Tumor Biol. (2014) 35:12561–12566 DOI 10.1007/s13277-014-2576-7
RESEARCH ARTICLE
Antitumor activities of ginseng polysaccharide in C57BL/6 mice with Lewis lung carcinoma Xiao Zhou & Huaiyin Shi & Gening Jiang & Yongan Zhou & Jianfang Xu
Received: 5 June 2014 / Accepted: 29 August 2014 / Published online: 11 September 2014 # International Society of Oncology and BioMarkers (ISOBM) 2014
Abstract In the present study, we prepared ginseng polysaccharide (GP) and evaluated its antitumor and immunomodulatory activities in C57BL/6 mice bearing with Lewis lung carcinoma (LLC). Administration of GP (50, 100, and 200 mg/kg) could not only significantly inhibit the growth of transplantable LLC tumor in C57BL/6 mice but also remarkably increase relative weight of spleen and thymus, splenocytes proliferation, and the ratio of CD4+/CD8+ T lymphocyte in peripheral blood in LLC-bearing mice. Furthermore, the serum IL-2 and IFN-γ production and NK cytolytic activity were also prompted in LLC-bearing mice in response to GP treatment at three doses. Additionally, GP showed no side effects such as weight loss in body weight and internal organs (lung, liver, kidney, and heart) as well as inactivity during the experiment. Therefore, GP might be conveniently exploited to be good immune-stimulating modifiers and had the potential value for tumor therapy. Xiao Zhou and Huaiyin Shi contributed equally to this work. X. Zhou : G. Jiang Department of Thoracic Surgery, Shanghai Pulmonary Hospital, Medical School of Tongji University, 507 Zhengmin Road, Shanghai 200433, People’s Republic of China H. Shi Department of Pathology, Chinese PLA General Hospital, Fuxing Road #28, 100853 Beijing, People’s Republic of China Y. Zhou (*) Department of Thoracic Surgery, Tangdu Hospital, the Fourth Military Medical University, 1 Xinsi Road, 710038 Xi’an, People’s Republic of China e-mail: [email protected] J. Xu (*) Department of Oncology, Shanghai Pulmonary Hospital, Tongji University School of Medicine, Tongji University Cancer Institute, 507 Zhengmin Road, 200433 Shanghai, People’s Republic of China e-mail: [email protected]
Keywords Ginseng polysaccharide . Antitumor . Immunomodulatory
Introduction Lung cancer is a world problem and the leading cause of cancer deaths in the world [1, 2]. Although there are various therapies to treat lung cancer including chemotherapy and radiotherapy, the clinical efficacy of current treatments is very limited [3]. The development and progression of lung cancer are associated with a variety of factors, including oxidative stress, apoptosis, immune factors disorders, dysfunction of lung epithelial cells, inflammation, etc. There is accumulating evidence highlighting the role of immunological responses in the pathogenesis of lung cancer [4, 5]. Therefore, there is a continuing need for development of new therapeutic drugs or novel strategies for the prevention and treatment of lung cancer. For this aim, using herbal immune stimulants like polysaccharide with good efficacy and few side effects is a good strategy in chemoprevention compared with allopathic medicine [6]. Ginseng, the dry extract prepared from Panax ginseng C.A. Meyer, has occupied an important place among the tonic remedies of oriental medicine for several thousand years with mysterious powers in Asian countries. Ginseng contains many active components, including ginsenosides, essential oil, peptidoglycans, polysaccharides, nitrogen-containing compounds, fatty acids, and phenolic compounds [7–9]. Among these components, ginseng polysaccharide (GP) have been demonstrated to have multiple functions, such as immunomodulation, antitumor, anti-adhesive, antioxidant, hypoglycemic activities, and so on [7–11]. However, the antitumor activity of GP against the growth of Lewis lung carcinoma (LLC) in mice has not been reported. Therefore, in the present study, we focused on GP for its antitumor and immunomodulatory activity in LLC-bearing C57BL/6 mice.
12562
The results will be helpful to develop novel drugs and functional foods.
Materials and methods Materials and chemicals The roots of P. ginseng were purchased from local herb stores in Xi’an (China). Concanavalin A (ConA) and lipopolysaccharide (LPS) were purchased from Jiancheng Biologic Project Co. (Nanjing, China). Fetal bovine serum (FBS) and RPMI-1640 tissue culture medium were purchased from Gibco BRL (Grand Island, NY, USA). Cytokine-detecting ELISA kits were purchased from Zhongshan Golden Bridge Biotechnology Co. (Beijing, China). Antimouse CD4+ and CD8+ monoclonal antibodies were obtained from Pharmingen (San Diego, CA). All other chemicals used in our experiments were of analytical reagent grade. Preparation for GP The air dried roots of P. ginseng was washed, dried at 60 °C and crushed up (50 mesh) and then weighed (200 g) and soaked with 95 % ethanol to remove the pigments and small lipophilic molecules, followed by filtration. The residue was dried in air and then dipped in 20 volumes of double-distilled water at 100 °C for three times (6 h for each). All water extracts were combined, filtrated, concentrated, and precipitated with three volumes of 95 % EtOH at 4 °C overnight to precipitate crude polysaccharides. The precipitate was collected by centrifugation and deproteinated by Sevag method [12]. Finally, the supernatant was lyophilized to give polysaccharides GP. Cell culture LLC cells were maintained in RPMI-1640 medium supplemented with 10 % FBS, 100 units/mL of penicillin, and 100 μg/mL of streptomycin in a humidified atmosphere containing 5 % CO2 at 37 °C. Lewis lung cancer model C57BL/6 male mice at the age of 6–8 weeks (weight, 18–22 g) were purchased from the Fourth Military Medical University, Xi’an, China. The animals were kept under a 12-h light/12-h dark cycle at 25 °C with a 60 % relative humidity and were provided water and standard chow ad libitium. One week later, the LLC cells (5×106 cells/mL) suspended in 0.1 ml phosphate-buffered saline (PBS) were implanted carefully intradermally into the left axila of the C57BL/6 mice. One day after LLC inoculation, the mice were randomly divided into vehicle control and GP treatment (50, 100, and
Tumor Biol. (2014) 35:12561–12566
200 mg kg−1 day−1) groups (n=5). All animals in GP group were administered orally with GP at doses of 50, 100, and 200 mg/kg in a volume of 0.1 mL per 20 g of body weight daily during the experimental period, while the control mice were administered PBS vehicle on the same schedule. The tumor volumes were measured every 3 days with a caliper and calculated according to the formula [(length×width2)/2], where length represents the larger tumor diameter and width stands for the smaller tumor diameter [13]. On the 21st day, body weights of the animals were recorded before sacrifice by cervical dislocation and the tumor, liver, kidney, heart, spleen, and lung were immediately removed, and their weights were determined on the balance. Blood samples were taken through orbital venous and collected in polystyrene tubes without the anticoagulant. The tumor growth inhibition rate was calculated using the following formula: [(mean tumor weight of control group-mean tumor weight of treatment group)/mean tumor weight of control group]×100 % [14]. The spleen and thymus weight were also measured, and their indexed were calculated as spleen or thymus weight (mg) divided by body weight (g) [15]. The experimental procedures were carried out in accordance with the National Institute of Health’s Guide for the Care and Use of Laboratory Animals, and their experimental use was approved by the Animal Ethics Committee of the university. Histological examination All the organs such as liver, heart, kidney, and spleen and tumor mass dissected from mice were weighed and fixed with 10 % neutral phosphate-buffered formalin for 4 h and then processed for paraffin embedding according to standard histological procedures. The embedded tissue was serially sectioned into sections in 5-μm thickness, and paraffin sections were deparaffinized in xylene, rehydrated with graded ethanol, and stained with hematoxylin and eosin (H&E). Lymphocyte proliferation assay Spleen collected from sacrificed mice under aseptic conditions were immediately minced into small pieces and passed through a fine steel mesh to obtain a homogeneous cell suspension. Recovered spleen cells were lysed with lysis buffer (0.15 M NH4Cl, pH 7.4) for 5 min to remove erythrocytes. After centrifugation (380×g at 4 °C for 10 min), the pelleted spleen cells were washed three times in PBS and resuspended in RPMI-1640 complete medium. Cell numbers and viability (over 95 %) were estimated by trypan blue dye exclusion technique, and cell proliferation was estimated based on the method of 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) [16]. Briefly, spleen lymphocytes (100 μl) were planted into 96-well plates (1× 106 cells/well) with or without ConA (final concentration of 1 μg/mL) or LPS (final concentration of 5 μg/mL) and
Tumor Biol. (2014) 35:12561–12566
a
2500
Tumor volume (mm3)
incubated at 37 °C in a humidified atmosphere containing 5 % CO2. The control was added same volume of RPMI-1640 medium. After 44 h, 20 μl MTT (5 mg/ml) were added to each well, and the plates were incubated for another 4 h. Thereafter, the plates were centrifuged (1,400×g, 5 min), and the supernatants were carefully removed by pipetting. Then 150 μl of dimethyl sulphoxide (DMSO) working solution was added to each well to dissolve purple formazan formed by MTT assay. Finally, the absorbance was read at 570 nm in an ELISA reader (Bio-Rad, USA) after 15 min.
12563
1500 1000 500 0 0
Anticoagulated blood from mice was treated with hemolysin to produce a lymphocyte suspension (1×106/mL), which was marked with PE-conjugated rat anti-mouse CD4+ or CD8+ mAbs in the dark for 30 min at 4 °C and collected by centrifugation at 380×g for 15 min. The percentage of positively stained cells, determined over 10,000 events, was analyzed using a FACS Calibur flow cytometer (Becton Dickinson, USA) and Cell Quest software. Cytolytic activity of natural killer cells in LLC-bearing C57BL/6 mice YAC-1, a Moloney virus-induced mouse T cell lymphoma of A/SN origin, was used as target cell in the 51Cr relase assay, and splenocytes prepared as described above were used as the effector cells. Effector and target cells were dispensed in triplicate into 96-well U-bottom microtiter plate at 1×106 cells/well and 2×104 cells/well, respectively, to give E/T ratio of 50:1. Plates were centrifuged at 400g for 5 min and incubated for 4 h at 37 °C in 5 % CO2 atmosphere. At the endpoints, 0.1 ml of the supernatant collected by centrifugation (600×g, 10 min) was collected for radioactivity counting. Spontaneous release was determined by adding 100 labeled target cells to 0.05 % Tween-20. Percentage of cytotoxicity, as measured by specific 51Cr release assay, was calculated by using the formula: (cpm experimental−cpmspontaneous)/ (cpm maximal−cpm spontaneous)×100.
9
12
15
18
21
Tumor weight (g)
4 3 2
** **
**
1
Control
50
100
200
(mg/kg)
GP
c
80
The tumor growth inhibition rate (%)
Flow cytometric analysis of differentiation antigens of CD4+ and CD8+ on peripheral blood T lymphocyte of LLC-bearing C57BL/6 mice
6
0
60 40 20 0 Control
50
100
200
(mg/kg)
GP
Fig. 1 Effects of GP on tumor volume (a), tumor weight (b), and tumor growth inhibition rate (c) in mice inoculated with Lewis lung carcinoma (LLC). Data are expressed as means±SD (n=5). **p
RESEARCH ARTICLE
Antitumor activities of ginseng polysaccharide in C57BL/6 mice with Lewis lung carcinoma Xiao Zhou & Huaiyin Shi & Gening Jiang & Yongan Zhou & Jianfang Xu
Received: 5 June 2014 / Accepted: 29 August 2014 / Published online: 11 September 2014 # International Society of Oncology and BioMarkers (ISOBM) 2014
Abstract In the present study, we prepared ginseng polysaccharide (GP) and evaluated its antitumor and immunomodulatory activities in C57BL/6 mice bearing with Lewis lung carcinoma (LLC). Administration of GP (50, 100, and 200 mg/kg) could not only significantly inhibit the growth of transplantable LLC tumor in C57BL/6 mice but also remarkably increase relative weight of spleen and thymus, splenocytes proliferation, and the ratio of CD4+/CD8+ T lymphocyte in peripheral blood in LLC-bearing mice. Furthermore, the serum IL-2 and IFN-γ production and NK cytolytic activity were also prompted in LLC-bearing mice in response to GP treatment at three doses. Additionally, GP showed no side effects such as weight loss in body weight and internal organs (lung, liver, kidney, and heart) as well as inactivity during the experiment. Therefore, GP might be conveniently exploited to be good immune-stimulating modifiers and had the potential value for tumor therapy. Xiao Zhou and Huaiyin Shi contributed equally to this work. X. Zhou : G. Jiang Department of Thoracic Surgery, Shanghai Pulmonary Hospital, Medical School of Tongji University, 507 Zhengmin Road, Shanghai 200433, People’s Republic of China H. Shi Department of Pathology, Chinese PLA General Hospital, Fuxing Road #28, 100853 Beijing, People’s Republic of China Y. Zhou (*) Department of Thoracic Surgery, Tangdu Hospital, the Fourth Military Medical University, 1 Xinsi Road, 710038 Xi’an, People’s Republic of China e-mail: [email protected] J. Xu (*) Department of Oncology, Shanghai Pulmonary Hospital, Tongji University School of Medicine, Tongji University Cancer Institute, 507 Zhengmin Road, 200433 Shanghai, People’s Republic of China e-mail: [email protected]
Keywords Ginseng polysaccharide . Antitumor . Immunomodulatory
Introduction Lung cancer is a world problem and the leading cause of cancer deaths in the world [1, 2]. Although there are various therapies to treat lung cancer including chemotherapy and radiotherapy, the clinical efficacy of current treatments is very limited [3]. The development and progression of lung cancer are associated with a variety of factors, including oxidative stress, apoptosis, immune factors disorders, dysfunction of lung epithelial cells, inflammation, etc. There is accumulating evidence highlighting the role of immunological responses in the pathogenesis of lung cancer [4, 5]. Therefore, there is a continuing need for development of new therapeutic drugs or novel strategies for the prevention and treatment of lung cancer. For this aim, using herbal immune stimulants like polysaccharide with good efficacy and few side effects is a good strategy in chemoprevention compared with allopathic medicine [6]. Ginseng, the dry extract prepared from Panax ginseng C.A. Meyer, has occupied an important place among the tonic remedies of oriental medicine for several thousand years with mysterious powers in Asian countries. Ginseng contains many active components, including ginsenosides, essential oil, peptidoglycans, polysaccharides, nitrogen-containing compounds, fatty acids, and phenolic compounds [7–9]. Among these components, ginseng polysaccharide (GP) have been demonstrated to have multiple functions, such as immunomodulation, antitumor, anti-adhesive, antioxidant, hypoglycemic activities, and so on [7–11]. However, the antitumor activity of GP against the growth of Lewis lung carcinoma (LLC) in mice has not been reported. Therefore, in the present study, we focused on GP for its antitumor and immunomodulatory activity in LLC-bearing C57BL/6 mice.
12562
The results will be helpful to develop novel drugs and functional foods.
Materials and methods Materials and chemicals The roots of P. ginseng were purchased from local herb stores in Xi’an (China). Concanavalin A (ConA) and lipopolysaccharide (LPS) were purchased from Jiancheng Biologic Project Co. (Nanjing, China). Fetal bovine serum (FBS) and RPMI-1640 tissue culture medium were purchased from Gibco BRL (Grand Island, NY, USA). Cytokine-detecting ELISA kits were purchased from Zhongshan Golden Bridge Biotechnology Co. (Beijing, China). Antimouse CD4+ and CD8+ monoclonal antibodies were obtained from Pharmingen (San Diego, CA). All other chemicals used in our experiments were of analytical reagent grade. Preparation for GP The air dried roots of P. ginseng was washed, dried at 60 °C and crushed up (50 mesh) and then weighed (200 g) and soaked with 95 % ethanol to remove the pigments and small lipophilic molecules, followed by filtration. The residue was dried in air and then dipped in 20 volumes of double-distilled water at 100 °C for three times (6 h for each). All water extracts were combined, filtrated, concentrated, and precipitated with three volumes of 95 % EtOH at 4 °C overnight to precipitate crude polysaccharides. The precipitate was collected by centrifugation and deproteinated by Sevag method [12]. Finally, the supernatant was lyophilized to give polysaccharides GP. Cell culture LLC cells were maintained in RPMI-1640 medium supplemented with 10 % FBS, 100 units/mL of penicillin, and 100 μg/mL of streptomycin in a humidified atmosphere containing 5 % CO2 at 37 °C. Lewis lung cancer model C57BL/6 male mice at the age of 6–8 weeks (weight, 18–22 g) were purchased from the Fourth Military Medical University, Xi’an, China. The animals were kept under a 12-h light/12-h dark cycle at 25 °C with a 60 % relative humidity and were provided water and standard chow ad libitium. One week later, the LLC cells (5×106 cells/mL) suspended in 0.1 ml phosphate-buffered saline (PBS) were implanted carefully intradermally into the left axila of the C57BL/6 mice. One day after LLC inoculation, the mice were randomly divided into vehicle control and GP treatment (50, 100, and
Tumor Biol. (2014) 35:12561–12566
200 mg kg−1 day−1) groups (n=5). All animals in GP group were administered orally with GP at doses of 50, 100, and 200 mg/kg in a volume of 0.1 mL per 20 g of body weight daily during the experimental period, while the control mice were administered PBS vehicle on the same schedule. The tumor volumes were measured every 3 days with a caliper and calculated according to the formula [(length×width2)/2], where length represents the larger tumor diameter and width stands for the smaller tumor diameter [13]. On the 21st day, body weights of the animals were recorded before sacrifice by cervical dislocation and the tumor, liver, kidney, heart, spleen, and lung were immediately removed, and their weights were determined on the balance. Blood samples were taken through orbital venous and collected in polystyrene tubes without the anticoagulant. The tumor growth inhibition rate was calculated using the following formula: [(mean tumor weight of control group-mean tumor weight of treatment group)/mean tumor weight of control group]×100 % [14]. The spleen and thymus weight were also measured, and their indexed were calculated as spleen or thymus weight (mg) divided by body weight (g) [15]. The experimental procedures were carried out in accordance with the National Institute of Health’s Guide for the Care and Use of Laboratory Animals, and their experimental use was approved by the Animal Ethics Committee of the university. Histological examination All the organs such as liver, heart, kidney, and spleen and tumor mass dissected from mice were weighed and fixed with 10 % neutral phosphate-buffered formalin for 4 h and then processed for paraffin embedding according to standard histological procedures. The embedded tissue was serially sectioned into sections in 5-μm thickness, and paraffin sections were deparaffinized in xylene, rehydrated with graded ethanol, and stained with hematoxylin and eosin (H&E). Lymphocyte proliferation assay Spleen collected from sacrificed mice under aseptic conditions were immediately minced into small pieces and passed through a fine steel mesh to obtain a homogeneous cell suspension. Recovered spleen cells were lysed with lysis buffer (0.15 M NH4Cl, pH 7.4) for 5 min to remove erythrocytes. After centrifugation (380×g at 4 °C for 10 min), the pelleted spleen cells were washed three times in PBS and resuspended in RPMI-1640 complete medium. Cell numbers and viability (over 95 %) were estimated by trypan blue dye exclusion technique, and cell proliferation was estimated based on the method of 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) [16]. Briefly, spleen lymphocytes (100 μl) were planted into 96-well plates (1× 106 cells/well) with or without ConA (final concentration of 1 μg/mL) or LPS (final concentration of 5 μg/mL) and
Tumor Biol. (2014) 35:12561–12566
a
2500
Tumor volume (mm3)
incubated at 37 °C in a humidified atmosphere containing 5 % CO2. The control was added same volume of RPMI-1640 medium. After 44 h, 20 μl MTT (5 mg/ml) were added to each well, and the plates were incubated for another 4 h. Thereafter, the plates were centrifuged (1,400×g, 5 min), and the supernatants were carefully removed by pipetting. Then 150 μl of dimethyl sulphoxide (DMSO) working solution was added to each well to dissolve purple formazan formed by MTT assay. Finally, the absorbance was read at 570 nm in an ELISA reader (Bio-Rad, USA) after 15 min.
12563
1500 1000 500 0 0
Anticoagulated blood from mice was treated with hemolysin to produce a lymphocyte suspension (1×106/mL), which was marked with PE-conjugated rat anti-mouse CD4+ or CD8+ mAbs in the dark for 30 min at 4 °C and collected by centrifugation at 380×g for 15 min. The percentage of positively stained cells, determined over 10,000 events, was analyzed using a FACS Calibur flow cytometer (Becton Dickinson, USA) and Cell Quest software. Cytolytic activity of natural killer cells in LLC-bearing C57BL/6 mice YAC-1, a Moloney virus-induced mouse T cell lymphoma of A/SN origin, was used as target cell in the 51Cr relase assay, and splenocytes prepared as described above were used as the effector cells. Effector and target cells were dispensed in triplicate into 96-well U-bottom microtiter plate at 1×106 cells/well and 2×104 cells/well, respectively, to give E/T ratio of 50:1. Plates were centrifuged at 400g for 5 min and incubated for 4 h at 37 °C in 5 % CO2 atmosphere. At the endpoints, 0.1 ml of the supernatant collected by centrifugation (600×g, 10 min) was collected for radioactivity counting. Spontaneous release was determined by adding 100 labeled target cells to 0.05 % Tween-20. Percentage of cytotoxicity, as measured by specific 51Cr release assay, was calculated by using the formula: (cpm experimental−cpmspontaneous)/ (cpm maximal−cpm spontaneous)×100.
9
12
15
18
21
Tumor weight (g)
4 3 2
** **
**
1
Control
50
100
200
(mg/kg)
GP
c
80
The tumor growth inhibition rate (%)
Flow cytometric analysis of differentiation antigens of CD4+ and CD8+ on peripheral blood T lymphocyte of LLC-bearing C57BL/6 mice
6
0
60 40 20 0 Control
50
100
200
(mg/kg)
GP
Fig. 1 Effects of GP on tumor volume (a), tumor weight (b), and tumor growth inhibition rate (c) in mice inoculated with Lewis lung carcinoma (LLC). Data are expressed as means±SD (n=5). **p
