Cancer Letters, 65 (1992) 79-84 Elsevier Scientific Publishers Ireland
79 Ltd.
Antitumor activity of polysaccharide from a Chinese medicinal herb, Acanfhopanax giraldii Harms 3.2. Wang, H. Tsumura”, Department of Pharmacology, &Japan)
K. Shimuraa
and H. Ito
asblnstitute of Labomtoy
Animals, Me Uniuersity School of Medicine, Tsu, Me 514,
(Received 20 September 1991) (Revision received 1 June 1992) (Accepted
2 June
1992)
Summary
Introduction
The results of experiments with Acanthopanax giraldii polysaccharide (AGP) demonstrated that it inhibited the growth of solid Sarcoma 180 and prolonged the survival time significantly. In tumor-bearing mice, AGP enhanced fhe phagocytosis and chemiluminescence of macrophages. By the immunofluorescent method, binding of the third component of complement (C3) cleavage product to macrophages and proportion of C3 positive cells were increased. In crossed immunoelectrophoresis, human serum C3 was converted by AGP and appeared as the 3rd peak. The height of the 3rd peak was directly proportional to doses of AGP. The residual Cl-f50 units of human serum decreased dosedependently. These results suggest that the antitumor activity of AGP is related to the enhancement of immune responses.
Several polysaccharides originating from higher plants, as biological response modifiers, have immunopotentiating and antitumor activities [1,3,7,16]. Acanthopanax (A.) giraldii Harms and A. senticosus Harms that are traditional Chinese medicinal herbs belonging to the same biological genus but different species. As tonic herbs in Chinese medicine, they are used to treat patients in debilitated conditions due to tumor and some chronic diseases. It has been reported that A. senticosus as well as its polysaccharide enhanced the phagocytosis of macrophages and inhibited the growth of transplanted tumor in mice [2,19]. However little is known about the effect of A. giraldii polysaccharide (AGP) on immune responses. In this paper, the effects of AGP on solid Sarcoma 180 (S180), phagocytosis of macrophages and alterations of complement C3 in human serum were studied.
Keywords:
Materials
Correspondence to: H. Ito, Department of Pharmacology, University School of Medicine, Tsu, Mie 514, Japan.
Animals Four-week-old female lCR/SLC mice were obtained from the Shizuoka Laboratory Animal Center, Hamamatsu, Japan.
Acanthopanax giraldii harms; anti tumor activity; macrophage activation; C3 cleavage
0304~3835/92/$05.00 Printed and Published
0 1992 Elsevier Scientific Publishers in Ireland
Ireland Ltd.
and Methods
80
Drugs AGP was isolated from A. Giraldii Harms roots (harvested in Gansu China) according to previous methods [4,9]. Briefly, the polysaccharide in the roots of A. giraldii Harms was extracted with boiling water. The extracts were filtered to remove insoluble materials. The filtrate was then dialyzed with a DIA filter. The non-dialysable fraction was concentrated in vacua and the crude polysaccharide was obtained by precipitation with 3 volumes of cold ethanol. The precipitate was dissolved in distilled water, applied to SHODEX lonpak S-804 column and rinsed with 0.01 M NaCI. The eluate and the washings were pooled and dried in vacua. The data of 13C NMR spectrum showed that AGP mainly consists of [ 1 - 3]-O-D-glucan chains. The preparation was passed through Detoxi-Gel columns to remove endotoxin. The polysaccharide was dissolved in physiological saline before administration. PSK (a protein-bound polysaccharide from Coriolus uersicolor of basidiomycetes) was supplied by Sankyo Pharmaceutical Co. Zymosan (Only suspension Tokyo, Japan. was used because of the poor solubility) was purchased from ICN Pharmaceuticals, Inc. Life Science Group, Cleveland, Ohio USA. PSK or zymosan was used as a reference agent. Luminol solution (Luminol 1 mM in 0.01 N/100 NaOH); buffer I (CaCI* 1 mM in Buffer II); buffer II (KC1 5 mM, NaCI 15 mM, glucose 5.5 mM in 10 mM Hepes with NaOH); FMLP (N-formy1-L-methiony1-~-leucy1-~-pheny1alanine) solution (FMLP 3 x 10 -6 M in buffer II with 0.1% &HsOH) was obtained from LANBO Science Co. Tokyo Japan. Antimouse C3 F(ab’)z was prepared by treating IgG (Cappel anti-mouse C3 rabbit Laboratories) with Pepsin [17]. Goat fluorescent anti-rabbit IgG was obtained from Miles Yeda Ltd Tokyo, Japan.
Evaluation of antitumor activity S180 cells (2 X lo6 cells/mouse)
were subcutaneously inoculated into 4-week-old ICR mice. AGP (5, 25 mg/kg), PSK (50
mg/kg) and zymosan (25 mg/kg) were administered intraperitoneally (i.p.) once daily for 10 days at 24 h after tumor inoculation, respectively. The same volume of saline was injected i.p. to the control mice. The tumor size (cm3) = 4/3r(a2b)/2 (a = long diameter; b = short diameter, mm) was determined on day 28. The survival time was determined on day 60 and expressed as the percentage of mean survival time of treated mice relative to that of control mice (T/C) [5].
Ingestion of polystyrene latex beads After 5 days of tumor implantation, the mice were i.p. injected with 5, 25 mg/kg AGP, 50 mg/kg PSK and 25 mg/kg zymosan daily for 5 days. The same volume of saline was injected i.p. to the control mice. Peritoneal macrophages were obtained from the mice at 2 h after the last injection by washing the peritoneal cavity with Eagle’s minimum essential medium (MEM) for determination of the intracellular number of polystyrene latex beads as described previously [ 171.
Luminol-dependent
chemiluminescence
of
macrophages The administration of AGP and the collection of peritoneal macrophages in tumorbearing mice were carried out as described above. A cell suspension (1 x lo7 cells/ml) was prepared in buffer II. Before assaying, the suspension was diluted with buffer I to a concentration of 1 x lo6 cells/ml. Then, 0.1 ml of the cell suspension and 0.1 ml of Luminol solution were mixed and incubated at 37OC for 1 min. After addition of 0.1 ml of FMLP solution, the maximum of chemiluminescence was measured with a LAB0 Science TD-4000 Lumiphotometer [ 151.
The third component of complement cleavage product binding assay
(C3)
The administration of AGP and the collection of peritoneal macrophages in tumorbearing mice were carried out as described above. A 0.2-ml portion of cell suspension (2 x lo6 cells/ml) was placed on a coverslip
81
in a Petri dish and incubated at 37OC for 30 min in a 5% CO2 incubator. The cells that had not adhered to the coverslip were removed by rinsing with Eagle’s MEM. The adherent cells on the coverslips were dried and fixed in 95% ethanol for 20 min, covered with antimoused C3 F (ab’)’ and reincubated for 1 h at 37OC. After washing with PBS, the cells were stained with goat FITC-anti-rabbit IgG at room temperature for 1 h, thoroughly washed again and examined by microscopy. Since the macrophages have receptors that specifically bind to C3b [14], only the cells binding to C3 cleavages could show immunofluorescence. The percentage of fluorescent (C3-positive) cells was calculated by counting of 200 adherent cells on each slide [17]. Crossed
immunoelectrophoresis
and residual
CH50 The C3 conversion activity of human serum (5 ~1) treated with AGP was assayed by twodimensional crossed immunoelectrophoresis according to the modified Laurel1 method [15]. An adequate amount of each polysaccharide in 0.05 ml of saline was added to 0.5 ml of human serum. The mixture was then incubated at 37OC for 1 h. For the control, 0.5 ml of human serum was incubated with 0.05 ml of saline. The first electrophoresis was performed on a 2.6 x 7.5 cm glass plate covered with 1.0% Agarose ME and electrophoresed in tricine Verona1 buffer (pH 8.6, u = 0.05) at 2 mA/cm for 100 min. The gel strip was separated and transferred to an immunoplate
(7.5 x 7.5 cm) which was prepared with antihuman whole serum. The second electrophoresis was carried out at 1.5 mA/cm for 5 h under the same conditions [16]. After a fresh serum was treated as described above, the residual CH50 was measured by lysis of sensitized sheep red blood cells according to the method of Mayer [8]. analysis Student’s t-test was used to determine the confidence limits in group comparisons. Statistical
Results and Discussion
The data in this paper proved that AGP not only inhibited the growth of solid S180 but also enhanced various immune responses in tumor-bearing mice. As shown in Table I, at doses of 5 and 25 mg/kg, AGP significantly inhibited the S180 tumor growth and prolonged the survival time of tumor bearing mice by 47.2% and 70.9%. There was a complete regression of the tumor in 3 out of 10 mice treated with 25 mg/kg AGP. At the same time, PSK and zymosan also exerted their antitumor effect on S180. It is well known that macrophages play an important role in non-specific immunity and tumor immunity [6]. As seen in Table II, AGP enhanced the phagocytosis of macrophages, which is also consistent with other antitumor polysaccharides [ 1 - 3,181. The observations of this study indicate that AGP has an enhancing effect on non-specific immunity. Since
Table I. Antitumor activity of AGP on solid Sarcoma 180. Each value represents the mean f S.D. of 10 mice. Statistically significant difference: 'P < 0.05, '*P < 0.01 as compared with controls. Group
Control AGP PSK Zymosan
Dose
Tumor size
Survival time
fmg/kg)
(cm31
(day)
-
38.9 f 11.3 30.1 f 14.4 13.3 l 10.5” 25.3 l 8.7’ 15.8 f 7.7”
32.0 47.1 >54.7 >50.4 >52.9
5 25 50 25
f f f f f
3.5 13.8’ 10.6” 10.0’ 12.0’
l
l
T/C (%I
Complete regression
100.0 147.2 170.9 157.5 165.3
o/10 o/10 3/10 l/10 3/10
82 Table II. Effect of AGP on activity of peritoneal macrophages in tumor-bearing mice. Each value represents the mean * S.D. of 5 mice. Statistically significant difference: ‘P < 0.01 as compared with controls. Group
Control AGP PSK Zymosan
Dose
Phagocytosis
(mg/kg)
(%)
-
30.3 55.7 77.6 65.4 67.6
5 25 50 25
zt f * zt zt
Intracellular number of latex beads/cell
5.8 7.5’ 4.5’ 6.5’ 9.0’
3.1 5.3 7.2 6.9 7.8
f 0.4 f 0.5 zt 0.7’ zt 0.8’ ztz 0.7’
AGP markedly stimulated phagocyte chemiluminescence and increased the proportion of C3 positive macrophages (Table III), the enhancement of phagocytosis is postulated to result from a series of metabolic and functional changes leading to the activated state of the macrophages in tumor-bearing mice. As peritoneal macrophages of mice have receptors which specifically bind to C3b cleavage, this allows the phagocytes to recognize their targets [ 13,141. Therefore, continued ability to bind C3 cleavage product via the C3b receptor could play an important role in the immune response [ 111. After intraperitoneal
Table III. Enhancing effect of AGP on chemiluminescence and induction of CS-positive macrophages in tumor-bearing mice. ( ) : times increase as compared with control. Each value represents the mean f S.D. of 5 mice. Statistically significant difference: ‘P < 0.01 as compared with controls. C3: the third component of complement. Group
Control AGP PSK Zymosan
Dose (mg/kg) 5 25 50 25
Maximum of chemiluminescence 0.05 0.51 0.88 0.65 0.61
f 0.02 ztz 0.18 * 0.17 zt 0.14 zt 0.10
(1.0) (10.2)’ (17.6)’ (13.0) (12.2)’
C3-positive cells (46)
l
8.3 78.8 94.5 90.5 93.6
f 2.5 ziz 5.5’ ztz 3.7’ zt 2.8’ zt 2.3’
administration, AGP significantly augmented the binding of C3 cleavage product to peritoneal macrophages and the proportion of C3 positive cells C3 (Table III). Since the enhancement of both phagocytosis and binding of C3 cleavage product was caused by administration of AGP, the enhanced phagocytosis is probably related to C3 activation. Crossed immunoelectrophoretic patterns of human serum treated with AGP is shown in Table IV. The converted C3 migrated to a larger mobility region appearing as a triple peak (Fig. 1). We designate the anodal peak as the 3rd peak. The height of the 3rd peak increased with decreasing height of native C3 peak in parallel to the doses of AGP. The residual CH50 units of serum were treated with AGP decreased dose-dependently. At a dose of 1000 pg/ml, AGP the residual hemolytic activity diminished completely. The results of CH50 of human serum treated with AGP in vitro indicated an inverse correlation with the change of the 3rd peak. Similar observations have been made by using other antitumor polysaccharides [ 161. It was reported that activated macrophages could inhibit the proliferation of tumor cells [6]. Several antitumor polysaccharides enhanced the phagocytosis and binding of C3 cleavage product to macrophages, activated C3 in human serum and exerted their suppressive
Table IV. Effect of AGP on C3 conversion and residual CH50 of human serum in vitro. Each value represents average value from five experiments. N.D.: not determined. Group
Control AGP
PSK Zymosan
Dose
3rd peak
(pg/ml)
(mm)
-
8.2 10.7 16.5 22.1 23.8 19.2 18.5
20 100 500 1000 500 500
zt 0.9 ztz 3.2 f 1.3 f 1.5 z+ 1.2 l 1.0 zt 1.5
Residual CH50 (unit/ml) 23.9 l 0.7 15.7 zt 1.6 8.6 f 1.5 3.5 f 1.5 0 N.D. N.D.
83
2
3
I 4
I
peak
3rd
m?
Albr
I 1
5
I
&
I
6
7
8
Fig. 1.
electrophoretic pattern of control electrophoretic pattern of C3 convetted by 500 pg/ml of AGP. Immunoelectrophoresis was carried out as described in text. Anode is to the right.
serum;
A: Crossed
B: crossed
effect on S180 with no direct cytocidal action [4,7,X2,16,18]. As similar results were obtained by use of AGP, the antitumor activity of AGP is likely to be due to the enhancement of host defense functions. In this paper, AGP was proved to possess an antitumor activity and an immunopotentiating activity, which corresponds to PSK [lo] and zymosan [ 161. The preliminary results of the present study are encouraging enough to warrant continued further investigation.
We wish to thank Prof. T. Tanaka (Department of Pharmacology, Mie University School of Medicine) for comments on the manuscript. References 1
Chu, D.T., Wong, W.L. and Mavligit, G.M. munotherapy with Chinese medicinal herbs
Co., Springfield, Illinois. 9
10
11
12
13
Acknowledgment
14
15 (1988) ImI. Immune
restoration of local xenogeneic graft-versus-host reaction in cancer patients by fractionated Astragalus membranaceus in vitro. J. Clin. Lab. fmmunol., 25, 119- 123. Fang, J.N., Proksch, A. and Wagner, H. (1985) Immunologically active polysaccharides of Acanfhopanax senticosus. Phytochemistry, 24, 2619-2622. Franz, G. (1989) Polysaccharides in pharmacy: current applications and future concepts. Plant. Med., 55, 493-497. ho, H. (1986) Effects of the antitumor agents from various natural sources on drug-metabolizing system, phagocytic activity and complement system in Sarcoma 180-bearing mice. Jpn. J. Pharmacol., 40, 435 -443. ho, H and Hidaka, H. (1983) Antitumor effect of a calmodulin antagonist on the growth of solid Sarcoma180. Cancer Lett., 19, 215-220. Krahenbuhl, J.L., Lambert, L.H. and Remigton, J.S. (1976) The effect of activated macrophages on tumor target cells: escape from cytosis. Cell Immunol., 25, 279 - 293. Lan, Z.F., Cheng, G.Q. Wang, F.L. and Xi, S.F. (1987) Effects of Radix hedysati polysaccharide on immunological function and transplanted tumors in mice. Chin. Pharmacol. Acta, 8, 275-277. Mayer, M.M. (1961) Procedure for the titration of complement. In: Experimental Immunochemistry, pp. 149 - 153. Editors: E.A. Kabat and M.M. Mayer. Thomas, Charles Miyazaki, T., Yadomae, T., Sugiura, M., ho, H., Fujii K., Naruse, S. and Kunhisa, M. (1974) Chemical structure of antitumor polysaccharfde, coriolan produced by Coriolus uenicofor. Chem. Pharm. Bull., 22, 1739- 1742. Nio, Y., Shiraishi, T., Tsubono, M., Morimoto, H. Tseng, C.C. Imai, S. and Tobe, T. (1991) In vitro immunomodulattng effect of protein-bound polysaccharide, PSK on peripheral blood, regional nodes and spleen lymphocytes in patient with gastric cancer. Cancer Immunol. immunother:, 32, 335 -341 Nishioka, K., Kawamura, K., Hirayama, T., Kawashima, T., Shimada K and Kogure M. (1976) The complement system in tumor immunity: significance of elevated levels of complement in tumor bearing hosts. Ann. NY. Acad. Sci., 276, 303 - 315. Okuda, T., Yoshioka, Y., Chiara, G. and Nioshioka, K. (1972) Anti complementary activity of antitumor polysaccharides. Nature. 238. 59-60. Rabellino, E.M., Ross, G.D. and Polley, M.J. (1978) Membrane receptors of mouse leukocytes I. Two types of complement receptors for different regions of C3. J. Immunol., 120, 879 - 885. Roitt, I.M., Brostoff, J. and Male, D.K. (1989) Adaptive and innate immunity In: Immunopharmacology pp. 1 - 5. Editors: I.M. Roitt, J. Brostoff and D.K. Male. Gower Medical Publishing, New-York-London. Steven, P. and Young, L.S. (1977) Quantitative granulocyte chemiluminescence in the rapid detection of impaired opsonization ofEscherichia cifi. Infect. fmmunoj., 16, 796 - 794.
84 16
17
Shimura, K., ho, H. and Hibasami, H. (1983) screening of host-mediated antitumor polysacchartdes by crossed immunoelectrophoresis using fresh humor serum. Jpn. J. Pharmacol., 33, 403 - 408. Shimura, H., ho, H., Hibasami, H., Shiomi, T., Hidaka, H. and Nishioka K. (1983) Enhancement of antibodyindependent phagocytosis by N-(6-aminohexyh-5-chloro1-naphthalene sulfonamide (W-7). Immunol. Commun., 12, 363 - 374.
18
19
Wang, J.Z., ho, H. and Shimura, K. (1989) Enhancing effect of antitumor polysaccharide from Astragalus or Radix hedysarum on C3 cleavage production of macrophages in mtce. Jpn. J. Pharmacol., 51, 432 -434. Xie, SS. (1989) lmmunoregulatory effect of polysacsenticosus (PAS) Imcharide of Acanthopanax munological mechanism of PAS against cancer. Chung Hua Chung Liu Tsa Chih, 11, 338-340.
79 Ltd.
Antitumor activity of polysaccharide from a Chinese medicinal herb, Acanfhopanax giraldii Harms 3.2. Wang, H. Tsumura”, Department of Pharmacology, &Japan)
K. Shimuraa
and H. Ito
asblnstitute of Labomtoy
Animals, Me Uniuersity School of Medicine, Tsu, Me 514,
(Received 20 September 1991) (Revision received 1 June 1992) (Accepted
2 June
1992)
Summary
Introduction
The results of experiments with Acanthopanax giraldii polysaccharide (AGP) demonstrated that it inhibited the growth of solid Sarcoma 180 and prolonged the survival time significantly. In tumor-bearing mice, AGP enhanced fhe phagocytosis and chemiluminescence of macrophages. By the immunofluorescent method, binding of the third component of complement (C3) cleavage product to macrophages and proportion of C3 positive cells were increased. In crossed immunoelectrophoresis, human serum C3 was converted by AGP and appeared as the 3rd peak. The height of the 3rd peak was directly proportional to doses of AGP. The residual Cl-f50 units of human serum decreased dosedependently. These results suggest that the antitumor activity of AGP is related to the enhancement of immune responses.
Several polysaccharides originating from higher plants, as biological response modifiers, have immunopotentiating and antitumor activities [1,3,7,16]. Acanthopanax (A.) giraldii Harms and A. senticosus Harms that are traditional Chinese medicinal herbs belonging to the same biological genus but different species. As tonic herbs in Chinese medicine, they are used to treat patients in debilitated conditions due to tumor and some chronic diseases. It has been reported that A. senticosus as well as its polysaccharide enhanced the phagocytosis of macrophages and inhibited the growth of transplanted tumor in mice [2,19]. However little is known about the effect of A. giraldii polysaccharide (AGP) on immune responses. In this paper, the effects of AGP on solid Sarcoma 180 (S180), phagocytosis of macrophages and alterations of complement C3 in human serum were studied.
Keywords:
Materials
Correspondence to: H. Ito, Department of Pharmacology, University School of Medicine, Tsu, Mie 514, Japan.
Animals Four-week-old female lCR/SLC mice were obtained from the Shizuoka Laboratory Animal Center, Hamamatsu, Japan.
Acanthopanax giraldii harms; anti tumor activity; macrophage activation; C3 cleavage
0304~3835/92/$05.00 Printed and Published
0 1992 Elsevier Scientific Publishers in Ireland
Ireland Ltd.
and Methods
80
Drugs AGP was isolated from A. Giraldii Harms roots (harvested in Gansu China) according to previous methods [4,9]. Briefly, the polysaccharide in the roots of A. giraldii Harms was extracted with boiling water. The extracts were filtered to remove insoluble materials. The filtrate was then dialyzed with a DIA filter. The non-dialysable fraction was concentrated in vacua and the crude polysaccharide was obtained by precipitation with 3 volumes of cold ethanol. The precipitate was dissolved in distilled water, applied to SHODEX lonpak S-804 column and rinsed with 0.01 M NaCI. The eluate and the washings were pooled and dried in vacua. The data of 13C NMR spectrum showed that AGP mainly consists of [ 1 - 3]-O-D-glucan chains. The preparation was passed through Detoxi-Gel columns to remove endotoxin. The polysaccharide was dissolved in physiological saline before administration. PSK (a protein-bound polysaccharide from Coriolus uersicolor of basidiomycetes) was supplied by Sankyo Pharmaceutical Co. Zymosan (Only suspension Tokyo, Japan. was used because of the poor solubility) was purchased from ICN Pharmaceuticals, Inc. Life Science Group, Cleveland, Ohio USA. PSK or zymosan was used as a reference agent. Luminol solution (Luminol 1 mM in 0.01 N/100 NaOH); buffer I (CaCI* 1 mM in Buffer II); buffer II (KC1 5 mM, NaCI 15 mM, glucose 5.5 mM in 10 mM Hepes with NaOH); FMLP (N-formy1-L-methiony1-~-leucy1-~-pheny1alanine) solution (FMLP 3 x 10 -6 M in buffer II with 0.1% &HsOH) was obtained from LANBO Science Co. Tokyo Japan. Antimouse C3 F(ab’)z was prepared by treating IgG (Cappel anti-mouse C3 rabbit Laboratories) with Pepsin [17]. Goat fluorescent anti-rabbit IgG was obtained from Miles Yeda Ltd Tokyo, Japan.
Evaluation of antitumor activity S180 cells (2 X lo6 cells/mouse)
were subcutaneously inoculated into 4-week-old ICR mice. AGP (5, 25 mg/kg), PSK (50
mg/kg) and zymosan (25 mg/kg) were administered intraperitoneally (i.p.) once daily for 10 days at 24 h after tumor inoculation, respectively. The same volume of saline was injected i.p. to the control mice. The tumor size (cm3) = 4/3r(a2b)/2 (a = long diameter; b = short diameter, mm) was determined on day 28. The survival time was determined on day 60 and expressed as the percentage of mean survival time of treated mice relative to that of control mice (T/C) [5].
Ingestion of polystyrene latex beads After 5 days of tumor implantation, the mice were i.p. injected with 5, 25 mg/kg AGP, 50 mg/kg PSK and 25 mg/kg zymosan daily for 5 days. The same volume of saline was injected i.p. to the control mice. Peritoneal macrophages were obtained from the mice at 2 h after the last injection by washing the peritoneal cavity with Eagle’s minimum essential medium (MEM) for determination of the intracellular number of polystyrene latex beads as described previously [ 171.
Luminol-dependent
chemiluminescence
of
macrophages The administration of AGP and the collection of peritoneal macrophages in tumorbearing mice were carried out as described above. A cell suspension (1 x lo7 cells/ml) was prepared in buffer II. Before assaying, the suspension was diluted with buffer I to a concentration of 1 x lo6 cells/ml. Then, 0.1 ml of the cell suspension and 0.1 ml of Luminol solution were mixed and incubated at 37OC for 1 min. After addition of 0.1 ml of FMLP solution, the maximum of chemiluminescence was measured with a LAB0 Science TD-4000 Lumiphotometer [ 151.
The third component of complement cleavage product binding assay
(C3)
The administration of AGP and the collection of peritoneal macrophages in tumorbearing mice were carried out as described above. A 0.2-ml portion of cell suspension (2 x lo6 cells/ml) was placed on a coverslip
81
in a Petri dish and incubated at 37OC for 30 min in a 5% CO2 incubator. The cells that had not adhered to the coverslip were removed by rinsing with Eagle’s MEM. The adherent cells on the coverslips were dried and fixed in 95% ethanol for 20 min, covered with antimoused C3 F (ab’)’ and reincubated for 1 h at 37OC. After washing with PBS, the cells were stained with goat FITC-anti-rabbit IgG at room temperature for 1 h, thoroughly washed again and examined by microscopy. Since the macrophages have receptors that specifically bind to C3b [14], only the cells binding to C3 cleavages could show immunofluorescence. The percentage of fluorescent (C3-positive) cells was calculated by counting of 200 adherent cells on each slide [17]. Crossed
immunoelectrophoresis
and residual
CH50 The C3 conversion activity of human serum (5 ~1) treated with AGP was assayed by twodimensional crossed immunoelectrophoresis according to the modified Laurel1 method [15]. An adequate amount of each polysaccharide in 0.05 ml of saline was added to 0.5 ml of human serum. The mixture was then incubated at 37OC for 1 h. For the control, 0.5 ml of human serum was incubated with 0.05 ml of saline. The first electrophoresis was performed on a 2.6 x 7.5 cm glass plate covered with 1.0% Agarose ME and electrophoresed in tricine Verona1 buffer (pH 8.6, u = 0.05) at 2 mA/cm for 100 min. The gel strip was separated and transferred to an immunoplate
(7.5 x 7.5 cm) which was prepared with antihuman whole serum. The second electrophoresis was carried out at 1.5 mA/cm for 5 h under the same conditions [16]. After a fresh serum was treated as described above, the residual CH50 was measured by lysis of sensitized sheep red blood cells according to the method of Mayer [8]. analysis Student’s t-test was used to determine the confidence limits in group comparisons. Statistical
Results and Discussion
The data in this paper proved that AGP not only inhibited the growth of solid S180 but also enhanced various immune responses in tumor-bearing mice. As shown in Table I, at doses of 5 and 25 mg/kg, AGP significantly inhibited the S180 tumor growth and prolonged the survival time of tumor bearing mice by 47.2% and 70.9%. There was a complete regression of the tumor in 3 out of 10 mice treated with 25 mg/kg AGP. At the same time, PSK and zymosan also exerted their antitumor effect on S180. It is well known that macrophages play an important role in non-specific immunity and tumor immunity [6]. As seen in Table II, AGP enhanced the phagocytosis of macrophages, which is also consistent with other antitumor polysaccharides [ 1 - 3,181. The observations of this study indicate that AGP has an enhancing effect on non-specific immunity. Since
Table I. Antitumor activity of AGP on solid Sarcoma 180. Each value represents the mean f S.D. of 10 mice. Statistically significant difference: 'P < 0.05, '*P < 0.01 as compared with controls. Group
Control AGP PSK Zymosan
Dose
Tumor size
Survival time
fmg/kg)
(cm31
(day)
-
38.9 f 11.3 30.1 f 14.4 13.3 l 10.5” 25.3 l 8.7’ 15.8 f 7.7”
32.0 47.1 >54.7 >50.4 >52.9
5 25 50 25
f f f f f
3.5 13.8’ 10.6” 10.0’ 12.0’
l
l
T/C (%I
Complete regression
100.0 147.2 170.9 157.5 165.3
o/10 o/10 3/10 l/10 3/10
82 Table II. Effect of AGP on activity of peritoneal macrophages in tumor-bearing mice. Each value represents the mean * S.D. of 5 mice. Statistically significant difference: ‘P < 0.01 as compared with controls. Group
Control AGP PSK Zymosan
Dose
Phagocytosis
(mg/kg)
(%)
-
30.3 55.7 77.6 65.4 67.6
5 25 50 25
zt f * zt zt
Intracellular number of latex beads/cell
5.8 7.5’ 4.5’ 6.5’ 9.0’
3.1 5.3 7.2 6.9 7.8
f 0.4 f 0.5 zt 0.7’ zt 0.8’ ztz 0.7’
AGP markedly stimulated phagocyte chemiluminescence and increased the proportion of C3 positive macrophages (Table III), the enhancement of phagocytosis is postulated to result from a series of metabolic and functional changes leading to the activated state of the macrophages in tumor-bearing mice. As peritoneal macrophages of mice have receptors which specifically bind to C3b cleavage, this allows the phagocytes to recognize their targets [ 13,141. Therefore, continued ability to bind C3 cleavage product via the C3b receptor could play an important role in the immune response [ 111. After intraperitoneal
Table III. Enhancing effect of AGP on chemiluminescence and induction of CS-positive macrophages in tumor-bearing mice. ( ) : times increase as compared with control. Each value represents the mean f S.D. of 5 mice. Statistically significant difference: ‘P < 0.01 as compared with controls. C3: the third component of complement. Group
Control AGP PSK Zymosan
Dose (mg/kg) 5 25 50 25
Maximum of chemiluminescence 0.05 0.51 0.88 0.65 0.61
f 0.02 ztz 0.18 * 0.17 zt 0.14 zt 0.10
(1.0) (10.2)’ (17.6)’ (13.0) (12.2)’
C3-positive cells (46)
l
8.3 78.8 94.5 90.5 93.6
f 2.5 ziz 5.5’ ztz 3.7’ zt 2.8’ zt 2.3’
administration, AGP significantly augmented the binding of C3 cleavage product to peritoneal macrophages and the proportion of C3 positive cells C3 (Table III). Since the enhancement of both phagocytosis and binding of C3 cleavage product was caused by administration of AGP, the enhanced phagocytosis is probably related to C3 activation. Crossed immunoelectrophoretic patterns of human serum treated with AGP is shown in Table IV. The converted C3 migrated to a larger mobility region appearing as a triple peak (Fig. 1). We designate the anodal peak as the 3rd peak. The height of the 3rd peak increased with decreasing height of native C3 peak in parallel to the doses of AGP. The residual CH50 units of serum were treated with AGP decreased dose-dependently. At a dose of 1000 pg/ml, AGP the residual hemolytic activity diminished completely. The results of CH50 of human serum treated with AGP in vitro indicated an inverse correlation with the change of the 3rd peak. Similar observations have been made by using other antitumor polysaccharides [ 161. It was reported that activated macrophages could inhibit the proliferation of tumor cells [6]. Several antitumor polysaccharides enhanced the phagocytosis and binding of C3 cleavage product to macrophages, activated C3 in human serum and exerted their suppressive
Table IV. Effect of AGP on C3 conversion and residual CH50 of human serum in vitro. Each value represents average value from five experiments. N.D.: not determined. Group
Control AGP
PSK Zymosan
Dose
3rd peak
(pg/ml)
(mm)
-
8.2 10.7 16.5 22.1 23.8 19.2 18.5
20 100 500 1000 500 500
zt 0.9 ztz 3.2 f 1.3 f 1.5 z+ 1.2 l 1.0 zt 1.5
Residual CH50 (unit/ml) 23.9 l 0.7 15.7 zt 1.6 8.6 f 1.5 3.5 f 1.5 0 N.D. N.D.
83
2
3
I 4
I
peak
3rd
m?
Albr
I 1
5
I
&
I
6
7
8
Fig. 1.
electrophoretic pattern of control electrophoretic pattern of C3 convetted by 500 pg/ml of AGP. Immunoelectrophoresis was carried out as described in text. Anode is to the right.
serum;
A: Crossed
B: crossed
effect on S180 with no direct cytocidal action [4,7,X2,16,18]. As similar results were obtained by use of AGP, the antitumor activity of AGP is likely to be due to the enhancement of host defense functions. In this paper, AGP was proved to possess an antitumor activity and an immunopotentiating activity, which corresponds to PSK [lo] and zymosan [ 161. The preliminary results of the present study are encouraging enough to warrant continued further investigation.
We wish to thank Prof. T. Tanaka (Department of Pharmacology, Mie University School of Medicine) for comments on the manuscript. References 1
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Acknowledgment
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