Endo-polysaccharide of Phellinus igniarius exhibited anti-tumor effect through enhancement of cell mediated immunity
Li Chen a ,Jingzhi Pan a ,Xue Li b ,Yong Zhou c ,Qinglong Meng a ,Qi Wang d ,?
a
College of Chinese Medicinal Materials,Jilin Agricultural University Changchun,Jilin,130118PR China b
Tianyao Limited,Jilin,130118PR China c
Xiuzheng Pharmaceutical Group,Jilin,130118PR China d
Engineering Research Center of Chinese Ministry of Education for Edible and Medicinal Fungi,Jilin Agricultural University Changchun,Jilin,130118PR China
a b s t r a c t
a r t i c l e i n f o Article history:
Received 22May 2009
Received in revised form 1November 2010Accepted 23November 2010
Available online 9December 2010Keywords:
Polysaccharide Cytokine Tumor Immunity
The purpose of this study was to assess the anti-tumor and immunomodulatory effects of PIE on tumor cells Sarcoma 180and Hepatoma 22in implanted mice.The monosaccharide composition of PIE was analyzed by GC.The results demonstrated that PIE monosaccharide composition was n(Xyl):n(Man):n (Fuc):n(Glc):n(Gal)=2.3:1:6.4:22.1:19.83.The oral administration of PIE characteristically inhibited the growth of S180and H22cells and increased the life span.The concentrations of serum IL-2and IL-18were signi ?cantly increased in the S180implanted mice fed with PIE in the doses of 500mg/kg and 250mg/kg compared with those in control (p b 0.01).The concentrations of serum IL-2were signi ?cantly increased in H22implanted mice in the doses of 500mg/kg and 250mg/kg compared with those in control (p b 0.01).
?2010Elsevier B.V.All rights reserved.
1.Introduction
Malignant tumor is one of the leading causes of death in the world despite newly developed tools and miracle drugs for treatment and diagnosis.The main cause is that immune systems were damaged in tumor treatment.Therefore,it is very important to investigate novel anti-tumor substances with improving immunity potential.Many polysaccharides isolated from mushroom are used as adjuvant in the treatment of cancer disease [1–4].Lentinan,Schizophyllan and Polysaccharide-K have been known to inhibit the growth of various transplantable tumors in experimental animals and increase the survival rate [5],and they have been accepted as immunoceuticals in several oriental countries [6–9].
Phellinus igniarius (Linnearus:Fries)Quelet is a well-known traditional Chinese medicinal fungi.Over the past several decades,researches have shown that the extract from the fruit body of P.igniarius has anti-in ?ammatory,antioxidative activities and also has the ability to be an anti-tumor [10–12].However,there are few reports focusing on the relationship between the anti-tumor and immunostimulatory activities of the endo-polysaccharide extracted from the submerged fermentation product of P.igniarius (PIE).In this study,PIE was tested for its potent effect to suppress the proliferation of tumor cells S180and H22in implanted mice.The serum cytokines of interleukin-2(IL-2),interleukin-18(IL-18)and tumor necrosis
factor-α(TNF-α)were also analyzed in order to obtain additional information on underlying mechanisms by which PIE might express the anti-tumor effects.2.Materials and methods 2.1.Experimental materials
Water-soluble crude polysaccharide was extracted from the cultured mycelium of P.igniarius .After ethanol precipitation and Sepharose G-100gel ?ltration the fraction of PIE was obtained with a yield of 14.78%.
https://www.doczj.com/doc/8118097776.html,positional analysis
The composition of PIE was analyzed by gas chro-matograph,the sample was alcoholized with methyl alcohol and silyllanized and then detected using a Varian 7890GC spectrometer (FID detector)with an SE-5415m ×0.2mm column.
The infrared spectrum of the sample was recorded on a Perkin-Elmer infrared spectrometer using the KBr disk technique.2.3.Mice
Six-weeks-old female ICR mice (18±2)g were purchased from Changchun Institute of Biological Products.The mice were kept in plastic cages.The mice were housed under normal laboratory
International Immunopharmacology 11(2011)255–259
?Corresponding author.
E-mail address:q_wang2006@https://www.doczj.com/doc/8118097776.html, (Q.
Wang).
1567-5769/$–see front matter ?2010Elsevier B.V.All rights reserved.doi:
10.1016/j.intimp.2010.11.033
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conditions (24±2°C,50±20%,12/12-h light –dark cycle)with free access to standard rodent chow and water.
2.4.Cell lines
S180and H22cell lines were purchased from the Jilin Institute of Tumor (Changchung,China).S180and H22cells were maintained as ascites in the Kunming mice by weekly passage [13].The Sarcoma 180cells,obtained from the peritoneal cavity of the tumor inoculated mice,were washed twice with phosphate buffered saline (PBS)and resuspended and adjusted to 1×106cells/ml in PBS.The cells were detached and adjusted to 5×106cells/ml in PBS.
2.5.Animal experiment
2.5.1.Solid tumor-bearing mice model
The implantation of S180and H22was conducted with the same methods [14].Brie ?y,the tumor cells (1.0×108cells/ml)were diluted to 4-fold with 0.9%NaCl.The diluted solution was inoculated to the right armpit of the mice subcutaneously for 0.2ml per mouse on day 0.The tumor bearing mice were divided into ?ve groups (ten mice each group),control group (normal saline),positive control group [Cyclo-phosphamide (CTX)30mg/kg],and three groups for PIE administration with dosages of 500,250,125mg/kg,respectively.Another ten mice were taken as a normal group treated with rodent chow.After administrating intragastrically once for 10days daily,tumor bearing mice were sacri ?ced on the 11th day,and sera,tumors,spleen and
thymus were collected.The inhibiting ratios of the tumor and organ indices were calculated using the following equation [14,15].
Inhibiting ratio (%)=[(C ?T)/C]×100%(C:average weight of tumor in model control group;T:average weight of tumor in medicine treatment group).Organ index=organ weight (in milligrams)/body weight (in grams).Each experiment was repeated three times.
Serum was obtained after incubating at room temperature and centrifugation at 3000rpm for 15min.IL-2,TNF-αand IL-18were examined with the commercial ELISA kits (Biowen,Canada).
2.5.2.Ascites tumor-bearing mice model
The tumor cells of S180and H22(1.0×108cells/ml)were diluted to 4-fold with 0.9%NaCl,respectively.The viability,assessed by the trypan blue dye exclusion method,was always found to be 95%or more.Mice were inoculated intraperitoneally (i.p.)for 0.2ml diluted solution on day 0.The tumor bearing mice were divided into ?ve groups,control group (normal saline),positive control group (CTX 30mg/kg),and three groups for PIE administration with dosages of 500,250,125mg/kg,respectively.The mice were administered intra-gastrically once for 10days daily,and recorded the survival days of mice.The increased life span (ILS)was calculated using the following equation [15].
ILS%=(T ?C)/C×100%(T:mean survival days of treated group;C:mean survival days of control group).2.6.Statistical analysis
The data were expressed as mean±standard deviation (SD).Treated and control groups were compared using Student's t -test.A signi ?cant difference was set up at P b 0.05.3.Results
3.1.Determination of PIE purity
The purity of PIE was analyzed by using HPLC and Sepharose CL-6B gel ?ltration.Fig.1showed that PIE had a single nearly narrow symmetrical peak,which indicated that it was https://www.doczj.com/doc/8118097776.html,position and physical property of PIE
PIE is a water-soluble grayish white powder,its molecular weight is 12kd,its monosaccharide composition,n(Xyl):n(Man):n(Fuc):n(Glc):n(Gal)=2.3:1:6.4:22.1:19.83,was analyzed by GC (Fig.2).
00.20.40.60.81
1.2
1.41.60
10
20
30
40
50
60
Tube number
O p t i c a l d e n s i t y
Fig.1.Chromatogram of PIE on Sepharose G-100.X-coordinate was tube number,y-coordinate was optical density.The purity of PIE was analyzed by using HPLC and Sepharose CL-6B gel ?ltration.PIE had a single nearly narrow symmetrical
peak.
Fig.2.Gas chromatogram of monosaccharide composition of PIE.The composition of PIE was analyzed by gas chro-matograph,the sample was alcoholized with methyl alcohol and silyllanized and then detected using a Varian 7890GC spectrometer (FID detector)with an SE-5415m ×0.2mm column.
256L.Chen et al./International Immunopharmacology 11(2011)255–259
PIE was masculine in Molish and showed special absorption peaks in IR.They did not contain the glucosidic bond of α(1→4),because of the negative reaction of I.No absorption peaks at 260nm and 280nm show that there were no nucleic acid and protein.3.3.Anti-tumor activity of solid tumor-bearing mice and the concentrations of serum TNF-α,IL-2,and IL-18
The growth of S180tumors in the mice treated orally with PIE was signi ?cantly suppressed compared with those treated with saline (p b 0.01).The inhibiting ratios of S180tumors were 43.91%,53.87%and 35.42%respectively.H22tumors were inhibited at 44.41%,40.47%and 38.16%respectively (Tables 1,2).Oral administration of PIE increased the spleen and thymus indexes of S180and H22-bearing mice,whereas the CTX treatment signi ?cantly decreased these indexes as compared with the control group (p b 0.05–0.01).(Figs.3,4).3.4.The concentrations of serum TNF-α,IL-2,IL-18
The concentrations of IL-2of S180-bearing mice and H22implanted mice were signi ?cantly enhanced during the administra-tion of PIE in the doses of 500mg/kg,250mg/kg (p b 0.01)and 125mg/kg (p b 0.05)compared with those in the control (Figs.5,6).The oral administration of PIE stimulated the production of serum IL-18in S180-bearing mice at doses of 500mg/kg and 250mg/kg (p b 0.01)(Fig.7).The concentrations of serum IL-18in H22-bearing mice were enhanced by treatment with PIE and CTX compared to those in the control group (Fig.8).However,no signi ?cant changes were found among the tested groups.The serum TNF-αhave also been enhanced,although these changes were not statistically signi ?cant among the tested groups (Figs.9,10).
3.5.Anti-tumor activity of ascites tumor-bearing mice
The mean survival days of the treated group were prolonged compared with the mean survival days of the control group.The life span of S180-bearing mouse model was signi ?cantly prolonged by PIE at 12.90%22.86%and 16.92%,respectively.The increased life span of H22-bearing mouse model was signi ?cantly prolonged by PIE at 44.86%,30.84%and 29.81%,respectively.The oral administration of PIE increased the life span of S180and H22bearing mice,while the
Table 1
Effects of orally administrated PIE on S180-bearing mice (n =10;ˉx ?s ).Group
Dose (mg/kg)
Weight (g)Tumor weight (g)
Inhibition rate (%)
Before treatment
After treatment Control
21.35±1.2931.25±2.72 2.71±0.64Positive control (CTX)3021.30±1.2328.15±2.60?0.85±0.26??68.63High dose group 50021.65±1.1033.10±1.78? 1.52±0.29??43.91Medium dose group 25021.40±1.2030.30±1.16 1.25±0.52??53.87Low dose group
125
21.30±1.18
33.35±3.11
1.75±0.51??
35.42
S180-bearing mice were orally administered saline (control group),CTX 30mg/kg (positive control,CTX)or PIE at 500,250or 125mg/kg once daily,for 10days.Each value is presented as mean±S.D.of the three independent experiments (n =10).Signi ?cant difference:*P b 0.05and **P b 0.01(Student's t -test).?P b 0.05to those of control.??P b 0.01to those of control.
Table 2
Effects of orally administrated PIE on H22-bearing mice (n =10;ˉx ?s ).Group
Dose (mg/kg)
Weight (g)Tumor weight (g)
Inhibition rate (%)
Before treatment
After treatment Control
22.29±1.3627.91±1.70 3.04±0.46Positive control (CTX)3022.58±1.7826.58±2.32?? 1.13±0.53??62.83High dose group 50021.58±1.7426.95±2.98 1.69±0.51??44.41Medium dose group 25021.88±1.2025.29±3.26 1.81±0.51??40.47Low dose group
125
22.08±1.66
27.82±2.03
1.88±0.57??
38.16
H22-bearing mice were orally administered saline (control group),CTX 30mg/kg (positive control,CTX)or PIE at 500,250or 125mg/kg for once daily,for 10days.Each value is presented as mean±S.D.of the three independent experiments (n =10).Signi ?cant difference:*p b 0.05and **p b 0.01(Student's t -test).*P b 0.05to those of control.**P b 0.01to those of control.
(m g /g )
Fig.3.Immune organ in S180-bearing mice.S180-bearing mice were orally adminis-tered saline (control group),CTX 30mg/kg positive control,CTX or PIE at 500,250or 125mg/kg once daily,for 10days.Each value is presented as mean ±S.D.of the three independent experiments (n =10).Signi ?cant difference:*p b 0.05and **p b 0.01(Student's t -test).
(m g /g )
Fig.4.Immune organ in H22-bearing mice.H22-bearing mice were orally administered saline (control group),CTX 30mg/kg positive control,CTX or PIE at 500,250or 125mg/kg for once daily,for 10days.Each value is presented as mean ±S.D.of the three independent experiments (n=10).Signi ?cant difference:*p b 0.05and **p b 0.01(Student's t -test).
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CTX treatment signi ?cantly decreased these indexes compared to the control group (P b 0.05–0.01)(Tables 3,4).4.Discussion and conclusions
The polysaccharides extracted from a large number of mushrooms have been found to be effective against Sarcoma 180tumors in mice [16].Many mushroom polysaccharides are accepted as anti-tumor drugs because of immunomodulating activities [17].For example,the in vitro study of Song showed that the ethanolic extract from the fruit body of P.igniarius had inhibited effects on human hepatocarcinoma SK-Hep-1cells and the rat heart vascular endothelial cells (RHE cells)[18].The study of Yang showed that the fungi of the P.igniarius extracts had obviously anti-tumor effects [19].However,a lot of the bene ?cial effects of the P.igniarius extracts have not been investigated and are largely overlooked at the biological and biochemical levels.
PIE from P.igniarius prolonged the survival time and inhibited the growth of the transplanted tumors,without causing a signi ?cant decrease in the body,spleen or thymus weight in mice.A signi ?cant increase in the relative thymus and spleen weight was observed compared with the control group.The relative spleen and thymus weight are important indexes for non-speci ?c immunity.The immunopotentiator could increase the spleen and thymus weights [20].PIE could restore the immunity of mice which was suppressed by the tumor cells.Thus,the anti-tumor activity of PIE could be due to immunostimulation.
In this study,serum IL-2was signi ?cantly increased in the tumor bearing mice after the oral administration of PIE.Therefore,the anti-tumor effects of PIE could be greatly attributed to the
enhancement
IL-2(S180)
(p g /m l )
Fig.5.Serum IL-2in S180-bearing mice.S180-bearing mice were orally administered saline (control group),CTX 30mg/kg positive control,CTX or PIE at 500,250or 125mg/kg once daily,for 10days.The mice in the normal group were not implanted tumor.Each value is presented as mean±S.D.of the three independent experiments (n =10).Signi ?cant difference:*p b 0.05and **p b 0.01(Student's t
-test).
IL-2 (H22)
(p g /m l )
Fig.6.Serum IL-2in H22-bearing mice.H22-bearing mice were orally administered saline (control group),CTX 30mg/kg positive control,CTX or PIE at 500,250or 125mg/kg once daily,for 10days.The mice in the normal group were not implanted tumor.Each value is presented as mean±S.D.of the three independent experiments (n =10).Signi ?cant difference:*p b 0.05and **p b 0.01(Student's t
-test).
IL-18 (S180)
(p g /m l )
Fig.7.Serum IL-18in S180-bearing mice.S180-bearing mice were orally administered saline (control group),CTX 30mg/kg positive control,CTX or PIE at 500,250or 125mg/kg once daily,for 10days.The mice in the normal group were not implanted tumor.Each value is presented as mean±S.D.of the three independent experiments (n =10).Signi ?cant difference:*p b 0.05and **p b 0.01(Student's t
-test).IL-18 (H22)
(p g /m l )
Fig.8.Serum IL-18in H22-bearing mice.H22-bearing mice were orally administered saline (control group),CTX 30mg/kg positive control,CTX or PIE at 500,250or 125mg/kg once daily,for 10days.The mice in the normal group were not implanted tumor.Each value is presented as mean ±S.D.of the three independent experiments (n =10).Signi ?cant difference:*p b 0.05and **p b 0.01(Student's t
-test).
TNF-α (S180)
(p g /m l )
Fig.9.Serum TNF-αin S180-bearing mice.S180-bearing mice were orally administered saline (control group),CTX 30mg/kg positive control,CTX or PIE at 500,250or 125mg/kg once daily,for 10days.The mice in the normal group were not implanted tumor.Each value is presented as mean ±S.D.of the three independent experiments (n =10).Signi ?cant difference:*p b 0.05and **p b 0.01(Student's t
-test).
TNF-α (H22)
(p g /m l )
Fig.10.Serum TNF-αin H22-bearing mice.H22-bearing mice were orally administered saline (control group),CTX 30mg/kg positive control,CTX or PIE at 500,250or 125mg/kg once daily,for 10days.The mice in the normal group were not implanted tumor.Each value is presented as mean ±S.D.of the three independent experiments (n =10).Signi ?cant difference:*p b 0.05and **p b 0.01(Student's t -test).
258L.Chen et al./International Immunopharmacology 11(2011)255–259
of IL-2production by PIE,which could enhance the cytolytic activity of NK cells and cytolytic T lymphocytes.The serum IL-18were also found to be increased in the mice exposed to CTX and PIE,especially more individuals in the mice fed high dose of PIE showed more serum IL-18.IL-18is a novel pro-in?ammatory cytokine that augments the natural killer cells activity in spleen cells.This cytokine can induce the TNF-αproduction of the T cells.On the other hand,TNF-αalso was able to induce apoptotic cell death,in?ammation and inhibits tu-morigenesis and viral replication with a different way from IL-2.The increase of serum TNF-αwas not found in S180and H22tumor-bearing mice after the treatment with PIE.Direct cytotoxic activity against S180and H22were not detected.These results suggest that PIE can also stimulate TNF-αrelated anti-tumor activation as well as the IL-2dependent,cell mediated immune response.Such multi-immune regulator effects of PIE should be analyzed by identifying how the components of the PIE interact with immune cells.The additional clinical evidence should be obtained to support the anti-tumor effects observed in the animal studies.Further studies should be done to know the effects of the PIE on host immunity. References
[1]Yang Na,Li De-Feng,Feng Lei,Xiang Ye,Liu Wei,Sun Hui,Wang Da-Cheng.
Structural basis for the tumor cell apoptosis-inducing activity of an antitumor lectin from the edible mushroom Agrocybe aegerita.J Mol Biol2009;387: 694–705.
[2]Wasser SP.Medicinal mushrooms as a source of antitumor and immunomodulat-
ing polysaccharides.Appl Microbiol Biotechnol2002;60:258–74.
[3]Hattori TS,Komatsu N,Shichijo S,Itoh K.Protein-bound polysaccharide K induced
apoptosis of the human Burkitt lymphoma cell line,Namalwa.Biomed Pharmac-other2004;58:22–230.
[4]Ooi VE,Liu F.Immunomodulation and anti-cancer activity of polysaccharide–
protein complexes.Curr Med Chem2000;7:715–29.
[5]Liu MQ,Li JZ,Kong FZ,Lin JY,Gao Y.Induction of immunomodulating cytokines by
a new polysaccharide–peptide complex from culture mycelia of Lentinus edodes.
Immunopharmacology1998;40:187–98.
[6]Borchers AT,Stern JS,Hackman RM,Keen CL,Gershwin ME.Mushrooms,tumors
and immunity.Proc Soc Exp Biol Med1999;221:281–93.
[7]Liu F,Ooi VE,Fung MC.Analysis of immunomodulating cytokine mRNAs in the
mouse induced by mushroom polysaccharides.Life Sci1999;64:1005–11.
[8]Ooi VEC,Liu F.A review of pharmacological activities of mushroom polysacchar-
ides.Int J Med Mushrooms1991:195–206.
[9]Kidd PM.The use of mushroom glucans and proteoglycans in cancer treatment.Alt
Med Rev2000;5(1):4–27.
[10]Wang Y,Shang XY,Wang SJ,Mo SY,Li S,Yang YC,et al.Structures,biogenesis,and
biological activities of pyrano[4,3-c]isochromen-4-one derivatives from the Fungus Phellinus igniarius.J Nat Prod(United States)2007;70(2):296–9.
[11]Yang Y,Ye LB,Zhang JS,Liu FF,Tang GJ.Structural analysis of a bioactive
polysaccharide,PISP1,from the medicinal mushroom Phellinus igniarius.Biosci Biotechnol Biochem(Jpn)2009;73(1):134–9.
[12]Yang Y,Zhang JS,Liu YF,Tang QJ,Zhao ZG,Xia WS.Structural elucidation of a3-O-
methyl-D-galactose-containing neutral polysaccharide from the fruiting bodies of Phellinus igniarius.Carbohydr Res(Netherlands)2007;342(8):1063–70.
[13]Harada S,Sera K,Futatsugawa S,Yanagisawa T,Takahashi T,Tamakawa Y.The two
inverse pathways:the apoptosis and the multinucleated cell switched by the magnesium/zinc balance in irradiated sarcoma in vivo.Nucl Instrum Meth Phys Res B1999;150:158–66.
[14]Xu SY,Bian RL,Chen X.Methodology of pharmacology experiment.Beijing:
People's Medical Publishing House;1991.p.1423–30.
[15]Li YK,Wang QM,Zhou JH.Methodology of pharmacology experiment in China.
Shanghai:Shanghai Scienti?c and Technical Publishers;1991.p.512–3.
[16]Jong SC,Donovick R.Antitumor substance from fungi.Adv Appl Microbiol
1989;34:183–7361.
[17]Okamura K,Suzuki M,Chihara T,Fujiwara A,Fukuda T,Goto S,et al.Clinical
evaluation of shizophyllan combined with irradiation in patients with cervical cancer.A randomized controlled study.Cancer1986;58:865–72.
[18]Song TY,Lin HC,Yang NC,Hu ML.Antiproliferative and antimetastatic effects of
the ethanolic extract of Phellinus igniarius(Linnearus:Fries)Quelet.J Ethnophar-macol(Ireland)2008;115(1):50–6.
[19]Yang Q,Hu XG,Wan Q,Wang WQ.Study on anti-tumor effect of medicinal fungi
Phellinus igniarius extracts.China J Chin Mater Med2006;31(20):1713–5. [20]Zheng R,Jie S,Hanchuan D,Moucheng W.Characterization and immunomodulat-
ing activities of polysaccharide from Lentinus edodes.Int immunopharmacol 1999;41:157–64.
Table3
Increased life span of oral administration of PIE on S180-bearing mice(n=10;ˉx?s). Group n Dose(mg/kg)Survival time(d)ILS(%)
Control1013.50±2.20
Positive control(CTX)103018.20±2.57??34.81 High dose group1050015.50±1.3112.90 Medium dose group1025017.50±2.78??22.86 Low dose group1012516.25±2.60?16.92
S180-bearing mice were orally administered saline(control group),CTX30mg/kg (positive control,CTX)or PIE at500,250or125mg/kg for once daily,for10days.Each value is presented as mean±S.D.of the three independent experiments(n=10). Signi?cant difference:*p b0.05and**p b0.01(Student's t-test).
?P b0.05to those of control.
??P b0.01to those of control.
Table4
Increased life span of oral administration of PIE on H22-bearing mice(n=10;ˉx?s). Group n Dose mg/kg Survival time(d)ILS(%)
Control1010.70±4.22
Positive control(CTX)103016.50±2.07??54.21 High dose group1050015.50±1.90??44.86 Medium dose group1025014.00±3.38??30.84 Low dose group1012513.89±2.62?29.81
H22-bearing mice were orally administered saline(control group),CTX30mg/kg (positive control,CTX)or PIE at500,250or125mg/kg for once daily,for10days.Each value is presented as mean±S.D.of the three independent experiments(n=10). Signi?cant difference:*p b0.05and**p b0.01(Student's t-test).
?P b0.05:to those of control.
??P b0.01to those of control.259
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