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Polysaccharides from Tricholoma matsutake and Lentinus edodes enhance 5-fluorouracil-mediated H22 cell growth inhibition
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J Tradit Chin Med 2014 June 15; 34(3): 309-316 ISSN 0255-2922 © 2014 JTCM. All rights reserved.
EXPERIMENTAL STUDY TOPIC
Polysaccharides from Tricholoma matsutake and Lentinus edodes enhance 5-fluorouracil-mediated H22 cell growth inhibition
Ming Ren, Lingyan Ye, Xiaoshi Hao, Zhixing Ren, Shuping Ren, Kun Xu, Juan Li aa Ming Ren, Lingyan Ye, Xiaoshi Hao, Zhixing Ren, Shuping Ren, Kun Xu, Juan Li, Department of Toxicology, School of Public Health, Jilin University, Changchun 130021, China Supported by Department of Science and Technology of Jilin Province (No. yyzx201123-2; No. 20130522056JH) Correspondence to: Prof. Shuping Ren and Prof. Kun Xu, Department of Toxicology, School of Public Health, Jilin University, Changchun 130021, China. [email protected]; [email protected] Telephone: +86-431-85619437 Accepted: February 27, 2014
5-FU, PL, PTM, PTM + PL, 5-FU + PL, and 5-FU + PTM groups, mice treated with 5-FU + COP showed: (a) significantly reduced tumor weight and volume (P< 0.05); (b) significantly higher serum levels of TNF-α, IL-2, and IFN-γ (P<0.05); (c) significantly increased CD4+ and CD8+ T cell frequencies in the spleen (P< 0.05); and (d) significantly increased splenic NK cell and CTL activities (P<0.05). The tumor weight and volume in mice treated with 5-FU+PL or 5-FU+PTM were significantly reduced compared with mice treated with 5-FU alone (P<0.05). Serum levels of TNF-α, IL-2, and IFN-γ, frequencies of CD4 + and CD8+ T cells in the spleen, and splenic NK and CTL activities were also significantly increased in mice treated with 5-FU+PL or 5-FU+PTM compared with mice treated with 5-FU alone (P<0.05).
Abstract OBJECTIVE: Few studies have investigated the effects produced by combinations of polysaccharides and chemotherapeutic drugs in cancer treatment. We hypothesized that a combination of polysaccharides (COP) from Lentinus edodes and Tricholoma matsutake would improve the efficacy of 5-fluorouracil (5-FU)-mediated inhibition of H22 cell growth.
CONCLUSION: Polysaccharides from Lentinus edodes and Tricholoma matsutake could enhance the efficacy of 5-FU-mediated H22 cell growth inhibition. © 2014 JTCM. All rights reserved.
METHODS: Mice were injected H22 cells and then treated with either 5-FU, polysaccharides from Tricholoma matsutake (PTM), polysaccharides from Lentinus edodes (PL), PTM + PL, 5-FU + PTM, 5-FU + PL, or 5-FU + COP. The tumor weight and volume, and splenic CD4 + and CD8 + T cell frequencies, were determined. Additionally, splenic natural killer (NK) cell and cytotoxic T lymphocyte (CTL) activities were assessed and the serum levels of tumor necrosis factor-α (TNF-α), Interleukin-2 (IL-2), and Interferon-γ (IFN-γ) were measured.
Key words: Polysaccharides; Immunomodulation; Shiitake mushrooms; Tricholoma matsutake; Fluorouracil; Growth inhibitors
INTRODUCTION Cancer is the one of the leading causes of death among adults in China.1 In clinical practice, cancer is treated by surgically removing the tumor and then administering chemotherapy.2 The disadvantages of chemotherapy are its lack of specificity and the destruction of lymphocytes, cells that play a pivotal role in combating tu-
RESULTS: Compared with mice from the control, JTCM | www. journaltcm. com
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mors.3 Moreover, immunosuppression has been detected in patients with cancers.4,5 Therefore, developing therapies to improve immune responses in cancer patients is critical for advancing cancer treatment. Many studies have reported that polysaccharides have immunomodulatory or antitumor activities, and that polysaccharides from different herbs have different activities.6-11 Byeon et al 12 observed that polysaccharides from Tricholoma matsutake (PTM) could activate splenic lymphocytes,13 while Harada et al 14 found that polysaccharides from Lentinus edodes (PL) had antitumor bioactivities. Thus, we hypothesized that a combination of polysaccharides (COP) that included PTM and PL might show better efficacy in inhibiting tumor cell growth. However, few studies have explored the effects induced by chemotherapeutic drugs with combinations of polysaccharides from different herbs on tumor cell growth. Hepatocellular carcinoma (HCC) is the third most frequent cause of cancer-related death worldwide, and most patients with HCC die within 1 year of diagnosis.15,16 5-fluorouracil (5-FU) is a chemotherapeutic drug that is frequently used in HCC treatment. The aim of the study was to investigate whether COP might enhance the efficacy of 5-FU in the growth inhibition of H22 cells, a mouse hepatoma line.
reacted dye was removed by aspiration and the blue MTT formazan precipitate was dissolved by adding 150 μL Dimethyl sulfoxide (DMSO) (Sigma-Aldrich, St. Louis, MO, USA) to each well. Absorbance at 490 nm was measured and results were expressed as percentages of the control. The effects of PTM on lymphocyte growth In a sterilized hood, spleens were removed from 8-week-old SPF-grade male C57BL/6 mice that weighed 18-22 g [Laboratory Animal Center of Jilin University, Jilin, China; Certificate of Quality No. SCXK (ji)2008-0005]. After spleens were mashed, suspended in PBS (Beyotime Biotechnology Co., Ltd., Jiangsu, China), and filtered through 200 mesh sieves (Beijing Solarbio Technology Co., Ltd., Beijing, China), ammonium chloride was added to lyse erythrocytes. After lysis, splenocytes were washed with PBS three times. The cell concentration was adjusted to 5× 106/mL with RPMI-1640 (Invitrogen, Carlsbad, CA, USA), and 5×105 cells per well were plated in 96-well plates (Corning Incorporated, Corning, NY, USA). PTM was added to the cells in triplicate at different doses, i.e., 0, 50, 100, 200, 300, 400, or 500 μg/mL. ConA (Sigma–Aldrich, St. Louis, MO, USA) was added to the cells at a final concentration of 5 μg/mL or RPMI-1640 alone was added as a control. Cells were incubated in an atmosphere of 5% CO2 at 37℃ for 12, 24, 36, 48, 60, or 72 h. The MTT assay to detect cell viability was conducted as described above. The Stimulation Index (SI) was calculated by the formula:
MATERIALS AND METHODS Reagents PL and PTM were provided by Bangbao Biotech., Co., Ltd. (Zhejiang, China). Polysaccharides were dissolved in phosphate-buffered saline (PBS). The 5-FU (25 mg/ mL) was from Tianjin Jinyao Amino Acids Co., Ltd. (Tianjin, China).
SI=
Animal experiments All protocols and procedures for animal experiments were approved by the Jilin University Institutional Animal Care and Use Committee. Male and female 8-week-old SPF-grade ICR (Institute of Cancer Research) mice that weighed 18-22 g were purchased from the Laboratory Animal Center of Jilin University, China [Certificate of Quality No. SCXK (ji) 2008-0005]. Prior to experiments, 80 mice were allowed to adapt to the environment for 1 week. Using a random number table method, mice were assigned to one of eight experimental groups: control, 5-FU (30 mg/ kg), PL, PTM, PL + PTM (COP), 5-FU + PL, 5-FU + PTM, or 5-FU + COP. Each group contained 10 mice that were treated as follows: Control, 1.5×105 H22 cells in 0.2 mL PBS were injected subcutaneously in the right thigh. 5-FU, 1.5×105 H22 cells in 0.2 mL PBS were injected subcutaneously in the right thigh; 24 h later, 5-FU was injected intraperitoneally following 10-day cycles with mice receiving injections on days 1-5. PL, 1.5 × 105 H22 cells in 0.2 mL PBS were injected subcutaneously in the right thigh; 24 h later, 1200 mg/
Cell culture P815 mastocytoma cells, YAC-1 cells, and H22 cells were cultured in RPMI-1640 (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS) (Invitrogen, Carlsbad, CA, USA) in an atmosphere of 5% CO2 at 37℃ . Cells in the logarithmic growth phase were used for experiments. Effects of PL on tumor cell growth H22 cells (a generous gift from the central lab of China-Japan Union Hospital of Jilin University, Jilin, China) in the logarithmic growth phase were plated in 96-well plates (Corning Incorporated, Corning, NY, USA) at 1×104 cells/well. When the confluence of cells was 80%-90% , PL was added to the cells in triplicate at different doses, i.e. 0, 50, 100, 200, 300, 400, or 500 μg/mL. Cells were incubated with PL for 12, 24, 36, 48, 60, or 72 h, respectively. At 4 h before the end of the incubation period, 20 μL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma-Aldrich, St. Louis, MO, USA) at a concentration of 5 mg/mL was added to each well. After 4 h, unJTCM | www. journaltcm. com
OD (cells stimulated by ConA) OD (cells without ConA)
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kg body weight PL was injected gastrically daily. PTM, 1.5×105 H22 cells in 0.2 mL PBS were injected subcutaneously in the right thigh; 24 h later, 1200 mg/ kg body weight PTM was injected gastrically daily. COP, 1.5×105 H22 cells in 0.2 mL PBS were injected subcutaneously in the right thigh; 24 h later, 1200 mg/ kg body weight PL+PTM (1∶1) was injected gastrically daily. 5-FU+PL, 1.5×105 H22 cells in 0.2 mL PBS were injected subcutaneously in the right thigh; 24 h later, 1200 mg/kg body weight PL was injected gastrically daily, and 5-FU was injected intraperitoneally following 10-day cycles with mice receiving injections on days 1-5. 5-FU+PTM, 1.5×105 H22 cells in 0.2 mL PBS were injected subcutaneously in the right thigh; 24 h later, 1200 mg/kg body weight PTM was injected gastrically daily, and 5-FU was injected intraperitoneally following 10-day cycles with mice receiving injections on days 1-5. 5-FU+COP, 1.5×105 H22 cells in 0.2 mL PBS were injected subcutaneously in the right thigh; 24 h later, 1200 mg/kg body weight PL+PTM (1∶1) was injected gastrically daily, and 5-FU was injected intraperitoneally following 10-day cycles with mice receiving injections on days 1-5.
China-Japan Union Hospital of Jilin University, Jilin, China) were used as target cells and splenocytes were used as effector cells at a ratio of effector cells to target cells of 20∶1. Cells were incubated for 24 h. After incubation, supernatants were collected and natural killer (NK) cell activities were determined using the LDH release assay according to the manufacturer's instructions (Nanjing Jiancheng Bioengineering Corp., Nanjing, China). The percentage of cytotoxicity was calculated as follows: (E-C)/(M-C) ×100, where C was the background release in medium alone, M was the maximal lysis obtained with Triton X-100, and E was the value measured for the experimental supernatant. CD4+ and CD8+ T cell quantification in the spleen Mice were anesthetized and killed and spleens were removed from mice of different groups in a sterilized hood. After spleens were mashed, suspended in PBS, and filtered through 200-micron mesh sieves, ammonium chloride was added to lyse erythrocytes. After lysis, splenocytes were washed with PBS three times. Cells were counted and 1×106 cells in 200 μL PBS were aliquoted into 1.5-mL microtubes. To each aliquot, 2.5 μL PE anti-mouse CD8 (BioLegend, San Diego, CA, USA) and 1 μL FITC anti-mouse CD4 (BioLegend) were added. Cells were incubated at 4℃ in the dark for 30 min. After incubation, cells were washed with PBS and the supernatant was discarded. Then, 500 μL PBS was added and CD4+ and CD8+ T cells were quantified by flow cytometry (Becton Dickinson, Franklin Lakes, NJ, USA). Measurements of tumor necrosis factor-α (TNF-α), Interleukin-2 (IL-2), and Interferon-γ (IFN-γ) levels in the serum After mice were anesthetized, blood was collected and serum was prepared. Serum levels of TNF-α, IL-2, and IFN-γ were measured by Enzyme linked immunosorbent assay (ELISA) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA).
Tumor weight and volume When all mice in the control group developed palpable tumors, mice from all groups were anesthetized and killed, and all tumors were removed and weighed. The volume of each tumor was calculated by the formula: tumor volume=1/2ab2, where 'a' is the largest diameter and 'b' is the perpendicular diameter.17 Assay for CTL activity C57BL/6 mice were killed and spleens were removed in a sterilized hood. After spleens were mashed, suspended in PBS, and filtered through 200-micron mesh sieves, ammonium chloride was added to lyse erythrocytes. After lysis, splenocytes were washed with PBS three times. Splenocytes were used as effector cells and P815 mastocytoma cells (a generous gift from the central lab of China-Japan Union Hospital of Jilin University, Jilin, China) were used as target cells at a ratio of effector cells to target cells of 20∶1 and incubated for 24 h. After incubation, supernatants were collected and CTL activity was measured using the lactate dehydrogenase (LDH) release assay according to the manufacturer's instructions (Nanjing Jiancheng Bioengineering Corp., Nanjing, China). The percentage of cytotoxicity was calculated as follows: (E-C)/(M-C) × 100, where C is the background release in medium alone, M is the maximal lysis obtained with Triton X-100 (Sigma – Aldrich, St. Louis, MO, USA), and E is the value measured for the experimental supernatant.
Statistical analyses The SPSS 13.0 (SPSS Inc., Chicago, IL, USA) software package was used for statistical analyses. All results were expressed as the mean ± standard deviation (SD). One-factor analysis of variance (ANOVA) was used for comparisons among groups and the least significant difference (LSD) test was used for comparisons between groups. Statistically significant differences were defined as P<0.05.
RESULTS Effects of PL on the growth of H22 cells The results of the MTT assay indicated that the viability of H22 cells incubated with PL decreased with time (Figure 1). Inhibition of H22 cell growth was seen at all PL concentrations ranging from 50 to 500 μg/mL,
Assays for natural killer cell activity YAC-1 cells (a generous gift from the central lab of JTCM | www. journaltcm. com
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and maximum inhibition occurred with 200 μg/mL between 36 and 48 h. These results indicated that PL could kill tumor cells.
with those in the control, PL, PTM, COP, 5-FU, 5-FU + PL, or 5-FU + PTM treatment groups (Figure 3; P<0.05). The tumor weight and volume in mice treated with 5-FU + PL or 5-FU + PTM were significantly reduced compared with those treated with 5-FU alone (P<0.05). The tumor weight and volume in mice treated with 5-FU alone were significantly reduced compared with those treated with PL, PTM, or COP (P<0.05).
Effects of PTM on lymphocytes in vitro The SI of lymphocytes incubated with PTM increased with time (Figure 2). Maximal stimulation was observed at 200 μg/mL PTM. Effects of 5-FU and COP on tumor volume and weight Tumor weight and volume in mice treated with 5-FU and COP were significantly reduced compared
Effects of 5-FU and COP on CTL and NK cell activities in vivo The CTL and NK cell activities in mice treated with
Figure 1 Changes in H22 cell viability after incubation with PL for the indicated times H22 cells were incubated with PL in triplicate. Inhibition of H22 cell growth was seen at all PL concentrations ranging from 50 to 500 μg/mL, and maximum inhibition occurred with 200 μg/mL between 36 and 48 h. These results indicated that PL could kill tumor cells. PL: polysaccharides from Lentinus edodes.
Figure 2 SI changes in lymphocytes incubated with PTM for indicated times Lymphocytes were plated in triplicate and incubated with PTM. The SI of lymphocytes incubated with PTM increased with time. Maximal stimulation was observed at 200 μg/mL PTM. SI: stimulation index; PTM: polysaccharides from tricholoma matsutake. JTCM | www. journaltcm. com
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5-FU + COP were significantly greater than in mice treated with 5-FU, 5-FU+PL, or 5-FU+PTM (Figure 4; P<0.05). Also, CTL and NK cell activities in mice treated with COP were significantly greater than in mice in the control, 5-FU, PL, PTM, 5-FU + PL, 5-FU+PTM, or 5-FU+COP treatment groups (Figure 4; P<0.05).
which indicated that COP treatment promoted an increase in the frequency of CD4+ and CD8+ T cells in the spleen. Effects of 5-FU and COP on serum cytokine levels Serum levels of TNF-α, IL-2, and IFN-γ in mice treated with 5-FU + COP were significantly higher than in mice treated with 5-FU, 5-FU + PL, or 5-FU + PTM (Figure 6). Also, serum levels of TNF-α, IL-2, and IFN-γ in mice treated with COP were significantly higher than in mice treated with PL, PTM, 5-FU, 5-FU+PL, 5-FU+PTM, or 5-FU+COP, suggesting that COP enhanced tumor cell killing by stimulating cytokine secretion (Figure 6).
Effects of 5-FU and COP on CD4+ and CD8+ T cells in spleen Mice treated with 5-FU+COP had significantly greater frequencies of CD4 + and CD8 + T cells in the spleen compared with mice treated with 5-FU, 5-FU + PL, or 5-FU+PTM (Figure 5; P<0.05). Also, mice treated with COP had significantly increased frequencies of CD4+ and CD8+ T cells in the spleen compared with mice treated with 5-FU, PL, PTM, 5-FU + PL, 5-FU + PTM, or 5-FU + COP (Figure 5; P<0.05),
DISCUSSION The major finding in our study was that COP could
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Figure 3 Comparison of tumor weight and volume in mice from different groups A: tumor weight; B: tumor volume. The indicated treatment groups received: control: PBS; 5-FU: 30 mg/kg 5-FU; PL: 1200 mg·kg-1· d-1 PL; PTM: 1200 mg·kg-1·d-1 PTM; COP: 1200 mg·kg-1·d-1 COP; 5-FU+PL: 30 mg/kg 5-FU and 1200 mg·kg-1·d-1 PL; 5-FU+PTM: 30 mg/kg 5-FU and 1200 mg·kg-1·d-1 PTM; and 5-FU+COP: 30 mg/kg 5-FU and 1200 mg·kg-1·d-1 COP. Mice were anesthetized and killed and tumors were removed, weighed, and measured; n=10 per group. 5-FU: 5-fluorouracil; PL: polysaccharides from Lentinus edodes; PTM: polysaccharides from Tricholoma matsutake; COP: combination of polysaccharides. aP<0.05, compared with the control group; bP<0.05, compared with the 5-FU group; cP<0.05, compared with the 5-FU+COP group. A
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Figure 4 Comparison of CTL and NK cell activities from different groups A: CTL; B: NK. The indicated treatment groups received: control: PBS; 5-FU: 30 mg/kg 5-FU; PL: 1200 mg·kg-1·d-1 PL; PTM: 1200 mg· kg-1·d-1 PTM; COP: 1200 mg·kg-1·d-1 COP; 5-FU+PL: 30 mg/kg 5-FU and 1200 mg·kg-1·d-1 PL; 5-FU+PTM: 30 mg/kg 5-FU and 1200 mg·kg-1·d-1 PTM; and 5-FU+COP: 30 mg/kg 5-FU and 1200 mg·kg-1·d-1 COP. Splenocytes from six mice per group were collected and tested for CTL and NK cell activities. CTL: cytotoxic T lymphocyte; NK: natural killer; 5-FU: 5-fluorouracil; PL: polysaccharides from Lentinus edodes; PTM: polysaccharides from Tricholoma matsutake; COP: combination of polysaccharides. aP<0.05, compared with the control group; bP<0.05, compared with the 5-FU group; cP<0.05, compared with the 5-FU+COP group. JTCM | www. journaltcm. com
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Figure 5 Comparison of frequency of splenic CD4+ and CD8+ T cells in different treatment groups A: CD4+; B: CD8+. The indicated treatment groups received: control: PBS; 5-FU: 30 mg/kg 5-FU; PL: 1200 mg·kg-1·d-1 PL; PTM: 1200 mg·kg-1·d-1 PTM; COP: 1200 mg·kg-1·d-1 COP; 5-FU+PL: 30 mg/kg 5-FU and 1200 mg·kg-1·d-1 PL; 5-FU+PTM: 30 mg/kg 5-FU and 1200 mg·kg-1·d-1 PTM; and 5-FU+COP: 30 mg/kg 5-FU and 1200 mg·kg-1·d-1 COP. Splenocytes from six mice per group were harvested and CD4+ and CD8+ T cells were quantified. 5-FU: 5-fluorouracil; PL: polysaccharides from Lentinus edodes; PTM: polysaccharides from Tricholoma matsutake; COP: combination of polysaccharides. aP<0.05, compared with the control group; bP< 0.05, compared with the 5-FU group; cP<0.05, compared with the 5-FU+COP group.
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Figure 6 Comparison of serum TNF-α, IL-2, and IFN-γ levels in mice in different treatment groups A: TNF-α; B: IL-2; C: IFN-γ. The indicated treatment groups received: control: PBS; 5-FU: 30 mg/kg 5-FU; PL: 1200 mg·kg-1·d-1 PL; PTM: 1200 mg·kg-1·d-1 PTM; COP: 1200 mg·kg-1·d-1 COP; 5-FU+PL: 30 mg/ kg 5-FU and 1200 mg·kg-1·d-1 PL; 5-FU + PTM: 30 mg/kg 5-FU and 1200 mg·kg-1·d-1PTM; and 5-FU+COP: 30 mg/kg 5-FU and 1200 mg· kg-1·d-1 COP. Serum was collected from 10 mice per group. TNF-α: tumor necrosis factor-α; IL-2: Interleukin-2; IFN-γ: Interferon-γ; 5-FU: 5-fluorouracil; PL: polysaccharides from Lentinus edodes; PTM: polysaccharides from Tricholoma matsutake; COP: combination of polysaccharides. aP<0.05, compared with the control group; bP<0.05, compared with the 5-FU group; cP<0.05, compared with the 5-FU+ COP group.
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enhance the efficacy of 5-FU-mediated growth inhibition of H22 cells. Also, tumor weight and volume were significantly reduced in mice treated with COP and 5-FU compared with mice treated with 5-FU alone. One of the mechanisms by which the combination of these polysaccharides increases the efficacy of 5-FU on H22 cell growth inhibition may be that the polysaccharides can increase the activities of NK cells and CTLs. In vivo, splenocytes from mice treated with COP significantly increased NK cell and CTL activities. As suggested by Ooi and Liu, polysaccharides might boost the host defense system by activating many types of immune cells that are vitally important for the maintenance of immune homeostasis.18 Our study confirmed that polysaccharides can strengthen host defense and increase the activities of NK cells and CTLs. Nevertheless, the immune activation properties of COP are probably not sufficient to justify its sole use to treat hepatoma, because COP alone-treated mice had significantly increased tumor weight and volume. COP can also act to inhibit cancer cell growth by stimulating the secretion of cytokines related to tumor cell death. In our in vivo experiments, mice that received PTM + PL had significantly higher levels of TNF-α, IFN-γ, and IL-2. These results are consistent with the findings of Cai et al,19 who reported that in vivo polysaccharide administration could stimulate the production of cytokines, as indicated by increased levels of serum TNF-α and IFN-γ. COP likely also enhanced 5-FU-mediated H22 cell growth inhibition by increasing the frequency of CD4 + and CD8 + T cells in the spleen. Lymphocytes are critical for inducing tumor cell death. In a study by Zhao et al,20 the frequency of CD4+ and CD8+ T cells was also increased by polysaccharides. Additionally, polysaccharides can help maintain the relative weight of the thymus and spleen, organs that supply the lymphocytes needed for the host to generate antitumor immune responses. In our study, the relative index of the spleen and thymus was significantly greater in mice treated with COP than in mice treated with 5-FU alone. In conclusion, COP can be used to enhance H22 cell growth inhibition when used in combination with 5-FU. This synergy suggests that these two polysaccharides can act more effectively together than singly to bolster the inhibitory effects of 5-FU on H22 cell growth.
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J Tradit Chin Med 2014 June 15; 34(3): 309-316 ISSN 0255-2922 © 2014 JTCM. All rights reserved.
EXPERIMENTAL STUDY TOPIC
Polysaccharides from Tricholoma matsutake and Lentinus edodes enhance 5-fluorouracil-mediated H22 cell growth inhibition
Ming Ren, Lingyan Ye, Xiaoshi Hao, Zhixing Ren, Shuping Ren, Kun Xu, Juan Li aa Ming Ren, Lingyan Ye, Xiaoshi Hao, Zhixing Ren, Shuping Ren, Kun Xu, Juan Li, Department of Toxicology, School of Public Health, Jilin University, Changchun 130021, China Supported by Department of Science and Technology of Jilin Province (No. yyzx201123-2; No. 20130522056JH) Correspondence to: Prof. Shuping Ren and Prof. Kun Xu, Department of Toxicology, School of Public Health, Jilin University, Changchun 130021, China. [email protected]; [email protected] Telephone: +86-431-85619437 Accepted: February 27, 2014
5-FU, PL, PTM, PTM + PL, 5-FU + PL, and 5-FU + PTM groups, mice treated with 5-FU + COP showed: (a) significantly reduced tumor weight and volume (P< 0.05); (b) significantly higher serum levels of TNF-α, IL-2, and IFN-γ (P<0.05); (c) significantly increased CD4+ and CD8+ T cell frequencies in the spleen (P< 0.05); and (d) significantly increased splenic NK cell and CTL activities (P<0.05). The tumor weight and volume in mice treated with 5-FU+PL or 5-FU+PTM were significantly reduced compared with mice treated with 5-FU alone (P<0.05). Serum levels of TNF-α, IL-2, and IFN-γ, frequencies of CD4 + and CD8+ T cells in the spleen, and splenic NK and CTL activities were also significantly increased in mice treated with 5-FU+PL or 5-FU+PTM compared with mice treated with 5-FU alone (P<0.05).
Abstract OBJECTIVE: Few studies have investigated the effects produced by combinations of polysaccharides and chemotherapeutic drugs in cancer treatment. We hypothesized that a combination of polysaccharides (COP) from Lentinus edodes and Tricholoma matsutake would improve the efficacy of 5-fluorouracil (5-FU)-mediated inhibition of H22 cell growth.
CONCLUSION: Polysaccharides from Lentinus edodes and Tricholoma matsutake could enhance the efficacy of 5-FU-mediated H22 cell growth inhibition. © 2014 JTCM. All rights reserved.
METHODS: Mice were injected H22 cells and then treated with either 5-FU, polysaccharides from Tricholoma matsutake (PTM), polysaccharides from Lentinus edodes (PL), PTM + PL, 5-FU + PTM, 5-FU + PL, or 5-FU + COP. The tumor weight and volume, and splenic CD4 + and CD8 + T cell frequencies, were determined. Additionally, splenic natural killer (NK) cell and cytotoxic T lymphocyte (CTL) activities were assessed and the serum levels of tumor necrosis factor-α (TNF-α), Interleukin-2 (IL-2), and Interferon-γ (IFN-γ) were measured.
Key words: Polysaccharides; Immunomodulation; Shiitake mushrooms; Tricholoma matsutake; Fluorouracil; Growth inhibitors
INTRODUCTION Cancer is the one of the leading causes of death among adults in China.1 In clinical practice, cancer is treated by surgically removing the tumor and then administering chemotherapy.2 The disadvantages of chemotherapy are its lack of specificity and the destruction of lymphocytes, cells that play a pivotal role in combating tu-
RESULTS: Compared with mice from the control, JTCM | www. journaltcm. com
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mors.3 Moreover, immunosuppression has been detected in patients with cancers.4,5 Therefore, developing therapies to improve immune responses in cancer patients is critical for advancing cancer treatment. Many studies have reported that polysaccharides have immunomodulatory or antitumor activities, and that polysaccharides from different herbs have different activities.6-11 Byeon et al 12 observed that polysaccharides from Tricholoma matsutake (PTM) could activate splenic lymphocytes,13 while Harada et al 14 found that polysaccharides from Lentinus edodes (PL) had antitumor bioactivities. Thus, we hypothesized that a combination of polysaccharides (COP) that included PTM and PL might show better efficacy in inhibiting tumor cell growth. However, few studies have explored the effects induced by chemotherapeutic drugs with combinations of polysaccharides from different herbs on tumor cell growth. Hepatocellular carcinoma (HCC) is the third most frequent cause of cancer-related death worldwide, and most patients with HCC die within 1 year of diagnosis.15,16 5-fluorouracil (5-FU) is a chemotherapeutic drug that is frequently used in HCC treatment. The aim of the study was to investigate whether COP might enhance the efficacy of 5-FU in the growth inhibition of H22 cells, a mouse hepatoma line.
reacted dye was removed by aspiration and the blue MTT formazan precipitate was dissolved by adding 150 μL Dimethyl sulfoxide (DMSO) (Sigma-Aldrich, St. Louis, MO, USA) to each well. Absorbance at 490 nm was measured and results were expressed as percentages of the control. The effects of PTM on lymphocyte growth In a sterilized hood, spleens were removed from 8-week-old SPF-grade male C57BL/6 mice that weighed 18-22 g [Laboratory Animal Center of Jilin University, Jilin, China; Certificate of Quality No. SCXK (ji)2008-0005]. After spleens were mashed, suspended in PBS (Beyotime Biotechnology Co., Ltd., Jiangsu, China), and filtered through 200 mesh sieves (Beijing Solarbio Technology Co., Ltd., Beijing, China), ammonium chloride was added to lyse erythrocytes. After lysis, splenocytes were washed with PBS three times. The cell concentration was adjusted to 5× 106/mL with RPMI-1640 (Invitrogen, Carlsbad, CA, USA), and 5×105 cells per well were plated in 96-well plates (Corning Incorporated, Corning, NY, USA). PTM was added to the cells in triplicate at different doses, i.e., 0, 50, 100, 200, 300, 400, or 500 μg/mL. ConA (Sigma–Aldrich, St. Louis, MO, USA) was added to the cells at a final concentration of 5 μg/mL or RPMI-1640 alone was added as a control. Cells were incubated in an atmosphere of 5% CO2 at 37℃ for 12, 24, 36, 48, 60, or 72 h. The MTT assay to detect cell viability was conducted as described above. The Stimulation Index (SI) was calculated by the formula:
MATERIALS AND METHODS Reagents PL and PTM were provided by Bangbao Biotech., Co., Ltd. (Zhejiang, China). Polysaccharides were dissolved in phosphate-buffered saline (PBS). The 5-FU (25 mg/ mL) was from Tianjin Jinyao Amino Acids Co., Ltd. (Tianjin, China).
SI=
Animal experiments All protocols and procedures for animal experiments were approved by the Jilin University Institutional Animal Care and Use Committee. Male and female 8-week-old SPF-grade ICR (Institute of Cancer Research) mice that weighed 18-22 g were purchased from the Laboratory Animal Center of Jilin University, China [Certificate of Quality No. SCXK (ji) 2008-0005]. Prior to experiments, 80 mice were allowed to adapt to the environment for 1 week. Using a random number table method, mice were assigned to one of eight experimental groups: control, 5-FU (30 mg/ kg), PL, PTM, PL + PTM (COP), 5-FU + PL, 5-FU + PTM, or 5-FU + COP. Each group contained 10 mice that were treated as follows: Control, 1.5×105 H22 cells in 0.2 mL PBS were injected subcutaneously in the right thigh. 5-FU, 1.5×105 H22 cells in 0.2 mL PBS were injected subcutaneously in the right thigh; 24 h later, 5-FU was injected intraperitoneally following 10-day cycles with mice receiving injections on days 1-5. PL, 1.5 × 105 H22 cells in 0.2 mL PBS were injected subcutaneously in the right thigh; 24 h later, 1200 mg/
Cell culture P815 mastocytoma cells, YAC-1 cells, and H22 cells were cultured in RPMI-1640 (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS) (Invitrogen, Carlsbad, CA, USA) in an atmosphere of 5% CO2 at 37℃ . Cells in the logarithmic growth phase were used for experiments. Effects of PL on tumor cell growth H22 cells (a generous gift from the central lab of China-Japan Union Hospital of Jilin University, Jilin, China) in the logarithmic growth phase were plated in 96-well plates (Corning Incorporated, Corning, NY, USA) at 1×104 cells/well. When the confluence of cells was 80%-90% , PL was added to the cells in triplicate at different doses, i.e. 0, 50, 100, 200, 300, 400, or 500 μg/mL. Cells were incubated with PL for 12, 24, 36, 48, 60, or 72 h, respectively. At 4 h before the end of the incubation period, 20 μL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma-Aldrich, St. Louis, MO, USA) at a concentration of 5 mg/mL was added to each well. After 4 h, unJTCM | www. journaltcm. com
OD (cells stimulated by ConA) OD (cells without ConA)
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kg body weight PL was injected gastrically daily. PTM, 1.5×105 H22 cells in 0.2 mL PBS were injected subcutaneously in the right thigh; 24 h later, 1200 mg/ kg body weight PTM was injected gastrically daily. COP, 1.5×105 H22 cells in 0.2 mL PBS were injected subcutaneously in the right thigh; 24 h later, 1200 mg/ kg body weight PL+PTM (1∶1) was injected gastrically daily. 5-FU+PL, 1.5×105 H22 cells in 0.2 mL PBS were injected subcutaneously in the right thigh; 24 h later, 1200 mg/kg body weight PL was injected gastrically daily, and 5-FU was injected intraperitoneally following 10-day cycles with mice receiving injections on days 1-5. 5-FU+PTM, 1.5×105 H22 cells in 0.2 mL PBS were injected subcutaneously in the right thigh; 24 h later, 1200 mg/kg body weight PTM was injected gastrically daily, and 5-FU was injected intraperitoneally following 10-day cycles with mice receiving injections on days 1-5. 5-FU+COP, 1.5×105 H22 cells in 0.2 mL PBS were injected subcutaneously in the right thigh; 24 h later, 1200 mg/kg body weight PL+PTM (1∶1) was injected gastrically daily, and 5-FU was injected intraperitoneally following 10-day cycles with mice receiving injections on days 1-5.
China-Japan Union Hospital of Jilin University, Jilin, China) were used as target cells and splenocytes were used as effector cells at a ratio of effector cells to target cells of 20∶1. Cells were incubated for 24 h. After incubation, supernatants were collected and natural killer (NK) cell activities were determined using the LDH release assay according to the manufacturer's instructions (Nanjing Jiancheng Bioengineering Corp., Nanjing, China). The percentage of cytotoxicity was calculated as follows: (E-C)/(M-C) ×100, where C was the background release in medium alone, M was the maximal lysis obtained with Triton X-100, and E was the value measured for the experimental supernatant. CD4+ and CD8+ T cell quantification in the spleen Mice were anesthetized and killed and spleens were removed from mice of different groups in a sterilized hood. After spleens were mashed, suspended in PBS, and filtered through 200-micron mesh sieves, ammonium chloride was added to lyse erythrocytes. After lysis, splenocytes were washed with PBS three times. Cells were counted and 1×106 cells in 200 μL PBS were aliquoted into 1.5-mL microtubes. To each aliquot, 2.5 μL PE anti-mouse CD8 (BioLegend, San Diego, CA, USA) and 1 μL FITC anti-mouse CD4 (BioLegend) were added. Cells were incubated at 4℃ in the dark for 30 min. After incubation, cells were washed with PBS and the supernatant was discarded. Then, 500 μL PBS was added and CD4+ and CD8+ T cells were quantified by flow cytometry (Becton Dickinson, Franklin Lakes, NJ, USA). Measurements of tumor necrosis factor-α (TNF-α), Interleukin-2 (IL-2), and Interferon-γ (IFN-γ) levels in the serum After mice were anesthetized, blood was collected and serum was prepared. Serum levels of TNF-α, IL-2, and IFN-γ were measured by Enzyme linked immunosorbent assay (ELISA) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA).
Tumor weight and volume When all mice in the control group developed palpable tumors, mice from all groups were anesthetized and killed, and all tumors were removed and weighed. The volume of each tumor was calculated by the formula: tumor volume=1/2ab2, where 'a' is the largest diameter and 'b' is the perpendicular diameter.17 Assay for CTL activity C57BL/6 mice were killed and spleens were removed in a sterilized hood. After spleens were mashed, suspended in PBS, and filtered through 200-micron mesh sieves, ammonium chloride was added to lyse erythrocytes. After lysis, splenocytes were washed with PBS three times. Splenocytes were used as effector cells and P815 mastocytoma cells (a generous gift from the central lab of China-Japan Union Hospital of Jilin University, Jilin, China) were used as target cells at a ratio of effector cells to target cells of 20∶1 and incubated for 24 h. After incubation, supernatants were collected and CTL activity was measured using the lactate dehydrogenase (LDH) release assay according to the manufacturer's instructions (Nanjing Jiancheng Bioengineering Corp., Nanjing, China). The percentage of cytotoxicity was calculated as follows: (E-C)/(M-C) × 100, where C is the background release in medium alone, M is the maximal lysis obtained with Triton X-100 (Sigma – Aldrich, St. Louis, MO, USA), and E is the value measured for the experimental supernatant.
Statistical analyses The SPSS 13.0 (SPSS Inc., Chicago, IL, USA) software package was used for statistical analyses. All results were expressed as the mean ± standard deviation (SD). One-factor analysis of variance (ANOVA) was used for comparisons among groups and the least significant difference (LSD) test was used for comparisons between groups. Statistically significant differences were defined as P<0.05.
RESULTS Effects of PL on the growth of H22 cells The results of the MTT assay indicated that the viability of H22 cells incubated with PL decreased with time (Figure 1). Inhibition of H22 cell growth was seen at all PL concentrations ranging from 50 to 500 μg/mL,
Assays for natural killer cell activity YAC-1 cells (a generous gift from the central lab of JTCM | www. journaltcm. com
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and maximum inhibition occurred with 200 μg/mL between 36 and 48 h. These results indicated that PL could kill tumor cells.
with those in the control, PL, PTM, COP, 5-FU, 5-FU + PL, or 5-FU + PTM treatment groups (Figure 3; P<0.05). The tumor weight and volume in mice treated with 5-FU + PL or 5-FU + PTM were significantly reduced compared with those treated with 5-FU alone (P<0.05). The tumor weight and volume in mice treated with 5-FU alone were significantly reduced compared with those treated with PL, PTM, or COP (P<0.05).
Effects of PTM on lymphocytes in vitro The SI of lymphocytes incubated with PTM increased with time (Figure 2). Maximal stimulation was observed at 200 μg/mL PTM. Effects of 5-FU and COP on tumor volume and weight Tumor weight and volume in mice treated with 5-FU and COP were significantly reduced compared
Effects of 5-FU and COP on CTL and NK cell activities in vivo The CTL and NK cell activities in mice treated with
Figure 1 Changes in H22 cell viability after incubation with PL for the indicated times H22 cells were incubated with PL in triplicate. Inhibition of H22 cell growth was seen at all PL concentrations ranging from 50 to 500 μg/mL, and maximum inhibition occurred with 200 μg/mL between 36 and 48 h. These results indicated that PL could kill tumor cells. PL: polysaccharides from Lentinus edodes.
Figure 2 SI changes in lymphocytes incubated with PTM for indicated times Lymphocytes were plated in triplicate and incubated with PTM. The SI of lymphocytes incubated with PTM increased with time. Maximal stimulation was observed at 200 μg/mL PTM. SI: stimulation index; PTM: polysaccharides from tricholoma matsutake. JTCM | www. journaltcm. com
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5-FU + COP were significantly greater than in mice treated with 5-FU, 5-FU+PL, or 5-FU+PTM (Figure 4; P<0.05). Also, CTL and NK cell activities in mice treated with COP were significantly greater than in mice in the control, 5-FU, PL, PTM, 5-FU + PL, 5-FU+PTM, or 5-FU+COP treatment groups (Figure 4; P<0.05).
which indicated that COP treatment promoted an increase in the frequency of CD4+ and CD8+ T cells in the spleen. Effects of 5-FU and COP on serum cytokine levels Serum levels of TNF-α, IL-2, and IFN-γ in mice treated with 5-FU + COP were significantly higher than in mice treated with 5-FU, 5-FU + PL, or 5-FU + PTM (Figure 6). Also, serum levels of TNF-α, IL-2, and IFN-γ in mice treated with COP were significantly higher than in mice treated with PL, PTM, 5-FU, 5-FU+PL, 5-FU+PTM, or 5-FU+COP, suggesting that COP enhanced tumor cell killing by stimulating cytokine secretion (Figure 6).
Effects of 5-FU and COP on CD4+ and CD8+ T cells in spleen Mice treated with 5-FU+COP had significantly greater frequencies of CD4 + and CD8 + T cells in the spleen compared with mice treated with 5-FU, 5-FU + PL, or 5-FU+PTM (Figure 5; P<0.05). Also, mice treated with COP had significantly increased frequencies of CD4+ and CD8+ T cells in the spleen compared with mice treated with 5-FU, PL, PTM, 5-FU + PL, 5-FU + PTM, or 5-FU + COP (Figure 5; P<0.05),
DISCUSSION The major finding in our study was that COP could
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Figure 3 Comparison of tumor weight and volume in mice from different groups A: tumor weight; B: tumor volume. The indicated treatment groups received: control: PBS; 5-FU: 30 mg/kg 5-FU; PL: 1200 mg·kg-1· d-1 PL; PTM: 1200 mg·kg-1·d-1 PTM; COP: 1200 mg·kg-1·d-1 COP; 5-FU+PL: 30 mg/kg 5-FU and 1200 mg·kg-1·d-1 PL; 5-FU+PTM: 30 mg/kg 5-FU and 1200 mg·kg-1·d-1 PTM; and 5-FU+COP: 30 mg/kg 5-FU and 1200 mg·kg-1·d-1 COP. Mice were anesthetized and killed and tumors were removed, weighed, and measured; n=10 per group. 5-FU: 5-fluorouracil; PL: polysaccharides from Lentinus edodes; PTM: polysaccharides from Tricholoma matsutake; COP: combination of polysaccharides. aP<0.05, compared with the control group; bP<0.05, compared with the 5-FU group; cP<0.05, compared with the 5-FU+COP group. A
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Figure 4 Comparison of CTL and NK cell activities from different groups A: CTL; B: NK. The indicated treatment groups received: control: PBS; 5-FU: 30 mg/kg 5-FU; PL: 1200 mg·kg-1·d-1 PL; PTM: 1200 mg· kg-1·d-1 PTM; COP: 1200 mg·kg-1·d-1 COP; 5-FU+PL: 30 mg/kg 5-FU and 1200 mg·kg-1·d-1 PL; 5-FU+PTM: 30 mg/kg 5-FU and 1200 mg·kg-1·d-1 PTM; and 5-FU+COP: 30 mg/kg 5-FU and 1200 mg·kg-1·d-1 COP. Splenocytes from six mice per group were collected and tested for CTL and NK cell activities. CTL: cytotoxic T lymphocyte; NK: natural killer; 5-FU: 5-fluorouracil; PL: polysaccharides from Lentinus edodes; PTM: polysaccharides from Tricholoma matsutake; COP: combination of polysaccharides. aP<0.05, compared with the control group; bP<0.05, compared with the 5-FU group; cP<0.05, compared with the 5-FU+COP group. JTCM | www. journaltcm. com
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Figure 5 Comparison of frequency of splenic CD4+ and CD8+ T cells in different treatment groups A: CD4+; B: CD8+. The indicated treatment groups received: control: PBS; 5-FU: 30 mg/kg 5-FU; PL: 1200 mg·kg-1·d-1 PL; PTM: 1200 mg·kg-1·d-1 PTM; COP: 1200 mg·kg-1·d-1 COP; 5-FU+PL: 30 mg/kg 5-FU and 1200 mg·kg-1·d-1 PL; 5-FU+PTM: 30 mg/kg 5-FU and 1200 mg·kg-1·d-1 PTM; and 5-FU+COP: 30 mg/kg 5-FU and 1200 mg·kg-1·d-1 COP. Splenocytes from six mice per group were harvested and CD4+ and CD8+ T cells were quantified. 5-FU: 5-fluorouracil; PL: polysaccharides from Lentinus edodes; PTM: polysaccharides from Tricholoma matsutake; COP: combination of polysaccharides. aP<0.05, compared with the control group; bP< 0.05, compared with the 5-FU group; cP<0.05, compared with the 5-FU+COP group.
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Figure 6 Comparison of serum TNF-α, IL-2, and IFN-γ levels in mice in different treatment groups A: TNF-α; B: IL-2; C: IFN-γ. The indicated treatment groups received: control: PBS; 5-FU: 30 mg/kg 5-FU; PL: 1200 mg·kg-1·d-1 PL; PTM: 1200 mg·kg-1·d-1 PTM; COP: 1200 mg·kg-1·d-1 COP; 5-FU+PL: 30 mg/ kg 5-FU and 1200 mg·kg-1·d-1 PL; 5-FU + PTM: 30 mg/kg 5-FU and 1200 mg·kg-1·d-1PTM; and 5-FU+COP: 30 mg/kg 5-FU and 1200 mg· kg-1·d-1 COP. Serum was collected from 10 mice per group. TNF-α: tumor necrosis factor-α; IL-2: Interleukin-2; IFN-γ: Interferon-γ; 5-FU: 5-fluorouracil; PL: polysaccharides from Lentinus edodes; PTM: polysaccharides from Tricholoma matsutake; COP: combination of polysaccharides. aP<0.05, compared with the control group; bP<0.05, compared with the 5-FU group; cP<0.05, compared with the 5-FU+ COP group.
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enhance the efficacy of 5-FU-mediated growth inhibition of H22 cells. Also, tumor weight and volume were significantly reduced in mice treated with COP and 5-FU compared with mice treated with 5-FU alone. One of the mechanisms by which the combination of these polysaccharides increases the efficacy of 5-FU on H22 cell growth inhibition may be that the polysaccharides can increase the activities of NK cells and CTLs. In vivo, splenocytes from mice treated with COP significantly increased NK cell and CTL activities. As suggested by Ooi and Liu, polysaccharides might boost the host defense system by activating many types of immune cells that are vitally important for the maintenance of immune homeostasis.18 Our study confirmed that polysaccharides can strengthen host defense and increase the activities of NK cells and CTLs. Nevertheless, the immune activation properties of COP are probably not sufficient to justify its sole use to treat hepatoma, because COP alone-treated mice had significantly increased tumor weight and volume. COP can also act to inhibit cancer cell growth by stimulating the secretion of cytokines related to tumor cell death. In our in vivo experiments, mice that received PTM + PL had significantly higher levels of TNF-α, IFN-γ, and IL-2. These results are consistent with the findings of Cai et al,19 who reported that in vivo polysaccharide administration could stimulate the production of cytokines, as indicated by increased levels of serum TNF-α and IFN-γ. COP likely also enhanced 5-FU-mediated H22 cell growth inhibition by increasing the frequency of CD4 + and CD8 + T cells in the spleen. Lymphocytes are critical for inducing tumor cell death. In a study by Zhao et al,20 the frequency of CD4+ and CD8+ T cells was also increased by polysaccharides. Additionally, polysaccharides can help maintain the relative weight of the thymus and spleen, organs that supply the lymphocytes needed for the host to generate antitumor immune responses. In our study, the relative index of the spleen and thymus was significantly greater in mice treated with COP than in mice treated with 5-FU alone. In conclusion, COP can be used to enhance H22 cell growth inhibition when used in combination with 5-FU. This synergy suggests that these two polysaccharides can act more effectively together than singly to bolster the inhibitory effects of 5-FU on H22 cell growth.
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