Int. J. Immunopharmac., Vol. 14, No. 8, pp. 1315-1320, 1992. Printed in Great Britain.

0192-0561/92 $5.00 + .00 Pergamon Press Ltd.

International Society for Immunopharmacology.

MITOGENIC AND TUMOR NECROSIS FACTOR PRODUCING ACTIVITIES OF PSEUDOSTELLARIA HETEROPHYLLA

C. K. WONG,* K. N. LEUNG,* K. P. FUNG,* P. K. T. PANG t and Y. M. CHOY**

*Department of Biochemistry, Chinese University of Hong Kong, Shatin, New Territories, Hong Kong; and tDepartment of Physiology, University of Alberta, Edmonton, Alberta, Canada

(Received 6 April 1992 and in final form 30 June 1992)

A b s t r a c t - - Separation of mitogenic fraction (PH-I) from Pseudostellaria heterophylla and characterization of its biological activities were investigated. PH-I was isolated as an alcohol-insoluble fraction from the hot water extract obtained by heating the roots of P. heterophylla in water at 80°C for 4 h. It is a water-soluble substance consisting of mainly carbohydrates (56.8°70) and a small amount of proteins (7.6°70). The incorporation of tritiated thymidine into the DNA of murine spleen lymphocytes could be stimulated by PH- I in a dose-dependent manner. PH-I could act as a priming agent for the release of tumor necrosis factor (TNF) in mice. Moreover, PH-I exhibited potent anti-tumor activities against Ehrlich ascites tumor (EAT) cells in vivo but not in vitro.

The roots of Pseudostellaria heterophylla is an Oriental mild crude drug with tonic effects. It is used to treat asthenia-syndrome cases which manifest as insufficiency of vital energy. It is generally believed that the application of tonic medicinal herbs may enhance the body resistance and is likely to be beneficial to the anti-neoplastic therapy. Recently, it has been found that many Chinese herbal medicines with tonic effects can modulate the immunologic function (Kumazawa, Mizunoe & Otsuka, 1982; Zhao, Mancini & Doria, 1990). Moreover, some of the Chinese herbs serve as an inhibitor or killer of the tumor cells (Kumazawa et al., 1982; Miyazaki, Oikawa, Yadomae, Yamada & Yamada, 1979; Ebina & Kohya, 1988). It is likely that most of the anti- tumor activities of Chinese herb are mediated by the immunological system of the host. For example, many anti-tumor natural polysaccharides were reported to induce tumor necrosis factor (Haranaka, Satomi, Sakurai, Haranaka, Okada & Kobayashi, 1985; Takahashi, Watanuki, Yamazaki & Abe, 1988) or activate macrophages (Luettig, Steinmuller, Gifford, Wagner, Lohmann-Matthes, 1989) in vivo.

Since the discovery of tumor necrosis factor (TNF) by Carswell and colleagues, bacteria were usually used as the priming agents for the release of TNF (Carswell, Old, Kassel, Green, Fiore & Williamson,

1975). Not until 1984, was a yeast polysaccharide, zymosan, found to be effective as a priming agent for the release of TNF (Haranaka, Satomi, Sakurai & Haranaka, 1984). In the present study, hot-water extracts from the roots of P. heterophylla were found to have a mitogenic effect on murine lymphocytes. Mitogenic activity was observed in the alcohol-insoluble and macromolecular fractions prepared from hot-water extracts. One of these fractions, PH-I, exhibited the most potent mitogenic activity on mouse splenocytes among all fractions being tested. In this paper, the effect of PH-I on TNF production was examined. In addition, the anti- tumor activity of PH-I on the growth of a murine tumor (Ehrlich ascites carcinoma) was also studied, both in vitro and in vivo.

E X P E R I M E N T A L P R O C E D U R E S

Extraction and fractionation o f P. heterophylla

The roots of P. heterophylla were imported from China. The roots (200 g) were broken down into small pieces and soaked in one liter of water at 4°C overnight. One liter of water was then added and heated at 80°C for 4 h. The precipitate was removed by high speed centrifugation (10,000 g, 30 min). The

*Author to whom correspondence should be addressed.

1315

1316 C. K. WONG et al.

supernatant was lyophilized. The lyophilized powder, which was designated as the crude extract (PH fraction), was subjected to stepwise alcohol precipitation. Briefly, the crude extract (PH) was dissolved in water (2.67o/0 w/v) and slowly added into an equal volume of 95o7o ethanol with continuous stirring. The precipitate was then centrifuged down. The resulting supernatant was slowly added into an equal volume of 95°7o ethanol. The precipitate was then centrifuged down and was designated as the PH-I fraction. The supernatant was again added slowly into an equal volume of 95o7o ethanol with continuous stirring. The precipitate was then centrifuged down and was designated as the PH-II fraction. The supernatant was then evaporated to dryness and then lyophilized. The lyophilized powder was designated as the PH-III fraction. The carbohydrate content of the isolated fractions was determined by the phenol sulfuric acid method (Dubois, Gilles, Hamilton, Rebers & Smith, 1956); while the protein content was assayed by the Lowry method (Lowry, Rosebrough, Farr & Randall, 1951).

Preparation of lymphocyte cultures

Spleen cell suspensions were prepared from the spleens of BALB/c mice. The spleen was gently cut, pressed gently through a 200-gage stainless steel sieve, and washed with RPMI medium (Sigma, U.S.A.) (1000 g, 2 min). Viable lymphocytes were obtained by F ico l l -Paque gradient centrifugation (3000g, 15 min) of spleen cell suspension. The lymphocytes were then washed twice with RPMI medium and resuspensed in RPMI medium supplemented with 10% fetal calf serum (FCS) (Gibco, U.S.A.).

Mitogenic assay

Murine spleen lymphocytes were co-cultured with PH-I in 96-well flat-bottomed microtiter plates (Nunc, Denmark) at a cell density of 5 × 105 cells/well in a total volume of 0.2 ml. Microtiter plates were incubated at 37°C in a humidified atmosphere with 5o/0 C02-950/0 air for 2 days. In the last 6 h of incubation, 0.5 of a microCurie (~Ci) of tritiated thymidine (3H-TdR) (Amersham, U.K.) in a 50 tal volume was added into each well. Lymphocytes were collected on glass fiber filters by a Titertek cell harvester (Flow Lab., U.K.). Incorporation of 3H-TdR into cells was determined by a liquid scintillation counter (Beckman, LS-1801, U.S.A.). Results are expressed as the arithmetic mean stimulation index (S.I.)_+ S.D. of triplicate

cultures (S.I. = counts/min in mitogen-treated cultures per counts/min in control cultures).

Induction of TNF in mice

On day 0, 5 mg zymosan or PH-I were injected i.p. into groups of three ICR mice. On day 6, 25/ag LPS or different doses of PH-I were injected intravenously (i.v.) into the primed mice. Two hours later, serum was obtained and the tumor necrosis factor (TNF) titer in mouse serum was determined by the mouse tumor necrosis factor ELISA test kit (Genzyme). Murine recombinant TNF-a was used as the standard.

Assay of anti-tumor activity

To study the effect of PH-I on the growth of Ehrlich ascites carcinoma (EAT) cells in vitro, EAT cells were cultured in microtiter plates at a cell density of 1 × 105 cells/well in a total volume of 0.2 ml in the presence or absence of PH-I. Microtiter plates were incubated at 37°C in a humidified atmosphere with 5°7o CO2-95O70 air for 2 days. In the last 6 h of incubation, 0.5/aCi 3H-TdR was added into each well. EAT cells were collected on glass fiber filters by a Titertek cell harvester. Incorporation of 3H-TdR into cells was determined by liquid scintillation counting. Results are expressed as the suppression (%) + S.D. of triplicate cultures. Suppression (%) = [100 x (control-treatment)/ control] %, where "control" is the 3H-TdR incorporation in the absence of PH-I and "treatment" is the 3H-TdR incorporation in the presence of PH-I.

To study the effect of PH-I on the growth of the EAT tumor in vivo, BALB/c mice in groups of five were injected intraperitoneally (i.p.) with 500/ag PH- I in 0.2 ml phosphate-buffered saline (PBS) for five consecutive days (days 0 - 4). The control group was injected with PBS only. On day 2, 1 × 105 EAT cells were inoculated i.p. into each mouse. On day 9, EAT cells in the peritoneal cavity of each mouse were harvested by exhaustive drainage. Cells were counted with a hemocytometer and viability was determined by the trypan blue exclusion method (Philip, 1973).

RESULTS

The roots of P. heterophylla were extracted by hot-water (80°C) and then by stepwise alcohol precipitation. The fraction PH-III showed a large yield (30.06%). In contrast, a relatively low yield was

Mitogen and TNF Production 1317

,--, 16 V - - o ( a )

~ 12 80 I"

.~ 60

. -~- .a. ..v 40

4 ~ 20

~ ' - - ~ " . . . . . 3' x _ I ~ I ,

0 100 200 300 400 500 ~ 0

"r

20 40 60 80 100

(~tg/ml)

Fig. 1. The mitogenic effect of different fractions PH, PH-I, PH-II, PH-III from Pseudostellaria heterophylla on murine lymphocytes. PH (0), PH-I (0 ) , PH-II (A), PH-III (11) in different concentrations were co-cultured with 5 × 105 BALB/c lymphocytes. After an incubation period of 48 h, cultured cells were pulsed with 0.5/aCi/well 3H-TdR and radioactivity incorporated was determined. Results were expressed as mean S.I. _+ S.D. Counts/min of

control cultures is 1015.6 _+ 55.7.

seen in f ract ions PH- I (0 .20%) and P H - I I (0 .24%). P H - I was found to con ta in mainly ca rbohydra t e (56.8%); a small a m o u n t of prote in (7 .6%) was also detected. F r o m the result, it seems obvious tha t PH- I is a polysacchar ide or proteoglycan.

Spleen lymphocytes f rom B A L B / c mice were cul tured with di f ferent f rac t ions separa ted f rom P. heterophylla. The results of typical exper iments are shown in Fig. 1. The results showed tha t f ract ions P H , PH- I , P H - I I bu t not P H - I I I caused the s t imula t ion of t r i t ia ted thymid ine inco rpo ra t ion into mur ine lymphocytes in a dose-dependent manner . A m o n g f rac t ions P H , PH- I and PH- I I , PH- I was found to exhibi t the mos t po ten t mitogenic activity on mur ine lymphocytes . The mitogenic effect of PH- I was unlikely due to the c o n t a m i n a t i o n of l ipopolysacchar ide (LPS). Lipid A was k n o w n to be the toxic and immunos t imu la t i ng par t in LPS (Burrell , 1990). In Fig. 2(a), t r ea tmen t of LPS with 1% acetic acid at 100°C for 90 min could completely abol ish the mi togenic effect o f LPS because the acetic acid could deplete the lipid A f rom LPS (Ha, Leung, Fung, Choy & Lee, 1985). On the o ther hand , in Fig. 2(b), acetic acid could not affect the mi togenic effect of PH-I . It indicated tha t the mi togenic effect of PH- I was not due to the c o n t a m i n a t i o n of LPS.

Table 1 shows t ha t PH- I could act as a pr iming agent with subsequent challenge with LPS for the induc t ion of t u m o r necrosis fac tor (TNF) in vivo.

LPS (p.g/ml) "D, _~ (b)

F 4 , 3 ~ ~

2

1

I I I I I 0 100 200 300 400 500

(gg/ml)

Fig. 2. (a) The mitogenic effect of LPS after the treatment of acetic acid on murine lymphocytes. LPS in different concentrations with (A) or without (@) the treatment of 1% acetic acid for 90 min were co-cultured with 5 x 105 BALB/c lymphocytes. After an incubation of 48 h, culture cells were given a pulse with 0.5 ~Ci/well 3H-TdR and radioactivity incorporated was determined. Results were expressed as mean S.I. _+ S.D. Counts/min of control cultures is 835.0 __ 95.2. (b) The mitogenic effect of PH-I after the treatment of acetic acid on murine lymphocytes. PH-I in different concentrations with (11) or without ( 0 ) the treatment of 1% acetic acid for 90 rain were co-cultured with 5 x 105 BALB/c lymphocytes. After an incubation of 48 h, cultures cells were given a pulse with 0.5 gCi/well 3H-TdR and radioactivity incorporated was determined. Results were expressed as mean S.I. _+ S.D.

Counts/rain of control cultures is 550.8 + 41.9.

However , PH-I could not act as an eliciting agent for TNF induc t ion in zymosan-pr imed mice. Figure 3 shows tha t 40% zymosan-pr imed E A T bear ing mice died on day 11 af ter LPS inject ion. However , none of the PH- I pr imed E A T bear ing mice died at the same time.

Figure 4 showed tha t PH- I could not suppress the E A T growth in vitro. However , Table 2 indicated tha t PH- I could significantly inhibi t the g rowth of E A T in vivo.

1 3 1 8 C . K . W O N G e t al.

Table 1. Induction of TNF by PH-I and zymosan in mice

Day 0 Day 6 TNF titer priming eliciting (ng/ml)

5 mg zymosan PBS 0.00 _ 0.00 5 mg PH-I PBS 0.00 _+ 0.00 PBS 25/ag LPS 0.97 _+ 0.49 5 mg zymosan 25 ,ug LPS 22.68 _+ 0.04* 5 mg PH-I 25/ag LPS 15.45 _+ 0.45* 5 mg zymosan 25/~g PH-I 0.00 _+ 0.00 5 mg zymosan 50/~g PH-I 0.00 + 0.00 5 mg zymosan 100/~g PH-I 0.00 + 0.00 5 mg zymosan 200/~g PH-I 0.00 _+ 0.00 5 mg zymosan 400 ~g PH-I 0.00 _+ 0.00 Nil 500 ~g PH-I 0.00 _+ 0.00 Nil 2 mg PH-I 0.00 _+ 0.00

On day 0, 5 mg zymosan or PH-I were injected i.p. in groups of three ICR mice. On day 6, 25 ~g LPS or different doses of PH-I were injected intravenously (i.v.) into the primed mice. Two hours later, serum was obtained and the tumor necrosis factor (TNF) titer in mouse serum was determined by the murine tumor necrosis factor ELISA test kit (Genzyme). Murine recombinant TNF-a was used as the s tandard. The differences between groups of priming control and treatments were determined by Student 's t-test. */9<0.001.

100

8 0

60

"~ 40

2 0

P • • • • • • • • • • • I A ~ A

I !__.

I - - L ~ A - - A

I I I I I 2 4 6 8 10

Days after LPS injection

Fig. 3. Survival test of EAT-bearing mice after the in vivo production of TNF. Group of 10 mice were inoculated 2 × 105 EAT on day 0. On day 1, 5 mg zymosan or PH-I was injected into each mouse i.p. On day 7, mice were injected i.v. with 25/~g LPS. The percentage survival were monitored on the indicated days. (O) EAT, PH-I, LPS;

(A) EAT, zymosan, LPS.

DISCUSSION

Severa l i m m u n o m o d u l a t o r y ac t iv i t ies h a v e been o b s e r v e d in the h i g h - m o l e c u l a r we igh t f r a c t i o n s o f

the h o t - w a t e r ex t r ac t s f r o m t he C h i n e s e h e r b s b u t

50 40 3o

~. 20

- 1 0

-20 r~ -30 -

-40 -

-50 0

L

- .1.

I I I I i 1 0 0 2 0 0 3 0 0 4 0 0 5 0 0

(~tg/ml)

Fig. 4. EAT cells were cultured at a cell density of 1 × 105 EAT cells/well in a total volume of 0.2 ml in the absence or presence of different concentrations of PH-I. After an incubation of 48 h, cultured cells were given a pulse with 0.5/~Ci/well 3H-TdR and radioactivity incorporated was determined. Results were expressed as mean

suppression _+ S.D.

Table 2. Suppression of EAT growth by PH-I in vivo

EAT number Treatment (× 10 8 cells) Suppression (%)

PBS 7.1 + 0.8 0.0 _+ 11.2 PH-I 2.1 + 0.2 69.8 _+ 6.6*

BALB/c mice in groups of five were injected i.p. with PH-I (500/~g 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 EAT (1 × 105 cells/mouse). The tumor cell number recoverable f rom the peritoneal cavity of each mouse was determined on day 9. The difference between groups o f control and treatment was determined by Student 's t-test. *P<0.001.

less o b s e r v e d in t he l o w - m o l e c u l a r we igh t f r a c t i o n s

( Y a m a d a , 1989). T h e i m m u n o p o t e n t i a t i n g f r a c t i o n s

h a v e been s h o w n to p o s s e s s a n t i - n e o p l a s t i c ac t iv i ty

( M i y a z a k i et al. , 1979; E b i n a & K o h y a , 1988;

C h i h a r a , H a m u r o , M a e d a , Ara i & F u k u o k a , 1970;

T a k a h a s h i , W a t a n u k i , Y a m a z a k i & A b e , 1988; L ien

& G a o , 1990; E b i n a & M u r a t a , 1990), s t i m u l a t i o n o f

T N F - i n d u c e d t u m o r cell d i f f e r e n t i a t i o n ( S a k a g a m i ,

I k e d a & K o n n o , 1989), i n d u c t i o n o f g a m m a -

i n t e r f e r o n a n d i n t e r l euk in -2 ( S a k a g a m i et al. , 1988),

i n d u c t i o n o f T N F ( H a r a n a k a et al. , 1984, 1985),

a n t i - c o m p l e m e n t a r y ac t iv i ty ( Y a m a d a , 1989; Y a m a d a et al. , 1990), a n t i - i n f l a m m a t o r y ac t iv i ty

( S a t o m i , S a k u r a i , l i m u r a , H a r a n a k a & H a r a n a k a ,

1989), e n h a n c e m e n t o f a n t i b o d y p r o d u c t i o n ( Z h a o et al. , 1990) a n d p o l y c l o n a l B-cell a c t i va t i ng ac t iv i ty

Mitogen and

(Kumazawa et al., 1982; Vacheron, Perin, Kodari, Smets, Zalisz & Guenounou, 1989).

Pseudostellaria heterophylla is widely used as a mild tonic herbal medicine in China. Our results showed that a macromolecular fraction (PH-I) separated from the roots of P. heterophylla by hot- water extraction and stepwise alcohol precipitation displayed mitogenic and TNF inducing activities. PH-I is believed to be a polysaccharide as it is found to have a high carbohydrate content (56.8°/o). The mitogenic activity of PH-I is probably due to the polysaccharides rather than due to contamination by LPS. It is because it is heat and mild acid stable [Figs 2(a) and (b)]. Moreover, its activity was completely abrogated by the treatment with periodate (data not shown). The absence of LPS in PH-I is again evidenced by the fact that polymyxin B showed no inhibitory effect on its mitogenic activity while polymyxin B strongly inhibited the mitogenicity of LPS (data not shown).

Priming agents for TNF production mostly come from bacteria. A non-bacterial priming agent, zymosan, had been described in 1984 (Haranaka et al., 1984). Many well-recognized priming agents such as zymosan can be used to induce TNF with a subsequent challenge with LPS in mice (Haranaka et al., 1984). In a previous study, traditional Chinese medicines (e.g. Angelica radix, C innamomum cortex) were given orally as reticuloendothelial stimulating agents to induce TNF production in vivo and prolonged the survival time of Meth A-bearing mice (Haranaka et al., 1985). The above crude Chinese herbs offer the advantages of low toxicity and ease of administration while comparing with other bacterial priming agents such as Bacillus C a m e t t e - Guerin (Carswell et al., 1975) and Listeria monocytogenes (Ha, Leung, Fung, Choy & Lee,

TNF Production 1319

1987). According to our results, TNF can also be induced in PH-I primed mice with subsequent challenge with LPS. However, the TNF titer was lower than that when using zymosan as the priming agent. The difference in the producibility of TNF might depend on the degree of stimulation of the reticuloendothelial system by the priming agent. The mechanism(s) whereby PH-I could exert its effect on the reticuloendothelial system is currently under investigation. PH-I was found to be better than zymosan in the treatment of tumor bearing mice. It is because the zymosan primed EAT-bearing mice died more easily after LPS injection than PH-I primed EAT-bearing mice.

Our data showed that PH-I could suppress EAT growth in vivo but not in vitro. This would indicate that the anti-tumor activity of PH-I was mediated by the immune system of the host. It is possible that PH-I could activate the reticuloendothelial system, natural killer cells or other cytokines release to kill the tumor. Experiments are currently in progress to examine these possibilities.

The carbohydrate content (56.8%) of fraction PH-I was considered to be lower than expected. It may be due to the fact that the phenol sulfuric acid method is not sensitive enough for determining these types of polysaccharides. Many previous studies indicated that the activity of the immunopotentiating fractions from Chinese herbs was strongly related to its carbohydrate structure (Miyazaki et al., 1979; Lien et al., 1990; Gonda, Tomoda, Shimizu & Kanari, 1990). The structure-activity relationship of PH-I will also be elucidated in the future.

Acknowledgements -- This work was supported in part by an endowment fund from the United College, The Chinese University of Hong Kong.

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