the immunostimulating activities of anti-tumor polysaccharides from pseudostellaria heterophylla

ELSEVIER Immunopharmacology 28 (1994) 47-54

Immunopharmacology

The immunostimulating activities of anti-tumor Polysaccharides from Pseudostellaria heterophylla

C.K. Wong, K.N. Leung, K.P. Fung, Y.M. Choy* Department of Biochemistry, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong

(Revision received 1 March 1994; accepted 15 March 1994)

Abstract

We have previously shown that a mitogenic fraction (PH-I) separated from Pseudostellaria heterophylla exhibits both immunomodulatory and anti-tumor activities. In the present study, PH-I was further purified by gel filtration chromato- graphy and the resulting three fractions (PH-I A, PH-I B and PH-I C) were assessed for their anti-tumor activity in vivo. It was found that fraction PH-I C from P. heterophylla could markedly suppress the growth of EAT cells in vivo. Mechanistic studies have shown that i.p. injection of PH-I C into mice could enhance the phagocytic activity of thioglycollate-elicited peritoneal macrophages. Moreover, PH-I C showed a potent activating effect on the cytotoxic activity of natural killer (NK) cells and alloreactive cytotoxic T cells (Tc) as well as increased the MurlL-2-induced lymphokine activated killer cell (LAK) activity in vitro. In addition, PH-I C could increase the number of tumor infiltrating lymphocytes (TILs) in the tumor site of WEHI-164-bearing mice. Finally, i.v. injection of PH-I C significantly elevated the levels of IFN- 7 and IL-4 in sera of EAT-bearing mice.

Key words." Anti - tumor polysaccharide; Immunost imula t ion; Pseudostellaria heterophylla

I. Introduction

Anti-tumor polysaccharides isolated from Tradi- tional Chinese Medicine (TCM) have many immu- nomodulating activities including activation of the cell mediated immunity. They have been shown to

* Corresponding author. Abbreviations: EAT, Ehrlich ascites tumor cells; ELISA, enzyme linked immunosorbent assay; i.p., intraperitoneally; i.v., intrave- nously; IFN-7, interferon-7; IL, interleukin; LAK, lymphokine activated killer cells; NK cells, natural killer cells; O.D., optical density; P. heterophylla, Pseudostellaria heterophylla; PBMC, peripheral blood mononuclear cells; Tc, cytotoxic T cells; TCM, traditional Chinese medicine; TILs, tumor infiltrating lymphocytes; TNF-~, tumor necrosis factor-7.

0162-3109/94/$7.00 Q 1994 Elsevier Science B.V. All rights reserved SSDI 0 1 6 2 - 3 1 0 9 ( 9 4 ) 0 0 0 1 3 - 6

cause the activation of various types of effector cells including B lymphocytes (Kumazawa et al., 1985a), T lymphocytes (Kumazawa et al., 1985b), Tc (Hamuro et al., 1978), macrophages (Adachi et al., 1990; Wang et al., 1993), polymorphonuclear cells (Kimuura et al., 1987; Morikawa et al., 1985), N K cells (Tsukagoshi et al., 1984), LAK cells (Hayashida et al., 1991) and TILs (Kariya et al., 1991; Mizutani and Yoshida, 1991). Besides the activation of cell-mediated immunity, many anti-tumor polysaccharides can also stimulate the production of cytokines in vivo with anti-tumor activities. A previous study showed that TNF-c~ could be induced in the solid M H 134 hepatoma by systemic administration of an anti-tumor polysaccharide, MGA, in mice

48 C.K. Wong et al. / Imrnunopharmacology 28 (1994) 47-54

(Takahashi et al., 1988). Another report indicated that a neutral glucan, schizophyllan, could elicit the production of IFN-y and IL-2 from the mitogen stimulated human peripheral blood mononuclear cells (PBMC) (Sakagami et al., 1988).

The roots ofPseudostellaria heterophylla are widely used in the Orient as a tonic drug for the treatment of asthenia-syndrome cases which manifest as an insufficiency of vital energy. It was previously shown to be a mitogen of murine spleen lymphocytes and a priming agent for TNF-~ release in mice (Wong et al., 1992). It exhibited anti-tumor activities against Ehrlich ascites tumor (EAT) cells in vivo but not in vitro (Wong et al., 1992). In the present study, attempts will be made to dissect out the underlying mechanisms by which P. heterophylla can exert its anti-tumor activities in vivo. In particular, the effects of the purified fractions from P. heterophylla on the activation of various effector cell types (e.g. Tc, macrophages, LAK, NK, TILs) will be evaluated. In addition, the ability of the anti-tumour polysaccharides from P. heterophylla to induce cytokine (e.g. IFN-y and IL-4) production in tumor-bearing mice will be examined.

tain the fractions PH-I A, PH-I B and PH-I C. Fractions of 12 ml were collected at a flow rate of 12 ml/h. The data of purification have been described in detail previously (Wong et al., 1994). The protein, carbohydrate and uronic acid of these fractions have also been described previously (Wong et al., 1994).

2.2. Suppression of EAT growth in mice by different fractions of P. heterophylla

Male ICR mice, about 30-35 g, were used in all experiments. EAT was maintained by weekly intra- peritoneal (i.p.) implantation in mice. ICR mice in groups of five were injected intraperitoneally with different fractions (500 #g per mouse) for 5 consecutive days (days 0-4). Control mice were injected with an equal volume of PBS. On day 2, all mice were injected i.p. with 1 × 105 EAT cells harvested from 7-day-old tumors in 0.2 ml PBS. The tumor cell number recoverable from the peritoneal cavity of each mouse was determined on day 9.

2.3. Assay of phagocytic activity of peritoneal macrophages

2. Materials and methods

2. l. Extraction and fractionation of P. heterophylla

The roots of P. heterophylla (200 g) were broken down into small pieces and soaked in one litre of water at 4°C overnight. One litre of water was then added and the fluid was heated at 80 °C for 4 h. The precipitate was removed by high speed centrifuga- tion (10000 g, 30 min). The supernatant was lyophilized. The lyophilized powder, which was designated as the crude extract (PH fraction), was subject to stepwise alcohol precipitation. Briefly, the crude extract (PH) was dissolved in water (2.67~o w/v) and slowly added into an equal volume of 95 ~o ethanol with continuous stirring. The precipitate was then centrifuged down. The resulting supernatant was slowly added into an equal volume of 95 ~o ethanol. The precipitate was then centrifuged down and was designated the PH-I fraction. The PH-I fraction was applied onto a Sephadex G-100 (Pharmacia) column (2.5 x 90 cm) and eluted with water to ob-

The spectrofluorometric method of Ragsdale and Grasso (1989) was used with slight modification. Peritoneal exudate cells (2 x 106) in 1 ml RPMI supplemented with 10~/o fetal calf serum (RPMI complete medium) were added to each well of 24- well plate and incubated at 37°C with 5~o CO2- 95 ~o air for 4 h. The non-adherent cells were washed away with RPMI medium. The macrophages were then incubated with 0.5 ml Zymosan A Biopartictes ® (S. cerevisiae), BODIPY ® FL conjugate (Molecular Probes) (16 /~g/ml in RPMI complete medium) at 37 ° C with 5 ~/o CO2-95 ~o air. After different periods of time, the macrophages were washed three times with PBS and lysed by addition of 0.5 ml of i~ o SDS. The lysate was mixed with 1.5 ml water and its fluorescence was measured by a Luminescence Spectrometer LS50 (Perkin Elmer) with an excita- tion wavelength of 488 mm and an emission wavelength of 515 nm. The protein concentration of the lysate was determined by the Lowry method. The phagocytic activity of macrophages was expressed as fluorescence per/~g protein of macrophages.

C.K. Wong et al. / Irnmunopharmacology 28 (1994) 47-54 49

2.4. Assay of natural killer cell activity

N K activity was measured by the 51Cr release method as described previously (Leung and Ada, 1981). The percentage specific cytolysis was calculated as follows:

% Specific cytolysis

Test culture counts - Spontaneous release counts

- Maximum release counts - Spontaneous release counts x 100%

2.5. Assay of alloreactive cytotoxic T cell activity

Alloreactive cytotoxic T cell activity was measured by the colorimetric method as described previously (Mtlllbacher et al., 1984). Briefly, C57BL/6J mice were immunized i.p. with 1 x 107 mitomycin C- treated L929 cells in 0.5 ml RPMI medium. Control mice were injected with 0.5 ml RPMI. After 5 days, splenic lymphocytes were prepared and act as a primary source of alloreactive cytotoxic T lymphocytes. Lymphocytes in RPMI complete medium (0.1 ml) were added onto a 96-well plate and mixed with 4 x 104 L929 cells so that the effector to target cell ratio was 50:1. The cell mixtures were incubated at 37°C for 24 h. The cytotoxicity of alloreactive cytotoxic T lymphocytes was measured by neutral red uptake method. The % cytotoxicity was expressed as the following formula:

C - T % cytotoxicity - x 100%

C

where C = m e a n O.D.540 of control wells (no effector cells were added) and T = m e a n O.D.540 of wells containing cytotoxic T cells.

2.6. Assay of lymphokine-activated killer cell activity

A modification of the colorimetric assay (Mtlll- bacher et al., 1984) was used to determine the lymphokine-activated killer (LAK) cell-mediated cytotoxicity. WEHI-164 cells, which are known to be LAK-sensitive but NK-resistant, were used as the target cells. WEHI-164 cells (4x 104) were

seeded onto each well of a 96-well microtiter plate and incubated at 37 °C overnight. The medium was flicked off and the cells were washed with RPMI.

Effector cells were generated from splenocytes which had been pretreated with recombinant IL-2 (50 U/ml) and/or different fractions (100/~g/ml) for 3 days at 37°C. Effector cells (106) in 0.1 ml volume were added to the target cells (4 x 104/well) so that the effector/target ratio was 25:1. After 24 h incubation at 37°C, the cytotoxicity of LAK cells was measured by neutral red uptake method. The absorbance in each well was read at 540 nm using a mi- croplate reader (Bio-Rad Model 3550, USA) and the results were expressed as the % cytotoxicity on WEHI-164 cells which was calculated as follows:

% cytotoxicity on WEHI-164 cells

C - T - x 100%

C

Where C = O.D.540 of control (without effector cells) and T = O.D.540 of test culture (with effector cells).

2.7. Assay of tumor-infiltrating lymphocytes

The method of Mizutani and Yoshida (Mizutani and Yoshida, 1991) was adopted with slight modification. Briefly, 2 x 105 WEHI-164 cells in 0.2 ml PBS was inoculated subcutaneously into the back of each BALB/c mouse on day 0. Purified fraction from P. heterophylla was then injected i.p. into each mouse from day 16 to day 22. On day 23, TILs were isolated from the sarcoma. Sarcoma was dissected out and TILs were isolated as described previously (Mizutani and Yoshida, 1991). Monoclonal anti-Thy 1.2 antibody (at a final dilution of 1:1000) was added to 1 ml TILs suspension (2 x 107 cells/ml). The mix- ture was then incubated at room temperature for 30 min. The cells were spun down and resuspended in 1 ml guinea pig complement (diluted 1:5 with RPMI medium) for 45 min at 37°C. The % of TILs of the cell population was determined by trypan blue dye exclusion method.

2.8. Effects of P. heterophylla polysaccharides on IFN- 7 and IL-4 production

Mice were inoculated i.p. with 1 x 105 EAT cells on day 0. On day 6, each mouse was injected intra- venously (i.v.) with 2 mg PH-I C in 0.2 ml PBS. Four mice from each group were bled from sub-

50 C.K. Wong et al. / Immunopharmacology 28 (1994) 47-54

clavian vessels to obtain the serum 2 h after injection of PH-I C. IFN- 7 and IL-4 titers in the pooled sera were determined by the murine IFN- 7 ELISA test kit (Genzyme) and murine IL-4 ELISA kit (Endogen) using recombinant murine IFN-7 and IL-4 as the standard respectively.

2.9. Statistical analysis

All results were expressed as the arithmetic mean + standard deviation (S.D.). The differences between control group and treatment groups were determined by the Student's t-test, p < 0.05 was re- garded as significantly different.

3.2. Effect of P. heterophylla fractions on the activation of macrophages

Since macrophages play an important role in in- nate and acquired immunity and are one of the major effector cell types involved in the host's defence against tumor invasion, therefore, the ability of various purified fractions from P. heterophylla to activate macrophages was examined. It was found that i.p. injection of PH-I C into mice could activate the effector functions of macrophages in vivo, as measured by the increased phagocytic activity of the thioglycollate-elicited peritoneal macrophages after incubated for 1 h with yeast in vitro (Fig. 1).

3. Results 3.3. Effects of P. heterophylla fractions on the activation of the host's cytotoxic effector cells

3.1. Effect of P. heterophylla fractions on the growth of E A T cells in vivo

Table 1 shows that i.p. injection of the purified fractions (PH-I B and PH-I C) from P. heterophylla significantly suppressed the growth of EAT cells in vivo. Among all the fractions being tested, PH-I C was found to exhibit the greatest anti-tumor activity in vivo.

Table 1

Suppression of EAT growth in mice by different fractions of P. heterophylla

Treatment EAT number Suppression (× lo ~) (%)

PBS 7.1+_2.6 -

PH-I A 5.0_+ 1.3 29.6 PH-I B 2.5 + 0.3 a 64.8 PH-I C 0.1 +0.1 b 98.6

ICR mice in groups of five were injected i.p. with different fractions (500/~g per mouse) for 5 consecutive days (days 0-4). Con- trol mice were injected with an equal volume of PBS. On day 2, all mice were injected i.p. with EAT (1 × 105 cells/mouse). The tumor cell number recoverable from the peritoneal cavity of each mousc was determined on day 9. The /° 0 suppression of EAT growth was obtained by comparison with the control group (mice injected with PBS). The differences between the control group and treatment groups were determined by Student's t-test. ~ P < 0.005, b P<0.001.

The anti-tumor activity ofP. heterophylla might be mediated through the activation of the host's cytotoxic anti-tumor cells. Therefore, the ability of various purified fractions from P. heterophylla to activate different types of non-specific cytotoxic cells such as NK cells and LAK cells as well as the antigen-

2 . 5

0

k 2 -0

r..) Z M

1.5

o

1.0 CONTROL PH-I A PH-I B PH-I C

Fig. 1. In vivo activation of the phagocytic activity ofthioglycol- late elicited peritoneal macrophages by P. heterophylla fractions. On day 0, P. heterophylla fractions (5 mg per mouse) were injected i.p. into groups of three C57BL/6J mice. On day 2, 2 ml thioglycollate was injected i.p. into each mouse. On day 5, the peritoneal macrophages were harvested. The phagocytic activity of macrophages of each group was determined by spectrofluorometric method after incubation with yeasts for 1 h at 37°C. The differences between control group and treatment groups were determined by Student's t-test. *p < 0.05.

C.K. Wong et al. / lmmunopharmacology 28 (1994) 47-54 51

Table 2

In vivo induction of natural killer cells by P. heterophylla fractions

Treatment % Specific 5tCr release from YAC-I target

0 24.59 -+ 7.09 36.72 _+ 25.47 61.31 + 28.80

PBS PH-I A PH-I B PH-I C

On day 0, different fractions (2 mg/mouse) in PBS were injected i.v. into groups of three BALB/c mice. On day 1, NK activity was assayed by a 4 h cytotoxic assay using 5tCr-labelled YAC-1 cells as target at an effector-to-target ratio of 50:1. The injection of PBS showed no significant effect on the activation of the NK cell activity.

specif ic c y t o t o x i c cells s u c h as a l l o r eac t ive T c w a s

s tud ied . I t w a s f o u n d t h a t i.v. i n j e c t i o n o f P H - I A,

P H - I B a n d P H - I C c o u l d a u g m e n t t he cy to tox i c

ac t iv i ty o f sp len ic N K cells ( T a b l e 2) a n d sl ightly

e n h a n c e t he cy to tox i c ac t iv i ty o f t he a l l o r eac t ive T c

( T a b l e 3). O n t he o t h e r h a n d , a l t h o u g h P H - I A c o u l d

n o t s t i m u l a t e t he a n t i - t u m o r ac t iv i ty o f L A K cells

in v i t ro , h o w e v e r , it c o u l d e n h a n c e the c y t o t o x i c ac-

t ivi ty o f I L - 2 - s t i m u l a t e d L A K cells (Fig. 2). In c o n -

t r a s t , P H - I B o r P H - I C a l o n e c o u l d s t i m u l a t e t he

a n t i - t u m o r ac t iv i ty o f L A K cel ls in vitro. I n a d d i t i o n ,

t hey a l so sl ightly a u g m e n t e d the I L - 2 - i n d u c e d L A K

act iv i ty in v i t ro (Fig. 2).

1 0 6

80

E-~

CD

60 O

O

>. r..) 4 0

2 0

x x x x x ~

X X x x x x x x X X × X X X X M N X X X X X X X X X X X

CONTROL PH-I • Pl l - I

X X xx x x _ ~

> ( x x x XTM

x x >(X

>(X XTM ~ M y x z

PH-I C IL-2 IL~II

P H i l •

KXN

K X ~ X X X X × × x x x x X X

k,,XAA x x

x x x x

x x I x x ' x x

K,/v~ x x

IL-2 IL-g

PII-I J PII-I e

Fig. 2. In vitro induction of lymphokine activated killer cell activity by P. heterophylla fractions. BALB/c lymphocytes (5 x 106/ml) were co-incubated with different fractions (100 #g/ml) and/or MurlL-2 (50 U/ml) in 10 ml RPMI complete medium for 3 days at 37°C. Effector ceils (1 x 106/0.1 ml) were added to the target (WEHI-164) cells so that the E/T ratio was 25:1. After 24 h incubation period at 37°C, the cell mixtures were washed and stained with neutral red. The dye uptake by target cells was determined by measuring the absorbance at 540 nm after lysing the cells in 1% SDS solution. The results were expressed as the O,/o cytotoxicity on WEHI-164 cells. The differences between control group and treatment groups were determined by the Student's t-test. *p<0.05, **p<0,001.

3.4. Effect o f P H - I C on the induction o f T ILs in tumor-bearing mice

Table 3

In vivo induction of alloreactive cytotoxic T cells by P. heterophylla fractions

Treatment °/,o Cytotoxicity

PBS 55.10_+ 1.53 PH-I A 63.54 + 3.28 a PH-I B 64.39 + 4.34 a PH-I C 61.01 -+ 2.19 ~

In v iew o f t he m o s t p o t e n t a n t i - t u m o r act iv i ty o f

P H - I C a m o n g the t h r e e pur i f ied f r a c t i o n s o f P,

heterophylla, t h e effect o f P H - I C o n t he i n d u c t i o n o f

Table 4

In vivo stimulation of TILs in WEHI-164 sarcoma-bearing mice by PH-I C

On days 0, 1, 2, 4, 5, 6, different fractions (200 gg/mouse) were injected i.p. into groups of three C57BL/6J mice. On day 3, l x 107 mitomycin C treated L929 cells were injected i.p. into each mouse. On day 7, the cytotoxicity of the alloreactive T cells was assayed by colorimetry. The differences between the control group and treatment groups were determined by Student's t-test. ~p<0.01.

Treatment % Number of TILs

PBS 19.78_+ 3.91 PH-I C 34.54 _+ 3.29 a

WEHI-164 sarcoma (2 x 105 cells in 0.2 ml PBS) were inoculated s.c. into each BALB/c mouse on day 0. PH-I C (500 #g in 0.5 ml PBS) was injected i.p. from day 16 to day 22 for 7 consecutive days. On day 23, TILs were isolated from the sarcoma and the og number of TILs was determined by trypan blue dye exclusion method. The difference between the control group and treatment groups was determined by the Student's t-test, ap<0.01.

52 C.K. Wong et al. / lmmunopharmacology 28 (1994) 47-54

Table 5

Effects of PH-I C on the induction of IFN- 7 and IL-4 in EAT- bearing mice

Treatment IFN- 7 Titer IL-4 titer (ng/ml) (ng/ml)

PBS U.D. U.D. PH-I C 0.70_+0.16 5.64+4.00

ICR mice in groups of four were inoculated i.p. with 105 EAT cells in 0.2 ml PBS on day 0. On day 6, each mouse was injected i.v. with PBS only or 2 mg PH-I C in 0.2 ml PBS. The mice were bled from subclavian vessels to obtain serum 2 h after the injection. IFN-7 and IL-4 titers in the pooled sera were determined by the murine IFN-7 ELISA test kit (Genzyme) and murine IL-4 ELISA kit (Endogen) using recombinant murine IFN-7 and IL-4 as the standard respectively. U.D. = undetectable.

TILs in tumor bearing mice was investigated. It was found that i.p. injection of fraction PH-I C could significantly increase the ~o of TILs in the WEHI- 164 sarcoma of BALB/c mice (Table 4).

3.5. Effect of PH-I C on the induction of lFN-~ and IL-4 formation in vivo

Since the anti-tumor activity of P. heterophylla could also be mediated by inducing the production of anti-tumor cytokines in vivo, therefore, the effect o fPH-I C on the production of two cytokines (IFN-~, and IL-4) in vivo was examined. Table 5 shows that i.v. injection with PH-I C induced the production of IFN-7 and IL-4 in the sera of EAT-bearing mice.

4. Discussion

PH-I, a mitogenic fraction separated from the roots of P. heterophylla, had previously been shown to act as a priming agent for TNF-~ release in vivo (Wong et al., 1992). It was found that PH-I could suppress EAT growth in vivo but not in vitro (Wong et al., 1992). In this paper, we showed that PH-I could be further purified into three fractions (PH-I A, PH-I B and PH-I C) by gel filtration chromato- graphy on Sephadex G-100 columns. Two of these fractions (PH-I B and PH-I C) could significantly suppress the growth of EAT cell in vivo. Based on the in vitro and in vivo anti-tumor assays, PH-I C was found to be the most potent anti-tumor fractions in P. heterophylla. Moreover, our data also showed

that i.p. injection of PH-I A, PH-I B and PH-I C into mice could enhance the phagocytic activity of peritoneal macrophages (Fig. 1). The mechanism(s) whereby P. heterophylla can enhance the phagocytic activity of macrophage is unclear but it may be due to the increased secretion of cytokines or the increased chemotactic response of macrophages.

PH-I C was also previously found to be a potent fraction for the induction of endogenous TNF-~ in vivo (data not shown). Other investigators had shown that TNF-~ could act in synergy with IFN- 7 in exhibiting cytotoxicity on tumor cells in vitro (Dubois et al., 1989; Williamson et al., 1983). It was suggested that an IFN-7-dependent increase in TNF-~ receptor expression might underlie this synergy (Aggarwal et al., 1985). Phase I clinical trial showed that the combination of TNF-~ and IFN-7 could induce synergistic therapeutic effects in cancer patients (Demetri et al., 1989). IL-4, on the other hand, was known to exert anti-tumor activities in transplantable, chemically induced and spontaneous tumors (Thomas and Balkwill, 1991). Our present results indicate that i.v. injection of anti-tumor fraction from P. heterophylla (PH-I C) could induce IFN-y and IL-4 production in vivo as elevated levels of IFN- 7 and IL-4 were detected in the sera of tumor-bearing mice shortly after the i.v. injection of PH-I C. The endogenous induction of IFN-y and IL-4 by P. heterophylla is probably one of the con- tributing factors for the observed anti-tumor activity of P. heterophylla in vivo (Table 1).

A recent report has shown that the protein-bound polysaccharide (PSK) can activate human natural killer cells independently of IFN-), or IL-2 (Kariya et al., 1992). Similarly, the present study shows that i.v. injection of PH-I C into mice could markedly activate the splenic NK cells activity in vivo. The mechanism by which P. heterophylla can activate NK activity remains obscure, but it could be due to the stimulation of cytokine production (e.g. IL-4, IFN-7 and TNF-~) by P. heterophylla. Moreover, the i.v. injection of PH-I A, PH-I B and PH-I C into mice could also slightly augment the induction of specific Tc activity in vivo. Besides NK cells, LAK cells also play an important role in cell mediated anti-tumor immunity. Previous studies have shown that Astragalus membranaceus polysaccharides (Chu et al., 1988), Krestin (Hayashida et al., 1991) and

C.K. Wong et al. / Irnmunopharmacology 28 (1994) 47-54 53

Lentinan (Yamasaki et al., 1989) could stimulate the IL-2 activated LAK cell activity in vitro. Data in this paper also strongly suggest that the PH-I A, PH-I B and PH-I C could potentiate the MurlL-2-induced LAK cell population in vitro. This potentiation of MurlL-2 activity by P. heterophylla may have clinical implications for the future use of rlL-2 in the treatment of cancer. Accumulating evidence has shown that a good correlation exists between LAK cell induction and anti-tumor effect following in vivo administration of high-dose rlL-2 in animals (Mule et al., 1984) as well as in humans (Yasumoto et al., 1987). However, 'adoptive immunotherapy' using high doses of rlL-2 and the massive reinfusion of LAK cells is often associated with high prohibi- tive toxicity such as high fever, weight gain, hypo- tension, pulmonary edema, and renal as well as he- patic toxicity (Gaspari, 1991; Rosenberg et al., 1987). As P. heterophylla can potentiate the induction of LAK activity both in vitro and in vivo, therefore the concomitant use of P. heterophylla along with rlL-2 in adoptive immunotherapy may reduce the require- ments for both the number of LAK cells and the dose of rlL-2 administration. This will decrease the toxicity without a loss in therapeutic efficacy and thus increase the therapeutic index of this newly developed treatment modality.

More recently, PSK has been shown to activate the in vitro autologous tumor killing activity of TILs (Kariya et al., 1991). Moreover, intralesional injection of OK-432 or PSK significantly augmented the cytotoxicity of TILs in cancer patients. The ratios of OKT8-positive (LAK), Leu7-positive (NK) cells were increased in the TIL subset by the injection of OK-432 (Gotoh et al., 1991). Our preliminary results show that i.p. injection of PH-I C into WEHI-164- sarcoma-bearing mice could activate a T lymphocyte response and augment the number of TILs in the tumor site. The ability of PH-I C to activate the infiltration of lymphocytes into tumor site suggests that activation of TILs may be one of the possible mechanisms by which P. heterophylla can exert its anti-tumor effect in vivo. In view of the important role of TILs in anti-tumor immunity, the in vitro activation of TILs by P. heterophylla will be investigated in the future. Moreover, the characterization and identification of the particular cell subpopulations in TILs with increased tumor reactivity are

also useful. Ideally these subpopulations could be selectively expanded and utilized for more effective adoptive immunotherapy.

In conclusion, the use of natural products such as P. heterophylla to induce IFN-7 and IL-4 for the treatment of cancer has many advantages. The polysaccharides of P. heterophylla are easily soluble in water and hence offer an easy way of administration. It can minimize the lethal side effects such as anorexia, cachexia caused by the direct injection of cytokines such as TNF-~ or IFN- 7 (Frei and Spriggs, 1989; Lenk et al., 1989). In addition, the anti-tumor polysaccharides from P. heterophylla can also stimulate many known host anti-tumor immune mechanisms in vivo such as the activation of Tc, LAK cells, NK cells, TILs and macrophages. More- over, the combined uses of anti-tumor polysaccharide ofP. heterophylla with cytokines such as TNF-c~ and IFN- 7 also give a profound effect on the anti- cancer therapy. Finally, the adoptive immunotherapy of tumor-bearing mice using the in vitro expansion of LAK cells and TILs which have previously been activated by P. heterophylla, followed by the concomitant application of P. heterophylla along with IL-2 will increase the therapeutic index of this newly developed anti-tumor treatment.

Acknowledgements

This work was supported in part by the Endowment fund from the United College, The Chinese Univer- sity of Hong Kong; and a small grant from the Chinese University UPGC.

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the immunostimulating activities of anti-tumor polysaccharides from pseudostellaria heterophylla

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