimulated the production of NO only at high concentrations
(200 and 400 μg/ml). To exclude the possible contamination of LPS in
RPS-1, RAW 264.7 cells were pretreated with PMB, a specific inhibitor
of LPS, followed by treatment with RPS-1 or LPS for 24 h. PMB could
significantly blocked the production of NO and TNF-α induced by LPS,
while pre-incubation with PMB only slightly attenuated RPS-1 induced
cytokine and NO secretion (Supplementary Fig. 3). These results sug-
gested that RPS-1 could activate murine macrophages RAW 264.7 cells.
bands attenuated with prolonged incubation time. In LPS-activated
macrophages, rapid phosphorylation of p38, JNK1/2 and ERK1/2 was
observed in 5 min. The protein levels of phosphorylated active form of
p38 and JNK gradually returned to the basal level after 30 min, whereas
ERK1/2 phosphorylation was maintained at high level even after
60 min. In summary, RPS-1 induced phosphorylation of all three
MAPKs in a time-dependent pattern which was different from LPS sti-
mulation.
Further, to clarify whether RPS-1 could activate NF-κB pathway, the
levels of IκBα and NF-κB p65 subunit were assessed in the cytosolic and
nuclear fractions, respectively. After exposure to RPS-1 for 30 min, the
red fluorescence of NF-κB p65 subunit was increased in nuclear regions
compared with control group (Fig. 7A). Upon RPS-1 stimulation, a
continuous reduction of IκBα in the cytosol and a concomitant increase
of NF-κB p65 in the nucleus were noted, which indicated that RPS-1
induced nuclear translocation of NF-κB p65 (Fig. 7B). According to the
data of luciferase reporter gene assay, a significantly increase of tran-
scriptional activity of NF-κB was observed in RPS-1 treated group as
well as in LPS stimulated group (Supplementary Fig. 4). These results
indicated that RPS-1 induced nuclear translocation and activation of
NF-κB in RAW 264.7 cells.
3.4. RPS-1 activated MAPKs and NF-κB pathways in RAW 264.7 cells
3.5. The roles of MAPKs and NF-κB pathways in macrophage activation
induced by RPS-1
There are three well-defined MAPK pathways: the extracellular-
signal-regulated kinase (ERK) pathway, the JUN N-terminal kinase
(JNK) pathway and the p38 pathway (Johnson & Lapadat, 2002). To
investigate whether the MAPK pathways were involved in RPS-1 in-
duced macrophage activation, we performed western blot analysis to
detect the phosphorylation of MAPKs in RAW 264.7 cells. As shown in
Fig. 6, upon challenged with RPS-1, the elevated phosphorylation of
MAPKs appeared after 15 min and the maximal phosphorylation of p38
and JNK1/2 occurred at 15 min and 30 min, respectively. After that, the
To confirm the roles of MAPKs and NF-κB signaling pathways in the
secretion of NO and TNF-α induced by RPS-1, RAW 264.7 cells were
preincubated with specific inhibitors for 1 h. As shown in
Supplementary Fig. 5, production of NO and TNF-α induced by RPS-1
was almost completely inhibited by BAY 11-7082, inhibitor of NF-κB.
The inhibitors of MAPKs could significantly weakened TNF-α release,
whereas SP600125 and PD98059 approximately suppressed half of NO
production. However, SB203580, inhibitor of phospho-p38, failed to
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CarbohydratePolymers198(2018)302–312
Fig. 4. 1H and 13C NMR spectra of RPS-1. A total of 10 mg of RPS-1 was dissolved in 0.5 ml of D2O. (A) 1H and (B) 13C NMR spectra of RPS-1 were recorded on a
Bruker AVANCE-300 MHz NMR spectrometer (Bruker, Germany) at 25 °C.
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CarbohydratePolymers198(2018)302–312
Fig. 5. RPS-1 stimulated the production of NO and TNF-α in RAW 264.7 cells. RAW 264.7 cells were treated with indicated concentrations of RPS-1 or LPS (100 ng/
ml) for 24 h. Supernatant (A) NO and (B) TNF-α levels were detected by Griess reagent and ELISA, respectively. Results were expressed as mean values
or n = 6, * p ≤ 0.05 versus control group).
SD (n = 3
show inhibitory effect on the NO production induced by RPS-1
(Supplementary Fig. 5A), which suggested that distinct expression
pattern of NO and TNF-α was involved in the activation of RAW 264.7
cells in response to RPS-1.
CT26 bearing mice was significantly inhibited in 200 mg/kg/day group
and increased immune organ indexes were simultaneous observed in
RPS-1 treatment group from 50 to 200 mg/kg/day (Table 2). We found
that treatment of RPS-1 resulted in higher secretion of Th1 cytokines,
including IL-2 and TNF-α, as well as immunoglobulin G in syngeneic
tumor model (Fig. 8A–C). RPS-1 and 5-FU, alone and combination,
tilted the balance of Bcl-2 and Bax towards proapoptosis functions in
tumor tissues (Fig. 8D). Moreover, combined administration of RPS-1
(200 mg/kg/day) and 5-FU (20 mg/kg/day) showed stronger antitumor
activity and partially alleviated immunosuppression induced by 5-FU
(Table 2 and Fig. 8).
3.6. RPS-1 suppressed the tumor growth in CT26 tumor-bearing mice
The macrophage activation induced by RPS-1 in vitro prompted us
to evaluate the antitumor and immunomodulatory properties in vivo.
Whether polysaccharides display biological activity depends on the
stable spatial configuration under physiological conditions (Masuda
et al., 2017; Zhang et al., 2005). RPS-1 was characterized as α-(1→6)-
branched α-(1→4)-glucan and the pancreatic α-amylase in small in-
testinal lumen may digest RPS-1 and impaired its potential im-
munoregulatory activity in vivo. Therefore, we tested the degradation
of RPS-1 treated with pancreatic α-amylase by particle size analysis. As
shown in Supplementary Fig. 6A, amylopectin from maize, highly-
branched α-1,4 glucan with α-1,6 branch, was digested in several hours
and further degraded at 12 h after treatment of pancreatic α-amylase.
Under the same conditions, the average diameter of RPS-1 was slightly
decreased until 12 h, from 59.31 nm to 56.25 nm (Supplementary
Fig. 6B and Supplementary Table 2). These data suggest that RPS-1 was
more resistant to pancreatic α-amylase than natural amylopectin. The
compact globular molecular conformations might partially render RPS-
1 resistant to α-amylase.
4. Discussion
For many years, harnessing the immune system to fight cancer is a
promising approach for the majority of cancer patients (Couzin-Frankel,
2013). Natural fungal polysaccharides possess immunomodulatory ac-
tivity and have been used to improve general health for decades in
Asian countries, especially in combination with cytotoxic-chemother-
apeutic agent for the treatment of cancers. In this study, we demon-
strated that R. nigricans polysaccharide RPS-1, α-(1→6)-branched α-
(1→4)-glucan, activated macrophage and significantly inhibited tumor
growth in CT26 tumor-bearing mice.
Growing evidences have revealed the crucial role of structure in the
bioactivity of polysaccharide (Li et al., 2016). Several structural fea-
tures were involved in the structure-activity relationship of poly-
saccharide, including monosaccharide composition, molecular weight,
We established subcutaneous CT26 tumor-bearing model in BALB/c
mice. After intragastric administration of RPS-1, the tumor growth of
Fig. 6. RPS-1 induced phosphorylation of p38, JNK and ERK in RAW 264.7 cells. After treated with RPS-1 or LPS for indicated time, levels of protein expression were
analyzed by western blot analysis.
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Fig. 7. RPS-1 induced nuclear translocation of NF-κB in RAW 264.7 cells. (A) After treated with RPS-1 (400 μg/ml) or LPS (100 ng/ml) for 30 min, cells were fixed
and incubated with primary antibody against NF-κB p65 and Cy-3 conjugated secondary antibody. After stained with DAPI, the prepared samples were photographed
by using fluorescence microscopy (Olympus, Japan). (B) RAW 264.7 cells were stimulated with RPS-1 or LPS for indicated time (5, 15, 30, 60 min) and nuclear and
cytoplasmic extracts were prepared. Levels of protein expression of NF-κB p65 and IκBα were measured by western blot analysis.
glycosidic linkage types and the conformation. Thus, structure char-
acteristics of RPS-1 were investigated to elucidate the basis of biological
activity. The weight-average molecular weight of RPS-1 was estimated
to be 1.617 × 107 Da and its RZ was 27.5 nm (Fig. 1D and Table 1),
which was similar to results reported from Ostrea edulis glycogen
(Fernandez, Rojas, & Nilsson, 2011). RPS-1 was identified as a homo-
geneous water-soluble glucan consisting of (1→4)-linked α-D-Glcp re-
sidues and (1→4, 6) linked α-D-Glcp branches (Figs. 2 and 4). The fine
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CarbohydratePolymers198(2018)302–312
Table 2
Effects of RPS-1 on the body weight, immune organ index and tumor growth of CT26 tumor-bearing mice (n = 6, * p ≤ 0.05 versus control group, # p ≤ 0.05 versus
5-FU group).
Group
Dosage
mg/kg/day)
Body weight
(g)
Spleen index (mg/g)
Thymus index (mg/g)
Tumor weight
(g)
Control
RPS-1
–
50
100
200
23.69
24.14
25.26
25.02
17.90
19.39
2.24
9.13
9.37
10.18
9.18
4.68
6.15
2.02
0.89
0.81
4.37
4.86
5.29
4.95
3.04
3.99
0.58
0.58
0.51
0.49
0.33
0.28
0.16
0.13
0.15
0.14
0.17*
0.12*
0.14*
1.45
0.67
1.12
2.95*
1.59*
0.61
0.62*
0.63
1.26
5-FU
20
1.38*
1.56*
0.69*
0.75#
Combined treatment
200 (RPS-1)
+ 20 (5-FU)
structures of these macromolecules including branching degree, mole-
cular weight and spatial structure play a key role in the physiological
activities (Kakutani et al., 2007). A polysaccharide (YCP) possessed
linear α-(1→4) bonded glucopyranoside main chain and α-(1→6) side
chain from marine filamentous fungus Phoma herbarum YS4108 in-
creased phagocytic activity of mice macrophages in vitro and in vivo
(Yang et al., 2005). However, the fragments of YCP with different
molecular weight showed different immunological activities and re-
ceptor signaling pathways (Ren, Yan, Yao, Jin, & Gao, 2010; Zhu et al.,
2014). The lengths of backbone and side chains as well as branches are
not known from this study and further research should be performed to
ascertain the primary structure and spatial configuration of RPS-1 in
physiological conditions.
backbone exhibit various physiological effects in vitro and vivo such as
anticancer and immunoregulation (Masuda et al., 2017; Takaya et al.,
1998). Response to biologically active polysaccharides, macrophage
released a variety of inflammatory cytokines, chemokines and other
immune-modulating mediators to modulate immune function and in-
creased capacity to eliminate invading pathogens and tumor cells (Lee
et al., 2015; Wang et al., 2014). RPS-1 promoted production of NO and
TNF-α in RAW 264.7 murine macrophages in a concentration-depen-
dent manner (Fig. 5) and pretreatment with PMB showed no obvious
effects (Supplementary Fig. 3). MAPKs are a group of serine/threonine
protein kinases and have an important role in variety cellular processes,
such as proliferation, differentiation, apoptosis and immune defence. A
number of immunomodulatory agents have been found to modulate
both innate and adaptive immunity by influencing MAPK signaling
cascade (Alamuru et al., 2014; Xu, Pan, Zhang, & Ashida, 2011). To
Recently, some natural and enzymatically synthesized poly-
saccharides consisting of highly α-(1→6)-branched α-(1→4)-glucan
Fig. 8. Effects of RPS-1 on serum cytokines levels and apoptotic related proteins expression levels. At the end of the experiment, orbital sinus blood sample was
collect and the concentrations of (A) TNF-α and (B) IL-2 were assayed by commercial ELISA kits (* p ≤ 0.05 versus control group, # p ≤ 0.05 versus 5-FU group).
(C) Tumor tissue lysates were prepared and levels of protein expression were analyzed by western blot analysis. Comb: combined treatment.
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explore the possible molecular mechanisms involved in the activation
of macrophage induced by RPS-1, we detected the expression of pro-
teins that are associated with MAPK signaling pathway. The elevated
phosphorylation of MAPKs was observed after treated with RPS-1 for
15 min in RAW 264.7 cells (Fig. 6). The inhibitors of JNK and ERK
significantly weakened NO and TNF-α release (Supplementary Fig. 5).
However, inhibitor of phospho-p38, SB203580, had no influence on the
NO production (Supplementary Fig. 5A). These results demonstrated
that MAPKs and NF-κB signaling pathways participated in the produc-
tion of NO and TNF-α induced by RPS-1.
In unstimulated cells, NF-κB is sequestered in the cell cytoplasm as
an inactive protein complex by IκB family proteins (e.g., IκBα, IκBβ,
and IκBε) (Ghosh, May, & Kopp, 1998). Response to external stimulus
with a variety of agonists, inhibitor of nuclear factor kappa-B kinase
(IKK) induced phosphorylation of IκBα leading to degradation via
ubiquitination pathway (Karin & Ben-Neriah, 2000). It is now generally
accepted that NF-κB signaling pathway participate in the expression of
inflammatory cytokines and mediators in macrophage. The recognition
of ligands by pattern recognition receptors on the cell surface or in the
cytoplasm of innate immune cells initiates activation of MAPKs and NF-
κB, leading to the expression of multiple genes that modulate the in-
flammatory response (Arthur & Ley, 2013; Hayden & Ghosh, 2004).
Consistent with such a role, NF-κB is employed to manipulate the signal
output of heterologous receptors of immune cells upon exposure to
immunoregulatory polysaccharides (Chen et al., 2017). After treated
with RPS-1, a continuous reduction of IκBα in the cytosol and a con-
comitant increase of NF-κB p65 in the nucleus were noted (Fig. 7A and
B). Consistent with this, RPS-1 significantly increased transcriptional
activity of NF-κB (Supplementary Fig. 4).
Conflict of interest statement
The authors have declared no conflict of interest.
Acknowledgements
This study was supported by Major Projects from the Science and
Technology Program of Shandong Province (the Key Technology) (No.
2015ZDJS04002), the Major State Basic Research Development
Program of China (973 Program) (No. 2012CB822102), the High
Technology Research and Development Program of China (863
Program) (No. 2012AA021501), National Natural Science Foundation
of China (NSFC) (No. 81702581) and the Scientific and Technological
Innovation Commission of Shenzhen (No. JCYJ20170306091400043).
Appendix A. Supplementary data
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.carbpol.2018.06.076.
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