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Anti-tumor and immunomodulatory activities of an exopolysaccharide from Rhizopus nigricans on CT26 tumor-bearing mice
International Immunopharmacology 36 (2016) 218–224
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International Immunopharmacology journal homepage: www.elsevier.com/locate/intimp
Anti-tumor and immunomodulatory activities of an exopolysaccharide from Rhizopus nigricans on CT26 tumor-bearing mice Lei Zhu a, Jianfeng Cao d, Guochuang Chen e, Yanghui Xu a, Jingbo Lu a, Fang Fang a, Kaoshan Chen a,b,c,e,⁎ a
Department of Pharmacy, Wannan Medical College, Wuhu 241000, China Anhui Province Key Laboratory of active biological macro-molecules, Wannan Medical College, Wuhu 241000, China Anhui Provincial Engineering Research Center for Polysaccharide Drugs, Wannan Medical College, Wuhu 241000, China d Department of Chemistry and life Science, Guizhou Normal College, Guiyang 550018, China e School of Life Science and National Glycoengineering Research Center, Shandong University, Jinan 250100, China b c
a r t i c l e
i n f o
Article history: Received 23 December 2015 Received in revised form 31 March 2016 Accepted 18 April 2016 Available online xxxx Keywords: Rhizopus nigricans Exopolysaccharide Immunomodulatory Antitumor activity
a b s t r a c t This study was aimed to investigate the anti-tumor and immunomodulatory activities of an exopolysaccharide (EPS) from Rhizopus nigricans. Our results showed EPS could significantly inhibit the tumor growth and increase the immune organs index of CT26 tumor-bearing mice. EPS treatment increased the productions of interleukin-2 (IL-2) and tumor necrosis factor-α (TNF-α) levels in serum. The increase of percentage of CD8+ cytotoxic T cells among total spleen T lymphocyte was also observed. Furthermore, EPS remarkably stimulate spleen lymphocytes proliferation in the absence or presence of mitogens. In addition, we found that EPS had synergistic effect with chemotherapy and improved immunosuppressive effect induced by 5-Fu. In summary, these findings indicated that the antitumor effects of EPS might be partly due to immune function activation and it might have potential to be used in the treatment for colorectal cancer. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Colorectal cancer is the third most common cancer and the third leading cause to cancer death in both men and women in the United States. In 2014, an estimated 71,830 men and 65,000 women would be diagnosed with colorectal cancer and 26,270 men and 24,040 women would die of the disease [1]. Although chemotherapy is one of the effective treatments methods against colon cancer, chemotherapeutic agent induce severe adverse effects while killing tumor cells [2]. Therefore, it is very crucial to find novel anti-tumor agents with high biologically activities and low toxicity to host. Polysaccharides possess various biological activities with low toxicity, and have attracted widespread attentions of scientists [3–6]. Recently, a number of studies on the action mechanisms of polysaccharides have demonstrated that polysaccharides could inhibit the tumor growth in vivo for their immunomodulatory activities [7–9]. They exert anti-tumor activity by boosting host's natural immune defense. As one of the most important part of the polysaccharides, fungi polysaccharide has drawn amount of attention due to their immunomodulatory and anti-tumor effects [10–13], some of those have been used in clinical as biological response modifiers (BRMs) and anti-tumor drugs for many
years, such as Lentinan [14]. Therefore, fungi polysaccharides will be potential useful for the development of anti-tumor drugs. Rhizopus nigricans is a filamentous fungus which is assigned to the class Zygomycetes, group Mucorales. It is widely used in pharmaceutical industry due to its activities of biotransformation and production of organic acids [15,16]. Our previous studies have shown that polysaccharides isolated from mycelia of liquid-cultured Rhizopus nigricans could inhibit the proliferation of human gastric cancer BGC-823 cells by inducing cell apoptosis and G2/M phase cell cycle arrest [17]. EPS was an exopolysaccharide isolated from fermentation broth Rhizopus nigricans and the relative chemical structure of EPS was reported previously, our previous studies also showed that EPS could induce S phase cycle arrest and apoptosis in human colorectal carcinoma HCT-116 cells, which is partly mediated by the mitochondrial apoptosis pathway [18]. However, its anti-tumor and immunomodulatory activities in vivo remain unknown. In this study, we found that EPS could improve the immunocompetence of CT26 tumor-bearing mice and possess a synergistic antitumor effect with 5-Fluorouracil (5-Fu). 2. Materials and methods 2.1. Materials
⁎ Corresponding author at: Department of Pharmacy, Wannan Medical College, No. 22 West Wenchang Road, Wuhu 241000, China. E-mail address: [email protected] (K. Chen).
http://dx.doi.org/10.1016/j.intimp.2016.04.033 1567-5769/© 2016 Elsevier B.V. All rights reserved.
EPS was extracted and purified as previously published method [18] and was free of lipopolysaccharide (LPS) tested by tachypleus amebocyte lysate method. Roswell Park Memorial Institute (RPMI) 1640
L. Zhu et al. / International Immunopharmacology 36 (2016) 218–224
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medium, fetal bovine serum, penicillin and streptomycin were purchased from Gibco (Gaithersburg, USA). 5-Fluorouracil (5-Fu) was purchased from Sigma (St. Louis, USA). CD3 and CD8a antibodies used for flow cytometry analyses were purchased from eBioscience (Santiago, USA). Concanavalin A (ConA), Lipopolysaccharide (LPS), 5-Fluorouracil and 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) were purchased from Sigma (St. Louis, USA).
200 mg/kg combined with 5-Fu at a dose of 20 mg/kg respectively for 10 days. The mice were weighed and sacrificed by cervical dislocation and the tumors, thymus, spleens, livers, kidneys and hearts were removed and weighed immediately.
2.2. Cell line
24 h after last drug administration, all the mice were sacrificed by cervical dislocation. The tumor, spleen and thymus were dissected and weighted immediately. The tumor inhibition ratio was calculated by following formula: inhibition ratio (%) = 100 [(A − B) / A] × 100%, where A is the average tumor weight of the model control group and B is that of the treatment group. The organ indexes of spleen, thymus, liver, kidney, heart were calculated as follows: organ index = average weight of organ (mg) / body weight (g).
The CT26 mouse colon cancer cell line was purchased from the Shanghai Institute of Cell Biology at the Chinese Academy of Sciences (Shanghai, China). They were cultured in RPMI-1640 medium, supplemented with 10% fetal bovine serum, 100 IU/ml penicillin and 100 μg/ml streptomycin and maintained in a humidified incubator (37 °C, 5% CO2).
2.6. Tumor inhibition rate and organ index
2.3. Animals
2.7. Serum cytokine levels assay
Female BALB/c mice weighing 18–22 g were purchased from Nanjing Qinglong mountain farm and acclimatized for 1 week before use. All the animal experiments followed the National Institute of Health Guide for the Care and Use of Laboratory Animals, and approved by the Animal Ethics Committee of Wannan Medical College. During the experiments the mice were housed in an air-condition room (temperature of 23 ± 2 °C, humidity of 50% ± 10%) on a 12-h light/12-h dark cycle with food and water provided ad libitum.
All the collected mice blood samples were centrifuged at 5000 ×g for 15 min to obtain serum. The concentrations of interleukin-2 (IL-2) and tumor necrosis factor-α (TNF-α) in the serum were determined by using ELISA kit (RayBiotech, USA) according to the instruction of the manufacturer.
2.4. In vitro anti-tumor activity
To prepare the splenocytes suspension, the spleens from the CT26 tumor-bearing mice were removed aseptically, placed in cold RPMI1640 medium. A nylon mesh (BD Falcon, USA) was placed on a sterile small beaker containing erythrocyte lysis buffer. The spleens were then teared apart and passed through the nylon mesh. The spleen cell suspensions were washed thrice and placed in RPMI-1640 medium containing 10% FBS. The cell viability of splenocytes was measured using a FACSVerse™ flow cytometer (Becton-Dickinson, USA) with a propidium iodide (PI)/RNAase stain. The cells (5 × 105 cells/ml, 300 μl) from the splenocytes were stained with 1.5 μl of FITC-labelled anti-mouse CD3 and 3.75 μl PE-labelled anti-mouse CD8a at 4 °C for 30 min in the dark. After incubation, unlabeled antibodies were washed with 3 ml phosphate buffer saline (PBS) including 0.05% sodium azide, and then resuspended in 200 μl of FACS buffer containing 2.0% FBS and 0.05% sodium azide (Sigma-Aldrich, USA). Cells were then analyzed with FACSVerse and the data were analyzed with FACSuite software (BD Biosciences, USA).
The cytotoxic effects of EPS on CT26 cells were examined in this experiment. The CT26 cells were seeded into a 96-well plate at concentration of 2 × 105 cells/ml in 90 μl RPMI-1640 containing 10% fetal bovine serum and incubated for 12 h. After the addition of exopolysaccharide solution at different concentrations (final concentration of polysaccharides were 0, 50, 100, 200, 400, 600, 800, 1000 μg/ml), the 96-well plate was incubated for 24 h. Subsequently, each well was added 20 μl (5 mg/ml) MTT and incubated for another 4 h. The supernatant was discarded and 150 μl of dimethyl sulfoxide (DMSO) was added in each well. The optical density (OD) of each well was detected at 570 nm using a microplate ELISA reader (Bio\\Rad, USA). 2.5. Experimental design and drug administration Experimental DesignI: direct treatment with EPS. Briefly, under aseptic conditions, 0.2 ml of CT26 cells suspension (2 × 106 cells/ml) was subcutaneously injected into the right axillary region of the BALB/c mice in all groups. After the inoculation of the CT26 cells for 24 h, mice were randomly divided into 5 groups (3 EPS treatment groups, 1 positive control group, 1 model control group) of 7 animals each. EPS was administered orally to each group at different dosages (50, 100, 200 mg/kg) in three EPS treated groups, respectively. 5-Fu was administered orally as positive control group at dose of 20 mg/kg and the model control group was treated with 0.9% normal saline. 7 normal mice were taken as normal control received same volume of physiologic saline, and another 7 mice were taken as normal EPS control received same volume of EPS (200 mg/kg). The anti-tumor activity of EPS was determined by measuring the tumor weight after 14 days treatment. The tumors, thymus and spleens were removed and weighed immediately, their blood samples were collected. Experimental Design II: treatment with EPS complexes carrying 5fluorouracil. The mouse tumor model was established by subcutaneous injection according to the method described by the method above. 24 h after the inoculation, the mice were administrated by 0.9% normal saline, 5-fluorouracil (5-Fu, 20 mg/kg) and EPS at the doses of 50, 100,
2.8. Flow cytometry analyses of CD8+ T lymphocytes
Fig. 1. The growth inhibitory effect of EPS on CT26 cells in vitro. CT26 cells were treated with EPS (50–1000 μg/ml) 24 h and the cell ability were tested by MTT method. The data presented are averages derived from at least triplicate experiments.
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3. Results
Table 1 Effects of EPS on the tumor weight, body weight and inhibitory rate. Group
Dosage (mg/kg)
Body weight (g)
Tumor weight (g)
Inhibitory rate (%)
3.1. The cytotoxicity of EPS on CT26 cells in vitro
Model control 5-Fu group EPS group
– 20 50 100 200
25.05 ± 2.35 15.50 ± 2.09*** 23.89 ± 1.41 24.94 ± 2.94 24.16 ± 1.25
2.89 ± 0.30 0.99 ± 0.25*** 2.38 ± 0.26 1.99 ± 0.35 1.48 ± 0.75**
– 65.74 17.65 31.14 48.79
In order to investigate the effects of EPS on the proliferation of CT26 cells in vitro, the cell viability was determined by the MTT assay. As shown in Fig. 1, EPS did not inhibit CT26 cell proliferation at concentrations of 50–1000 μg/ml in vitro.
Data were expressed as mean ± SD (n = 7). Values were significantly different compared with the model group by Dunnett's test: **P b 0.01, ***P b 0.001.
2.9. Lymphocytes proliferation assay Lymphocytes proliferation was performed according to the method described previously with some modifications [19]. The spleens isolated from sacrificed mice were aseptically removed and chopped into small pieces using scissors and forceps. The spleen cells were passed though sterilized meshes (200 mesh), and suspended in lysis buffer (0.15 M NH4Cl, pH 7.4) for 5 min to remove erythrocytes. The remaining cells were washed twice with PBS and resuspended in RPMI-1640 complete medium. The splenocytes were seed into a 96-well plate at 1 × 106 cells/ml in a 100 μl RPMI-1640 medium and cultured with concanavalin A (ConA, 5.0 μg/ml) and lipopolysaccharide (LPS, 10 μg/ml), or RPMI-1640 medium in a final volume of 200 μl at 37 °C with 5% CO2. After 44 h of incubation, 20 μl MTT solutions (5 mg/ml) were added to each well and incubated for another 4 h. The plate was centrifuged at 200 ×g for 15 min and the supernatant was discarded, then a total of 150 μl DMSO was added to each well. After all crystals dissolved, the absorbance was determined at 570 nm using an automatic ELISA plate reader.
2.10. Statistical analysis All statistical analyses by using SPSS version 11.0 and all measured variables were presented as means ± SD. Dunnett's test was used to test the differences between treatment groups and control group. A two-tailed P value of 0.05 was considered to be statistically significant.
3.2. Effects of EPS on tumor growth and body weight in tumor-bearing mice To investigate whether EPS had inhibitory effects on CT26 tumor growth in mice, the CT26-bearing BALB/c mice were treated by oral administration of 50, 100 and 200 mg/kg dosages of EPS or 5-Fu (20 mg/kg). As shown in Table 1 and Fig. 2, EPS (200 mg/kg) significantly shrank the tumor compared with the model group (P b 0.01). As expected, 5-Fu dramatically suppressed tumor growth (P b 0.001) but simultaneously reduced the body weight of mice compared to the model control group (P b 0.001), which indicated that 5-Fu had a strong toxicity on the body. Furthermore, the body weights of all the EPStreated mice in EPS groups were higher than that of the 5-Fu group, which suggested that the EPS had no toxicity to the organism. 3.3. Effects of EPS on immune organ index As shown in Fig. 3, the spleen index and thymus index of mice in 5-Fu group were remarkably lower than model group (P b 0.001), the dose of EPS (100 mg/kg) significantly increased spleen index compared with the model group (P b 0.05). Although there were no obvious changes in the thymus index, all the EPS-treated mice had higher thymus index than the model group. These results indicated that EPS had beneficial effects on the immune organs. 3.4. Effects of EPS on cytokine levels in serum EPS treatment (100 and 200 mg/kg) evidently increased the cytokine secretion (P b 0.05, P b 0.01). As shown in Fig. 4, the levels of IL-2 and TNF-α were significantly decreased in 5-Fu group compared with the model group (P b 0.01), which indicated that 5-Fu could damage immune system and induce immunosuppression. In contrast, the levels of IL-2 and TNF-α in serum were remarkably higher in the EPS treatment groups than model group in a dose-dependent manner.
Fig. 2. The anti-tumor effects of EPS on the growth of CT26 tumor (n = 7). Solid tumor blocks were removed from the mice by using surgical scissors immediately. Experimenter operated carefully to ensure the integrity of the tumor blocks. A: model group, in which tumor-bearing mice received 0.9% normal saline. B: EPS treated group, in which tumor-bearing mice received EPS (50 mg/kg). C: EPS treated group, in which tumor-bearing mice received EPS (100 mg/kg). D: EPS treated group, in which tumor-bearing mice received EPS (200 mg/kg). E: 5-Fu group, in which tumor-bearing mice received 5-Fu (20 mg/kg).
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Fig. 3. Effects of EPS on immune organ index in CT26 tumor-bearing mice. The organ index was measured as the ratio of organ weight (mg) to bodyweight (g). Normal saline: normal control, in which non tumor-bearing mice received 0.9% normal saline. Normal EPS: normal EPS control, in which non tumor-bearing mice received EPS (200 mg/kg). Model: negative control, in which tumor-bearing mice received 0.9% normal saline. 5Fu: positive control, in which tumor-bearing mice received 5-Fu (20 mg/kg). EPS: EPS treated groups, in which tumor-bearing mice received EPS (50, 100 or 200 mg/kg). Data were presented as means ± SD (n = 7). Values were significantly different compared with the model group by Dunnett's test: *P b 0.05, ***P b 0.001.
3.5. Effects of EPS on the percentage of CD8+ T cells among total splenic T cells The data of flow cytometry analyses revealed that the EPS (100 mg/kg and 200 mg/kg) dramatically increased the percentage of CD8+ T cells among total T lymphocytes from 28.77% to 35.78% and 37.21% respectively (P b 0.05), whereas 5-Fu slightly decreased the levels of CD8+ T cells (Fig. 5).
Fig. 4. Levels of serum cytokines in CT26 tumor-bearing mice. Data were presented as means ± SD (n = 7). Normal saline: normal control, in which non tumor-bearing mice received 0.9% normal saline. Normal EPS: normal EPS control, in which non tumorbearing mice received EPS (200 mg/kg). Model: negative control, in which tumorbearing mice received 0.9% normal saline. 5-Fu: positive control, in which tumorbearing mice received 5-Fu (20 mg/kg). EPS: EPS treated groups, in which tumorbearing mice received EPS (50, 100 or 200 mg/kg). Values were significantly different compared with the CT26 model group by Dunnett's test: *P b 0.05, **P b 0.01.
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Fig. 5. Percentage of CD8+ T cells among total T lymphocytes in different groups of CT26 tumor-bearing mice. All values were mean ± SD (n = 7). Normal saline: normal control, in which non tumor-bearing mice received 0.9% normal saline. Normal EPS: normal EPS control, in which non tumor-bearing mice received EPS (200 mg/kg). Model: negative control, in which tumor-bearing mice received 0.9% normal saline. 5-Fu: positive control, in which tumor-bearing mice received 5-Fu (20 mg/kg). EPS: EPS treated groups, in which tumor-bearing mice received EPS (50, 100 or 200 mg/kg). Values were significantly different compared with the CT26 model group by Dunnett's test: *P b 0.05.
3.6. Effects of EPS on the lymphocyte proliferation in vivo We investigated the effects of EPS on lymphocyte proliferation in tumor-bearing mice. The effects of EPS by oral administration on the proliferation of lymphocyte challenged with mitogens (LPS and ConA) were demonstrated in Fig. 6. The lymphocyte proliferation without mitogen stimulation was significantly higher compared with the model group in a dose-dependent manner (P b 0.05, P b 0.01). In the present of ConA (5.0 μg/ml) or LPS (10.0 μg/ml), the proliferation of lymphocyte was dramatically increased in the EPS treatment groups (200 mg/kg) compared with model group (P b 0.01), but proliferation of lymphocyte was decreased in the 5-Fu group (P b 0.05).
Fig. 6. The effect of EPS on lymphocyte proliferation of CT26 tumor-bearing mice. Data were expressed as mean ± SD (n = 7). Normal saline: normal control, in which non tumor-bearing mice received 0.9% normal saline. Normal EPS: normal EPS control, in which non tumor-bearing mice received EPS (200 mg/kg). Model: negative control, in which tumor-bearing mice received 0.9% normal saline. 5-Fu: positive control, in which tumor-bearing mice received 5-Fu (20 mg/kg). EPS: EPS treated groups, in which tumor-bearing mice received EPS (50, 100 or 200 mg/kg). Values were significantly different compared with the CT26 model group by Dunnett's test: *P b 0.05, **P b 0.01.
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Table 2 Anti-tumor activity of EPS combined with 5-Fu against CT26 subcutaneous transplantation tumor. Group
Dose (mg/kg)
Body weight (g)
Tumor weight (g)
Inhibitory rate (%)
Model 5-Fu Drug combination
– 20 EPS (50) + 5-Fu (20) EPS (100) + 5-Fu (20) EPS (200) + 5-Fu (20)
25.54 ± 1.34 17.4 ± 1.56*** 22.83 ± 1.86*### 19.40 ± 2.51** 20.39 ± 2.34**##
2.99 ± 0.89 1.24 ± 0.62*** 1.09 ± 0.36*** 0.89 ± 0.51*** 0.83 ± 0.49***
– 58.53% 63.55% 70.23% 72.24%
Data were expressed as mean ± SD (n = 7). Values were significantly different compared with the Model group by Dunnett's test: *P b 0.05, **P b 0.01, *** P b 0.001. Values were significantly different compared with the 5-Fu group by Dunnett's test: ##P b 0.05, ###P b 0.001.
indicated that EPS could improve immunosuppressive effect induced by 5-Fu (P b 0.05, P b 0.01).
3.7. Effect of EPS in combination with 5-Fu on tumor growth of CT26-bearing mice The effect of EPS in combination with 5-Fu on the tumor growth of CT26-bearing mice was studied. As shown in Table 2, all the treatment groups could significantly reduce tumor weight. In the combination of EPS with 5-Fu group, the tumor weight was significantly lower than the 5-Fu group in a dose-dependent manner and their inhibitions were 63.55%, 70.23% and 72.24%, respectively, which were higher than the inhibition ratio of only 5-Fu (58.53%), which suggested that EPS possess a synergistic effect on 5-Fu. Compared with the model group, the average weight of the mice in each group were significantly decreased, in which the only 5-Fu group decreased most significantly (P b 0.001), indicating that only 5-Fu administration shows most hazardous to organism. It was worth noting that the average weight of mice in the combination treatment groups was distinctly higher than the 5-Fu group (P b 0.01, P b 0.001).
3.8. Effect of EPS in combination with 5-Fu on immune organ index The spleen and thymus indexes could reflect the immune function of the organism directly. As shown in Table 3 as expected, the 5-Fu group exhibited an evidently decrease in the both immune organ index and immune organ weight compared with the model group, which indicated that 5-Fu could suppress the immune function (P b 0.05, P b 0.001). However, compared with the 5-Fu group, the index and weight of immune organ in combination treatment groups showed the significant increase at the dose of 50 and 200 mg/kg of EPS respectively, which
3.9. Effect of EPS in combination with 5-Fu on heart, liver and kidney index in CT26-bearing mice The results were summarized in Table 4, the organ index in each treatment group were lower than the model groups and 5-Fu group exhibited a significantly decrease in liver and kidney indexes (P b 0.05), but all of the organ indexes in the combination treatment groups showed a slight increase compared with the 5-Fu treated group, which supported the alexipharmac effect of EPS in 5-Fu cancer therapy. 4. Discussion In recent decades, many reports had demonstrated that various polysaccharides from natural sources have a variety of biological functions, especially anti-tumor and immune-enhancing activities [20]. Since Brander reported the Zymosan have anti-tumor effect, fungi polysacchrides were attracted widespread attention [21]. Our previous studies showed that Rhizopus nigricans polysaccharides could inhibit the proliferation of human gastric cancer BGC-823 cells by inducing cell apoptosis and G2/M phase cell cycle arrest and suppress proliferation of human colorectal carcinoma HCT-116 cells by triggering S phase cell cycle arrest and apoptosis in vitro. However, the antitumor effects of EPS in vivo have not been elucidated. Thus we investigate the anti-tumor activity of EPS using CT26 tumor-bearing mice. The results indicated that EPS has a strong antitumor effect and
Table 3 Effects of combined with 5-Fu on immune organ of CT26-bearing mice. Group
Dose (mg/kg)
Spleen weight (g)
Spleen index (mg/g)
Thymus weight (g)
Thymus index (mg/g)
Model 5-Fu Drug combination
– 20 EPS (50) + 5-Fu (20) EPS (100) + 5-Fu (20) EPS (200) + 5-Fu (20)
0.209 ± 0.069 0.050 ± 0.023*** 0.126 ± 0.035**## 0.060 ± 0.032*** 0.081 ± 0.028***
8.18 ± 2.33 2.87 ± 1.41*** 5.52 ± 1.33*# 3.09 ± 1.20*** 3.97 ± 1.05***
0.129 ± 0.024 0.070 ± 0.006*** 0.116 ± 0.031## 0.084 ± 0.024** 0.104 ± 0.027#
5.05 ± 1.02 4.02 ± 0.28* 5.08 ± 0.96# 4.33 ± 0.98 5.10 ± `0.99#
Data were expressed as mean ± SD (n = 7). Values were significantly different compared with the Model group by Dunnett's test: *P b 0.05, **P b 0.01, ***P b 0.001. Values were significantly different compared with the 5-Fu group by Dunnett's test: #P b 0.05, ##P b 0.01.
Table 4 Effects of EPS combined with 5-Fu on organ index of CT26-bearing mice. Group
Dose (mg/kg)
Heart index (mg/g)
Liver index (mg/g)
Kidney index (mg/g)
Model 5-Fu Drug combination
– 20 EPS (50) + 5-Fu (20) EPS (100) + 5-Fu (20) EPS (200) + 5-Fu (20)
5.52 ± 0.30 4.74 ± 0.27 5.21 ± 0.24 5.27 ± 0.29 5.18 ± 0.34
51.87 ± 1.77 39.50 ± 2.18* 43.08 ± 2.10 44.34 ± 2.46 44.68 ± 2.02
11.88 ± 0.90 7.60 ± 0.84* 9.12 ± 0.50 9.38 ± 0.73 9.49 ± 0.72
Data were expressed as mean ± SD (n = 7). Values were significantly different compared with the Model group by Dunnett's test: *P b 0.05.
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immunomodulatory activity in vivo, but it could not inhibit proliferation of CT26 cells directly in vitro. This result agreed with the previous literatures which reports that many polysaccharides usually did not have any anti-tumor activity in vitro [22–24]. The spleen and thymus index were important index for immunity, the development of immune organs directly affects immune function and disease resistance. It was well known that as the main immune organs thymus and spleen play an important role in the immunity of the host. Therefore, a higher organ index indicated a stronger immune capability [25]. Consistent with the antitumor activity, our data showed that EPS-treated mice had higher immune organ index than the model group. Compared with the 5-Fu group, a significant increase was observed at the dose of 50 and 200 mg/kg of EPS in combination of EPS and 5-Fu treatment groups. These results indicated that EPS had a beneficial effect on the immunity of CT26 tumor-bearing mice and improve the immune organ damaged by 5-Fu. Serum cytokines are important in immune system and play a vital role in mediating the host defense [23,26] The IL-2 can promote the proliferation of responsive T cells and TNF-α also can induce apoptosis and tumor necrosis. In the present experiment, EPS could significantly increase the two cytokines at dose of 100 and 200 mg/kg compared with the model group or 5-Fu. The data implied that EPS might exert anti-tumor effect by promoting secretion of cytokines in CT26 tumorbearing mice. As cytotoxic cell, CD8+ T cell plays an important role in antivirus and anti-tumor immunity [27,28]. Recently immunotherapy for cancer has recently become a key treatment modality and many new modalities of immunotherapies targeting cytotoxic T cells [29]. Our results showed that EPS could increase the percentage of CD8+ T cells and the up-regulated ratio of CD8+ T cells might participate in the anti-tumor activity of EPS in vivo. The ability of lymphocytes response to mitogen by undergoing mitotic proliferation reflects the immune potential of the organism [30]. It was generally accepted that splenocyte proliferation response induced by ConA and LPS in vitro was used as a conventional method to evaluate T lymphocyte activity and B lymphocyte activity respectively [9,23,31]. Cellular proliferation induced by ConA is commonly used to detect T lymphocyte immunity, and LPS-induced activation of B cells indicates B lymphocyte immunity [32]. The results in the study demonstrated that EPS could significantly activate potential of T and B lymphocyte cells, which indicated that EPS could enhance the potential organism immunity. Heidelberger et al reported the anti-tumor activity of 5-Fu in 1957. Fifty years after the first synthesis of 5-Fu, it is still a standard component of adjuvant and palliative therapy having a proven impact on survival time in patients with colorectal cancer [33]. It has been one of the most commonly used chemotherapeutic drugs in the treatment of various types of cancer for over 40 years with approximately two million patients treated worldwide each year [34]. However, it also destroys the normal cells of the body, which lead to cancer patients suffering from recurrent infections induced by immunosuppression [35]. In recent years, chemotherapy combined with immune adjuvant therapy is employed to improve the anti-tumor effect of chemotherapy [36–38]. Many reports indicated that polysaccharides possess a synergistic effect on chemotherapy [19,39]. In this study, we tested the adjuvant antitumor activity of EPS in combination with 5-Fu in CT26 tumor-bearing mice. The results demonstrated that EPS combined with 5-FU could increase the tumor inhibitory rate, immune organ index and weight in the tumor-bearing mice. These data indicated that EPS could enhance the anti-tumor effect and improved the suppressed immunity in tumor bearing mice subject to 5-Fu chemotherapy. It is reported that 10–30% of patients receiving 5-Fu develop a severe to internal organ toxic reaction and women with a low body mass index were at the highest risk for acute organ toxicity [40,41]. In order to evaluate the attenuated effect of EPS on organ toxicity induced by 5-Fu, the body weight, liver, kidney and heart index were determined. Although the body weight and organ index were deceased due to the toxicity of 5-Fu in combination
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EPS and 5-Fu groups, it was increased in a certain extent compared with 5-Fu group. The result suggested that EPS could alleviate the toxicity induced by 5-Fu in CT26 tumor-bearing mice. Take together, the mixture of EPS and 5-Fu produced a synergistic effect on antitumor activity and the results illustrated that EPS possess a synergistic effect on 5-Fu may be achieved by enhancing the immune system functions and alleviating the toxicity of chemotherapy drugs. In conclusion, these findings can be suggested that the anti-tumor effects of EPS may be in part due to immune function activation and it can be the potential use on clinical application to treat colorectal cancer. Acknowledgements This study was supported by Anhui Provincial Natural Science Foundation (NO. 1408085MH197), the Key Project of Education Department of Anhui Province (NO. KJ2015A199), Scientific Research Fund of Guizhou Provincial Science and Technology Department, China (NO. [2013]2233) and Key Technology Research of Burdock Fructooligosaccharide Scale Production in Anhui Provincial Engineering Research Center for Polysaccharide Drugs (NO. 2012jc14). References [1] R. Siegel, C. Desantis, A. Jemal, Colorectal cancer statistics, 2014, CA Cancer J. Clin. 64 (2014) 104–117. [2] C.G. Drake, E.S. Antonarakis, Update: Immunological strategies for prostate cancer, Curr. Urol. Rep. 11 (2010) 202–207. [3] L. Shao, Z. Wu, H. Zhang, W. Chen, L. Ai, B. Guo, Partial characterization and immunostimulatory activity of exopolysaccharides from Lactobacillus rhamnosus KF5, Carbohydr. Polym. 107 (2014) 51–56. [4] J. Wang, X. Zhao, Y. Yang, A. Zhao, Z. Yang, Characterization and bioactivities of an exopolysaccharide produced by Lactobacillus plantarum YW32, Int. J. Biol. Macromol. 74C (2014) 119–126. [5] W. Zhang, J. Yang, J. Chen, Y. Hou, X. Han, Immunomodulatory and antitumour effects of an exopolysaccharide fraction from cultivated Cordyceps sinensis (Chinese caterpillar fungus) on tumour-bearing mice, Biotechnol. Appl. Biochem. 42 (2005) 9–15. [6] T. Huang, J. Lin, J. Cao, P. Zhang, Y. Bai, G. Chen, K. Chen, An exopolysaccharide from Trichoderma pseudokoningii and its apoptotic activity on human leukemia K562 cells, Carbohydr. Polym. 89 (2012) 701–708. [7] J. Yang, X. Li, Y. Xue, N. Wang, W. Liu, Anti-hepatoma activity and mechanism of corn silk polysaccharides in H22 tumor-bearing mice, Int. J. Biol. Macromol. 64 (2014) 276–280. [8] B. Yang, B. Xiao, T. Sun, Antitumor and immunomodulatory activity of Astragalus membranaceus polysaccharides in H22 tumor-bearing mice, Int. J. Biol. Macromol. 62 (2013) 287–290. [9] G. Zeng, Y. Ju, H. Shen, N. Zhou, L. Huang, Immunopontentiating activities of the purified polysaccharide from evening primrose in H22 tumor-bearing mice, Int. J. Biol. Macromol. 52 (2013) 280–285. [10] W.J. Li, Y. Chen, S.P. Nie, M.Y. Xie, M. He, S.S. Zhang, K.X. Zhu, Ganoderma atrum polysaccharide induces anti-tumor activity via the mitochondrial apoptotic pathway related to activation of host immune response, J. Cell. Biochem. 112 (2011) 860–871. [11] L. Zhao, Y. Chen, S. Ren, Y. Han, H. Cheng, Studies on the chemical structure and antitumor activity of an exopolysaccharide from Rhizobium sp. N613, Carbohydr. Res. 345 (2010) 637–643. [12] S.K. Mallick, S. Maiti, S.K. Bhutia, T.K. Maiti, Food Chem. Toxicol. 48 (2010) 2115–2121. [13] L. Chen, J. Pan, X. Li, Y. Zhou, Q. Meng, Q. Wang, Endo-polysaccharide of Phellinus igniarius exhibited anti-tumor effect through enhancement of cell mediated immunity, Int. Immunopharmacol. 11 (2011) 255–259. [14] R. Zheng, S. Jie, D. Hanchuan, W. Moucheng, Characterization and immunomodulating activities of polysaccharide from Lentinus edodes, Int. Immunopharmacol. 5 (2005) 811–820. [15] G. Schmeda-Hirschmann, C. Aranda, M. Kurina, J.A. Rodriguez, C. Theoduloz, Biotransformations of imbricatolic acid by aspergillus Niger and Rhizopus nigricans cultures, Molecules 12 (2007) 1092–1100. [16] D.X. Wu, Y.X. Guan, H.Q. Wang, S.J. Yao, 11 alpha-Hydroxylation of 16 alpha,17epoxyprogesterone by Rhizopus nigricans in a biphasic ionic liquid aqueous system, Bioresour. Technol. 102 (2011) 9368–9373. [17] G.C. Chen, P.Y. Zhang, T.T. Huang, W.Q. Yu, J. Lin, P. Li, K.S. Chen, Polysaccharides from Rhizopus nigricans mycelia induced apoptosis and G2/M arrest in BGC-823 cells, Carbohydr. Polym. 97 (2013) 800–808. [18] W. Yu, G. Chen, P. Zhang, K. Chen, Purification, partial characterization and antitumor effect of an exopolysaccharide from Rhizopus nigricans, Int. J. Biol. Macromol. 82 (2016) 299–307. [19] Z. Wang, B. Wu, X. Zhang, M. Xu, H. Chang, X. Lu, X. Ren, Purification of a polysaccharide from Boschniakia rossica and its synergistic antitumor effect combined with 5Fluorouracil, Carbohydr. Polym. 89 (2012) 31–35.
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Contents lists available at ScienceDirect
International Immunopharmacology journal homepage: www.elsevier.com/locate/intimp
Anti-tumor and immunomodulatory activities of an exopolysaccharide from Rhizopus nigricans on CT26 tumor-bearing mice Lei Zhu a, Jianfeng Cao d, Guochuang Chen e, Yanghui Xu a, Jingbo Lu a, Fang Fang a, Kaoshan Chen a,b,c,e,⁎ a
Department of Pharmacy, Wannan Medical College, Wuhu 241000, China Anhui Province Key Laboratory of active biological macro-molecules, Wannan Medical College, Wuhu 241000, China Anhui Provincial Engineering Research Center for Polysaccharide Drugs, Wannan Medical College, Wuhu 241000, China d Department of Chemistry and life Science, Guizhou Normal College, Guiyang 550018, China e School of Life Science and National Glycoengineering Research Center, Shandong University, Jinan 250100, China b c
a r t i c l e
i n f o
Article history: Received 23 December 2015 Received in revised form 31 March 2016 Accepted 18 April 2016 Available online xxxx Keywords: Rhizopus nigricans Exopolysaccharide Immunomodulatory Antitumor activity
a b s t r a c t This study was aimed to investigate the anti-tumor and immunomodulatory activities of an exopolysaccharide (EPS) from Rhizopus nigricans. Our results showed EPS could significantly inhibit the tumor growth and increase the immune organs index of CT26 tumor-bearing mice. EPS treatment increased the productions of interleukin-2 (IL-2) and tumor necrosis factor-α (TNF-α) levels in serum. The increase of percentage of CD8+ cytotoxic T cells among total spleen T lymphocyte was also observed. Furthermore, EPS remarkably stimulate spleen lymphocytes proliferation in the absence or presence of mitogens. In addition, we found that EPS had synergistic effect with chemotherapy and improved immunosuppressive effect induced by 5-Fu. In summary, these findings indicated that the antitumor effects of EPS might be partly due to immune function activation and it might have potential to be used in the treatment for colorectal cancer. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Colorectal cancer is the third most common cancer and the third leading cause to cancer death in both men and women in the United States. In 2014, an estimated 71,830 men and 65,000 women would be diagnosed with colorectal cancer and 26,270 men and 24,040 women would die of the disease [1]. Although chemotherapy is one of the effective treatments methods against colon cancer, chemotherapeutic agent induce severe adverse effects while killing tumor cells [2]. Therefore, it is very crucial to find novel anti-tumor agents with high biologically activities and low toxicity to host. Polysaccharides possess various biological activities with low toxicity, and have attracted widespread attentions of scientists [3–6]. Recently, a number of studies on the action mechanisms of polysaccharides have demonstrated that polysaccharides could inhibit the tumor growth in vivo for their immunomodulatory activities [7–9]. They exert anti-tumor activity by boosting host's natural immune defense. As one of the most important part of the polysaccharides, fungi polysaccharide has drawn amount of attention due to their immunomodulatory and anti-tumor effects [10–13], some of those have been used in clinical as biological response modifiers (BRMs) and anti-tumor drugs for many
years, such as Lentinan [14]. Therefore, fungi polysaccharides will be potential useful for the development of anti-tumor drugs. Rhizopus nigricans is a filamentous fungus which is assigned to the class Zygomycetes, group Mucorales. It is widely used in pharmaceutical industry due to its activities of biotransformation and production of organic acids [15,16]. Our previous studies have shown that polysaccharides isolated from mycelia of liquid-cultured Rhizopus nigricans could inhibit the proliferation of human gastric cancer BGC-823 cells by inducing cell apoptosis and G2/M phase cell cycle arrest [17]. EPS was an exopolysaccharide isolated from fermentation broth Rhizopus nigricans and the relative chemical structure of EPS was reported previously, our previous studies also showed that EPS could induce S phase cycle arrest and apoptosis in human colorectal carcinoma HCT-116 cells, which is partly mediated by the mitochondrial apoptosis pathway [18]. However, its anti-tumor and immunomodulatory activities in vivo remain unknown. In this study, we found that EPS could improve the immunocompetence of CT26 tumor-bearing mice and possess a synergistic antitumor effect with 5-Fluorouracil (5-Fu). 2. Materials and methods 2.1. Materials
⁎ Corresponding author at: Department of Pharmacy, Wannan Medical College, No. 22 West Wenchang Road, Wuhu 241000, China. E-mail address: [email protected] (K. Chen).
http://dx.doi.org/10.1016/j.intimp.2016.04.033 1567-5769/© 2016 Elsevier B.V. All rights reserved.
EPS was extracted and purified as previously published method [18] and was free of lipopolysaccharide (LPS) tested by tachypleus amebocyte lysate method. Roswell Park Memorial Institute (RPMI) 1640
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medium, fetal bovine serum, penicillin and streptomycin were purchased from Gibco (Gaithersburg, USA). 5-Fluorouracil (5-Fu) was purchased from Sigma (St. Louis, USA). CD3 and CD8a antibodies used for flow cytometry analyses were purchased from eBioscience (Santiago, USA). Concanavalin A (ConA), Lipopolysaccharide (LPS), 5-Fluorouracil and 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) were purchased from Sigma (St. Louis, USA).
200 mg/kg combined with 5-Fu at a dose of 20 mg/kg respectively for 10 days. The mice were weighed and sacrificed by cervical dislocation and the tumors, thymus, spleens, livers, kidneys and hearts were removed and weighed immediately.
2.2. Cell line
24 h after last drug administration, all the mice were sacrificed by cervical dislocation. The tumor, spleen and thymus were dissected and weighted immediately. The tumor inhibition ratio was calculated by following formula: inhibition ratio (%) = 100 [(A − B) / A] × 100%, where A is the average tumor weight of the model control group and B is that of the treatment group. The organ indexes of spleen, thymus, liver, kidney, heart were calculated as follows: organ index = average weight of organ (mg) / body weight (g).
The CT26 mouse colon cancer cell line was purchased from the Shanghai Institute of Cell Biology at the Chinese Academy of Sciences (Shanghai, China). They were cultured in RPMI-1640 medium, supplemented with 10% fetal bovine serum, 100 IU/ml penicillin and 100 μg/ml streptomycin and maintained in a humidified incubator (37 °C, 5% CO2).
2.6. Tumor inhibition rate and organ index
2.3. Animals
2.7. Serum cytokine levels assay
Female BALB/c mice weighing 18–22 g were purchased from Nanjing Qinglong mountain farm and acclimatized for 1 week before use. All the animal experiments followed the National Institute of Health Guide for the Care and Use of Laboratory Animals, and approved by the Animal Ethics Committee of Wannan Medical College. During the experiments the mice were housed in an air-condition room (temperature of 23 ± 2 °C, humidity of 50% ± 10%) on a 12-h light/12-h dark cycle with food and water provided ad libitum.
All the collected mice blood samples were centrifuged at 5000 ×g for 15 min to obtain serum. The concentrations of interleukin-2 (IL-2) and tumor necrosis factor-α (TNF-α) in the serum were determined by using ELISA kit (RayBiotech, USA) according to the instruction of the manufacturer.
2.4. In vitro anti-tumor activity
To prepare the splenocytes suspension, the spleens from the CT26 tumor-bearing mice were removed aseptically, placed in cold RPMI1640 medium. A nylon mesh (BD Falcon, USA) was placed on a sterile small beaker containing erythrocyte lysis buffer. The spleens were then teared apart and passed through the nylon mesh. The spleen cell suspensions were washed thrice and placed in RPMI-1640 medium containing 10% FBS. The cell viability of splenocytes was measured using a FACSVerse™ flow cytometer (Becton-Dickinson, USA) with a propidium iodide (PI)/RNAase stain. The cells (5 × 105 cells/ml, 300 μl) from the splenocytes were stained with 1.5 μl of FITC-labelled anti-mouse CD3 and 3.75 μl PE-labelled anti-mouse CD8a at 4 °C for 30 min in the dark. After incubation, unlabeled antibodies were washed with 3 ml phosphate buffer saline (PBS) including 0.05% sodium azide, and then resuspended in 200 μl of FACS buffer containing 2.0% FBS and 0.05% sodium azide (Sigma-Aldrich, USA). Cells were then analyzed with FACSVerse and the data were analyzed with FACSuite software (BD Biosciences, USA).
The cytotoxic effects of EPS on CT26 cells were examined in this experiment. The CT26 cells were seeded into a 96-well plate at concentration of 2 × 105 cells/ml in 90 μl RPMI-1640 containing 10% fetal bovine serum and incubated for 12 h. After the addition of exopolysaccharide solution at different concentrations (final concentration of polysaccharides were 0, 50, 100, 200, 400, 600, 800, 1000 μg/ml), the 96-well plate was incubated for 24 h. Subsequently, each well was added 20 μl (5 mg/ml) MTT and incubated for another 4 h. The supernatant was discarded and 150 μl of dimethyl sulfoxide (DMSO) was added in each well. The optical density (OD) of each well was detected at 570 nm using a microplate ELISA reader (Bio\\Rad, USA). 2.5. Experimental design and drug administration Experimental DesignI: direct treatment with EPS. Briefly, under aseptic conditions, 0.2 ml of CT26 cells suspension (2 × 106 cells/ml) was subcutaneously injected into the right axillary region of the BALB/c mice in all groups. After the inoculation of the CT26 cells for 24 h, mice were randomly divided into 5 groups (3 EPS treatment groups, 1 positive control group, 1 model control group) of 7 animals each. EPS was administered orally to each group at different dosages (50, 100, 200 mg/kg) in three EPS treated groups, respectively. 5-Fu was administered orally as positive control group at dose of 20 mg/kg and the model control group was treated with 0.9% normal saline. 7 normal mice were taken as normal control received same volume of physiologic saline, and another 7 mice were taken as normal EPS control received same volume of EPS (200 mg/kg). The anti-tumor activity of EPS was determined by measuring the tumor weight after 14 days treatment. The tumors, thymus and spleens were removed and weighed immediately, their blood samples were collected. Experimental Design II: treatment with EPS complexes carrying 5fluorouracil. The mouse tumor model was established by subcutaneous injection according to the method described by the method above. 24 h after the inoculation, the mice were administrated by 0.9% normal saline, 5-fluorouracil (5-Fu, 20 mg/kg) and EPS at the doses of 50, 100,
2.8. Flow cytometry analyses of CD8+ T lymphocytes
Fig. 1. The growth inhibitory effect of EPS on CT26 cells in vitro. CT26 cells were treated with EPS (50–1000 μg/ml) 24 h and the cell ability were tested by MTT method. The data presented are averages derived from at least triplicate experiments.
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3. Results
Table 1 Effects of EPS on the tumor weight, body weight and inhibitory rate. Group
Dosage (mg/kg)
Body weight (g)
Tumor weight (g)
Inhibitory rate (%)
3.1. The cytotoxicity of EPS on CT26 cells in vitro
Model control 5-Fu group EPS group
– 20 50 100 200
25.05 ± 2.35 15.50 ± 2.09*** 23.89 ± 1.41 24.94 ± 2.94 24.16 ± 1.25
2.89 ± 0.30 0.99 ± 0.25*** 2.38 ± 0.26 1.99 ± 0.35 1.48 ± 0.75**
– 65.74 17.65 31.14 48.79
In order to investigate the effects of EPS on the proliferation of CT26 cells in vitro, the cell viability was determined by the MTT assay. As shown in Fig. 1, EPS did not inhibit CT26 cell proliferation at concentrations of 50–1000 μg/ml in vitro.
Data were expressed as mean ± SD (n = 7). Values were significantly different compared with the model group by Dunnett's test: **P b 0.01, ***P b 0.001.
2.9. Lymphocytes proliferation assay Lymphocytes proliferation was performed according to the method described previously with some modifications [19]. The spleens isolated from sacrificed mice were aseptically removed and chopped into small pieces using scissors and forceps. The spleen cells were passed though sterilized meshes (200 mesh), and suspended in lysis buffer (0.15 M NH4Cl, pH 7.4) for 5 min to remove erythrocytes. The remaining cells were washed twice with PBS and resuspended in RPMI-1640 complete medium. The splenocytes were seed into a 96-well plate at 1 × 106 cells/ml in a 100 μl RPMI-1640 medium and cultured with concanavalin A (ConA, 5.0 μg/ml) and lipopolysaccharide (LPS, 10 μg/ml), or RPMI-1640 medium in a final volume of 200 μl at 37 °C with 5% CO2. After 44 h of incubation, 20 μl MTT solutions (5 mg/ml) were added to each well and incubated for another 4 h. The plate was centrifuged at 200 ×g for 15 min and the supernatant was discarded, then a total of 150 μl DMSO was added to each well. After all crystals dissolved, the absorbance was determined at 570 nm using an automatic ELISA plate reader.
2.10. Statistical analysis All statistical analyses by using SPSS version 11.0 and all measured variables were presented as means ± SD. Dunnett's test was used to test the differences between treatment groups and control group. A two-tailed P value of 0.05 was considered to be statistically significant.
3.2. Effects of EPS on tumor growth and body weight in tumor-bearing mice To investigate whether EPS had inhibitory effects on CT26 tumor growth in mice, the CT26-bearing BALB/c mice were treated by oral administration of 50, 100 and 200 mg/kg dosages of EPS or 5-Fu (20 mg/kg). As shown in Table 1 and Fig. 2, EPS (200 mg/kg) significantly shrank the tumor compared with the model group (P b 0.01). As expected, 5-Fu dramatically suppressed tumor growth (P b 0.001) but simultaneously reduced the body weight of mice compared to the model control group (P b 0.001), which indicated that 5-Fu had a strong toxicity on the body. Furthermore, the body weights of all the EPStreated mice in EPS groups were higher than that of the 5-Fu group, which suggested that the EPS had no toxicity to the organism. 3.3. Effects of EPS on immune organ index As shown in Fig. 3, the spleen index and thymus index of mice in 5-Fu group were remarkably lower than model group (P b 0.001), the dose of EPS (100 mg/kg) significantly increased spleen index compared with the model group (P b 0.05). Although there were no obvious changes in the thymus index, all the EPS-treated mice had higher thymus index than the model group. These results indicated that EPS had beneficial effects on the immune organs. 3.4. Effects of EPS on cytokine levels in serum EPS treatment (100 and 200 mg/kg) evidently increased the cytokine secretion (P b 0.05, P b 0.01). As shown in Fig. 4, the levels of IL-2 and TNF-α were significantly decreased in 5-Fu group compared with the model group (P b 0.01), which indicated that 5-Fu could damage immune system and induce immunosuppression. In contrast, the levels of IL-2 and TNF-α in serum were remarkably higher in the EPS treatment groups than model group in a dose-dependent manner.
Fig. 2. The anti-tumor effects of EPS on the growth of CT26 tumor (n = 7). Solid tumor blocks were removed from the mice by using surgical scissors immediately. Experimenter operated carefully to ensure the integrity of the tumor blocks. A: model group, in which tumor-bearing mice received 0.9% normal saline. B: EPS treated group, in which tumor-bearing mice received EPS (50 mg/kg). C: EPS treated group, in which tumor-bearing mice received EPS (100 mg/kg). D: EPS treated group, in which tumor-bearing mice received EPS (200 mg/kg). E: 5-Fu group, in which tumor-bearing mice received 5-Fu (20 mg/kg).
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Fig. 3. Effects of EPS on immune organ index in CT26 tumor-bearing mice. The organ index was measured as the ratio of organ weight (mg) to bodyweight (g). Normal saline: normal control, in which non tumor-bearing mice received 0.9% normal saline. Normal EPS: normal EPS control, in which non tumor-bearing mice received EPS (200 mg/kg). Model: negative control, in which tumor-bearing mice received 0.9% normal saline. 5Fu: positive control, in which tumor-bearing mice received 5-Fu (20 mg/kg). EPS: EPS treated groups, in which tumor-bearing mice received EPS (50, 100 or 200 mg/kg). Data were presented as means ± SD (n = 7). Values were significantly different compared with the model group by Dunnett's test: *P b 0.05, ***P b 0.001.
3.5. Effects of EPS on the percentage of CD8+ T cells among total splenic T cells The data of flow cytometry analyses revealed that the EPS (100 mg/kg and 200 mg/kg) dramatically increased the percentage of CD8+ T cells among total T lymphocytes from 28.77% to 35.78% and 37.21% respectively (P b 0.05), whereas 5-Fu slightly decreased the levels of CD8+ T cells (Fig. 5).
Fig. 4. Levels of serum cytokines in CT26 tumor-bearing mice. Data were presented as means ± SD (n = 7). Normal saline: normal control, in which non tumor-bearing mice received 0.9% normal saline. Normal EPS: normal EPS control, in which non tumorbearing mice received EPS (200 mg/kg). Model: negative control, in which tumorbearing mice received 0.9% normal saline. 5-Fu: positive control, in which tumorbearing mice received 5-Fu (20 mg/kg). EPS: EPS treated groups, in which tumorbearing mice received EPS (50, 100 or 200 mg/kg). Values were significantly different compared with the CT26 model group by Dunnett's test: *P b 0.05, **P b 0.01.
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Fig. 5. Percentage of CD8+ T cells among total T lymphocytes in different groups of CT26 tumor-bearing mice. All values were mean ± SD (n = 7). Normal saline: normal control, in which non tumor-bearing mice received 0.9% normal saline. Normal EPS: normal EPS control, in which non tumor-bearing mice received EPS (200 mg/kg). Model: negative control, in which tumor-bearing mice received 0.9% normal saline. 5-Fu: positive control, in which tumor-bearing mice received 5-Fu (20 mg/kg). EPS: EPS treated groups, in which tumor-bearing mice received EPS (50, 100 or 200 mg/kg). Values were significantly different compared with the CT26 model group by Dunnett's test: *P b 0.05.
3.6. Effects of EPS on the lymphocyte proliferation in vivo We investigated the effects of EPS on lymphocyte proliferation in tumor-bearing mice. The effects of EPS by oral administration on the proliferation of lymphocyte challenged with mitogens (LPS and ConA) were demonstrated in Fig. 6. The lymphocyte proliferation without mitogen stimulation was significantly higher compared with the model group in a dose-dependent manner (P b 0.05, P b 0.01). In the present of ConA (5.0 μg/ml) or LPS (10.0 μg/ml), the proliferation of lymphocyte was dramatically increased in the EPS treatment groups (200 mg/kg) compared with model group (P b 0.01), but proliferation of lymphocyte was decreased in the 5-Fu group (P b 0.05).
Fig. 6. The effect of EPS on lymphocyte proliferation of CT26 tumor-bearing mice. Data were expressed as mean ± SD (n = 7). Normal saline: normal control, in which non tumor-bearing mice received 0.9% normal saline. Normal EPS: normal EPS control, in which non tumor-bearing mice received EPS (200 mg/kg). Model: negative control, in which tumor-bearing mice received 0.9% normal saline. 5-Fu: positive control, in which tumor-bearing mice received 5-Fu (20 mg/kg). EPS: EPS treated groups, in which tumor-bearing mice received EPS (50, 100 or 200 mg/kg). Values were significantly different compared with the CT26 model group by Dunnett's test: *P b 0.05, **P b 0.01.
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Table 2 Anti-tumor activity of EPS combined with 5-Fu against CT26 subcutaneous transplantation tumor. Group
Dose (mg/kg)
Body weight (g)
Tumor weight (g)
Inhibitory rate (%)
Model 5-Fu Drug combination
– 20 EPS (50) + 5-Fu (20) EPS (100) + 5-Fu (20) EPS (200) + 5-Fu (20)
25.54 ± 1.34 17.4 ± 1.56*** 22.83 ± 1.86*### 19.40 ± 2.51** 20.39 ± 2.34**##
2.99 ± 0.89 1.24 ± 0.62*** 1.09 ± 0.36*** 0.89 ± 0.51*** 0.83 ± 0.49***
– 58.53% 63.55% 70.23% 72.24%
Data were expressed as mean ± SD (n = 7). Values were significantly different compared with the Model group by Dunnett's test: *P b 0.05, **P b 0.01, *** P b 0.001. Values were significantly different compared with the 5-Fu group by Dunnett's test: ##P b 0.05, ###P b 0.001.
indicated that EPS could improve immunosuppressive effect induced by 5-Fu (P b 0.05, P b 0.01).
3.7. Effect of EPS in combination with 5-Fu on tumor growth of CT26-bearing mice The effect of EPS in combination with 5-Fu on the tumor growth of CT26-bearing mice was studied. As shown in Table 2, all the treatment groups could significantly reduce tumor weight. In the combination of EPS with 5-Fu group, the tumor weight was significantly lower than the 5-Fu group in a dose-dependent manner and their inhibitions were 63.55%, 70.23% and 72.24%, respectively, which were higher than the inhibition ratio of only 5-Fu (58.53%), which suggested that EPS possess a synergistic effect on 5-Fu. Compared with the model group, the average weight of the mice in each group were significantly decreased, in which the only 5-Fu group decreased most significantly (P b 0.001), indicating that only 5-Fu administration shows most hazardous to organism. It was worth noting that the average weight of mice in the combination treatment groups was distinctly higher than the 5-Fu group (P b 0.01, P b 0.001).
3.8. Effect of EPS in combination with 5-Fu on immune organ index The spleen and thymus indexes could reflect the immune function of the organism directly. As shown in Table 3 as expected, the 5-Fu group exhibited an evidently decrease in the both immune organ index and immune organ weight compared with the model group, which indicated that 5-Fu could suppress the immune function (P b 0.05, P b 0.001). However, compared with the 5-Fu group, the index and weight of immune organ in combination treatment groups showed the significant increase at the dose of 50 and 200 mg/kg of EPS respectively, which
3.9. Effect of EPS in combination with 5-Fu on heart, liver and kidney index in CT26-bearing mice The results were summarized in Table 4, the organ index in each treatment group were lower than the model groups and 5-Fu group exhibited a significantly decrease in liver and kidney indexes (P b 0.05), but all of the organ indexes in the combination treatment groups showed a slight increase compared with the 5-Fu treated group, which supported the alexipharmac effect of EPS in 5-Fu cancer therapy. 4. Discussion In recent decades, many reports had demonstrated that various polysaccharides from natural sources have a variety of biological functions, especially anti-tumor and immune-enhancing activities [20]. Since Brander reported the Zymosan have anti-tumor effect, fungi polysacchrides were attracted widespread attention [21]. Our previous studies showed that Rhizopus nigricans polysaccharides could inhibit the proliferation of human gastric cancer BGC-823 cells by inducing cell apoptosis and G2/M phase cell cycle arrest and suppress proliferation of human colorectal carcinoma HCT-116 cells by triggering S phase cell cycle arrest and apoptosis in vitro. However, the antitumor effects of EPS in vivo have not been elucidated. Thus we investigate the anti-tumor activity of EPS using CT26 tumor-bearing mice. The results indicated that EPS has a strong antitumor effect and
Table 3 Effects of combined with 5-Fu on immune organ of CT26-bearing mice. Group
Dose (mg/kg)
Spleen weight (g)
Spleen index (mg/g)
Thymus weight (g)
Thymus index (mg/g)
Model 5-Fu Drug combination
– 20 EPS (50) + 5-Fu (20) EPS (100) + 5-Fu (20) EPS (200) + 5-Fu (20)
0.209 ± 0.069 0.050 ± 0.023*** 0.126 ± 0.035**## 0.060 ± 0.032*** 0.081 ± 0.028***
8.18 ± 2.33 2.87 ± 1.41*** 5.52 ± 1.33*# 3.09 ± 1.20*** 3.97 ± 1.05***
0.129 ± 0.024 0.070 ± 0.006*** 0.116 ± 0.031## 0.084 ± 0.024** 0.104 ± 0.027#
5.05 ± 1.02 4.02 ± 0.28* 5.08 ± 0.96# 4.33 ± 0.98 5.10 ± `0.99#
Data were expressed as mean ± SD (n = 7). Values were significantly different compared with the Model group by Dunnett's test: *P b 0.05, **P b 0.01, ***P b 0.001. Values were significantly different compared with the 5-Fu group by Dunnett's test: #P b 0.05, ##P b 0.01.
Table 4 Effects of EPS combined with 5-Fu on organ index of CT26-bearing mice. Group
Dose (mg/kg)
Heart index (mg/g)
Liver index (mg/g)
Kidney index (mg/g)
Model 5-Fu Drug combination
– 20 EPS (50) + 5-Fu (20) EPS (100) + 5-Fu (20) EPS (200) + 5-Fu (20)
5.52 ± 0.30 4.74 ± 0.27 5.21 ± 0.24 5.27 ± 0.29 5.18 ± 0.34
51.87 ± 1.77 39.50 ± 2.18* 43.08 ± 2.10 44.34 ± 2.46 44.68 ± 2.02
11.88 ± 0.90 7.60 ± 0.84* 9.12 ± 0.50 9.38 ± 0.73 9.49 ± 0.72
Data were expressed as mean ± SD (n = 7). Values were significantly different compared with the Model group by Dunnett's test: *P b 0.05.
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immunomodulatory activity in vivo, but it could not inhibit proliferation of CT26 cells directly in vitro. This result agreed with the previous literatures which reports that many polysaccharides usually did not have any anti-tumor activity in vitro [22–24]. The spleen and thymus index were important index for immunity, the development of immune organs directly affects immune function and disease resistance. It was well known that as the main immune organs thymus and spleen play an important role in the immunity of the host. Therefore, a higher organ index indicated a stronger immune capability [25]. Consistent with the antitumor activity, our data showed that EPS-treated mice had higher immune organ index than the model group. Compared with the 5-Fu group, a significant increase was observed at the dose of 50 and 200 mg/kg of EPS in combination of EPS and 5-Fu treatment groups. These results indicated that EPS had a beneficial effect on the immunity of CT26 tumor-bearing mice and improve the immune organ damaged by 5-Fu. Serum cytokines are important in immune system and play a vital role in mediating the host defense [23,26] The IL-2 can promote the proliferation of responsive T cells and TNF-α also can induce apoptosis and tumor necrosis. In the present experiment, EPS could significantly increase the two cytokines at dose of 100 and 200 mg/kg compared with the model group or 5-Fu. The data implied that EPS might exert anti-tumor effect by promoting secretion of cytokines in CT26 tumorbearing mice. As cytotoxic cell, CD8+ T cell plays an important role in antivirus and anti-tumor immunity [27,28]. Recently immunotherapy for cancer has recently become a key treatment modality and many new modalities of immunotherapies targeting cytotoxic T cells [29]. Our results showed that EPS could increase the percentage of CD8+ T cells and the up-regulated ratio of CD8+ T cells might participate in the anti-tumor activity of EPS in vivo. The ability of lymphocytes response to mitogen by undergoing mitotic proliferation reflects the immune potential of the organism [30]. It was generally accepted that splenocyte proliferation response induced by ConA and LPS in vitro was used as a conventional method to evaluate T lymphocyte activity and B lymphocyte activity respectively [9,23,31]. Cellular proliferation induced by ConA is commonly used to detect T lymphocyte immunity, and LPS-induced activation of B cells indicates B lymphocyte immunity [32]. The results in the study demonstrated that EPS could significantly activate potential of T and B lymphocyte cells, which indicated that EPS could enhance the potential organism immunity. Heidelberger et al reported the anti-tumor activity of 5-Fu in 1957. Fifty years after the first synthesis of 5-Fu, it is still a standard component of adjuvant and palliative therapy having a proven impact on survival time in patients with colorectal cancer [33]. It has been one of the most commonly used chemotherapeutic drugs in the treatment of various types of cancer for over 40 years with approximately two million patients treated worldwide each year [34]. However, it also destroys the normal cells of the body, which lead to cancer patients suffering from recurrent infections induced by immunosuppression [35]. In recent years, chemotherapy combined with immune adjuvant therapy is employed to improve the anti-tumor effect of chemotherapy [36–38]. Many reports indicated that polysaccharides possess a synergistic effect on chemotherapy [19,39]. In this study, we tested the adjuvant antitumor activity of EPS in combination with 5-Fu in CT26 tumor-bearing mice. The results demonstrated that EPS combined with 5-FU could increase the tumor inhibitory rate, immune organ index and weight in the tumor-bearing mice. These data indicated that EPS could enhance the anti-tumor effect and improved the suppressed immunity in tumor bearing mice subject to 5-Fu chemotherapy. It is reported that 10–30% of patients receiving 5-Fu develop a severe to internal organ toxic reaction and women with a low body mass index were at the highest risk for acute organ toxicity [40,41]. In order to evaluate the attenuated effect of EPS on organ toxicity induced by 5-Fu, the body weight, liver, kidney and heart index were determined. Although the body weight and organ index were deceased due to the toxicity of 5-Fu in combination
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EPS and 5-Fu groups, it was increased in a certain extent compared with 5-Fu group. The result suggested that EPS could alleviate the toxicity induced by 5-Fu in CT26 tumor-bearing mice. Take together, the mixture of EPS and 5-Fu produced a synergistic effect on antitumor activity and the results illustrated that EPS possess a synergistic effect on 5-Fu may be achieved by enhancing the immune system functions and alleviating the toxicity of chemotherapy drugs. In conclusion, these findings can be suggested that the anti-tumor effects of EPS may be in part due to immune function activation and it can be the potential use on clinical application to treat colorectal cancer. Acknowledgements This study was supported by Anhui Provincial Natural Science Foundation (NO. 1408085MH197), the Key Project of Education Department of Anhui Province (NO. KJ2015A199), Scientific Research Fund of Guizhou Provincial Science and Technology Department, China (NO. [2013]2233) and Key Technology Research of Burdock Fructooligosaccharide Scale Production in Anhui Provincial Engineering Research Center for Polysaccharide Drugs (NO. 2012jc14). References [1] R. Siegel, C. Desantis, A. Jemal, Colorectal cancer statistics, 2014, CA Cancer J. Clin. 64 (2014) 104–117. [2] C.G. Drake, E.S. Antonarakis, Update: Immunological strategies for prostate cancer, Curr. Urol. Rep. 11 (2010) 202–207. [3] L. Shao, Z. Wu, H. Zhang, W. Chen, L. Ai, B. Guo, Partial characterization and immunostimulatory activity of exopolysaccharides from Lactobacillus rhamnosus KF5, Carbohydr. Polym. 107 (2014) 51–56. [4] J. Wang, X. Zhao, Y. Yang, A. Zhao, Z. Yang, Characterization and bioactivities of an exopolysaccharide produced by Lactobacillus plantarum YW32, Int. J. Biol. Macromol. 74C (2014) 119–126. [5] W. Zhang, J. Yang, J. Chen, Y. Hou, X. Han, Immunomodulatory and antitumour effects of an exopolysaccharide fraction from cultivated Cordyceps sinensis (Chinese caterpillar fungus) on tumour-bearing mice, Biotechnol. Appl. Biochem. 42 (2005) 9–15. [6] T. Huang, J. Lin, J. Cao, P. Zhang, Y. Bai, G. Chen, K. Chen, An exopolysaccharide from Trichoderma pseudokoningii and its apoptotic activity on human leukemia K562 cells, Carbohydr. Polym. 89 (2012) 701–708. [7] J. Yang, X. Li, Y. Xue, N. Wang, W. Liu, Anti-hepatoma activity and mechanism of corn silk polysaccharides in H22 tumor-bearing mice, Int. J. Biol. Macromol. 64 (2014) 276–280. [8] B. Yang, B. Xiao, T. Sun, Antitumor and immunomodulatory activity of Astragalus membranaceus polysaccharides in H22 tumor-bearing mice, Int. J. Biol. Macromol. 62 (2013) 287–290. [9] G. Zeng, Y. Ju, H. Shen, N. Zhou, L. Huang, Immunopontentiating activities of the purified polysaccharide from evening primrose in H22 tumor-bearing mice, Int. J. Biol. Macromol. 52 (2013) 280–285. [10] W.J. Li, Y. Chen, S.P. Nie, M.Y. Xie, M. He, S.S. Zhang, K.X. Zhu, Ganoderma atrum polysaccharide induces anti-tumor activity via the mitochondrial apoptotic pathway related to activation of host immune response, J. Cell. Biochem. 112 (2011) 860–871. [11] L. Zhao, Y. Chen, S. Ren, Y. Han, H. Cheng, Studies on the chemical structure and antitumor activity of an exopolysaccharide from Rhizobium sp. N613, Carbohydr. Res. 345 (2010) 637–643. [12] S.K. Mallick, S. Maiti, S.K. Bhutia, T.K. Maiti, Food Chem. Toxicol. 48 (2010) 2115–2121. [13] L. Chen, J. Pan, X. Li, Y. Zhou, Q. Meng, Q. Wang, Endo-polysaccharide of Phellinus igniarius exhibited anti-tumor effect through enhancement of cell mediated immunity, Int. Immunopharmacol. 11 (2011) 255–259. [14] R. Zheng, S. Jie, D. Hanchuan, W. Moucheng, Characterization and immunomodulating activities of polysaccharide from Lentinus edodes, Int. Immunopharmacol. 5 (2005) 811–820. [15] G. Schmeda-Hirschmann, C. Aranda, M. Kurina, J.A. Rodriguez, C. Theoduloz, Biotransformations of imbricatolic acid by aspergillus Niger and Rhizopus nigricans cultures, Molecules 12 (2007) 1092–1100. [16] D.X. Wu, Y.X. Guan, H.Q. Wang, S.J. Yao, 11 alpha-Hydroxylation of 16 alpha,17epoxyprogesterone by Rhizopus nigricans in a biphasic ionic liquid aqueous system, Bioresour. Technol. 102 (2011) 9368–9373. [17] G.C. Chen, P.Y. Zhang, T.T. Huang, W.Q. Yu, J. Lin, P. Li, K.S. Chen, Polysaccharides from Rhizopus nigricans mycelia induced apoptosis and G2/M arrest in BGC-823 cells, Carbohydr. Polym. 97 (2013) 800–808. [18] W. Yu, G. Chen, P. Zhang, K. Chen, Purification, partial characterization and antitumor effect of an exopolysaccharide from Rhizopus nigricans, Int. J. Biol. Macromol. 82 (2016) 299–307. [19] Z. Wang, B. Wu, X. Zhang, M. Xu, H. Chang, X. Lu, X. Ren, Purification of a polysaccharide from Boschniakia rossica and its synergistic antitumor effect combined with 5Fluorouracil, Carbohydr. Polym. 89 (2012) 31–35.
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