Pergamon Int. J. lmmunopharmac., Vol. 16, No. 4, pp. 2 7 1 - 2 7 7 , 1994

Elsevier Science Ltd Copyright ~e~ 1994 International Society for lmmunopharmacology

Printed in Great Britain. All rights reserved 0 1 9 2 - 0 5 6 1 / 9 4 $6.00 + .00

0 1 9 2 - 0 5 6 1 ( 9 3 ) E 0 0 1 8 - 5

TUMOR NECROSIS FACTOR ELICITING FRACTIONS SEPARATED FROM PSEUDOSTELLARIA H E T E R O P H Y L L A

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

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

(Received 12 August 1993 and in final form 2 December 1993)

Abstract - - Tumor necrosis factor (TNF) could be induced in the serum of ICR mice bearing Ehrlich ascites tumor (EAT) cells by the i.v. administration of antitumor fractions (PH-I A, PH-I B, PH-I C, PH-I Ca, PH-I Cb) separated from the roots of Pseudostellaria heterophylla. TNF could also be detected in the ascitic fluid of EAT-bearing mice 2 h after i.v. injection with PH-I C, PH-I Ca or PH-I Cb, The proliferation of EAT cells was found to be significantly suppressed after the administration of these fractions. Our results suggest that the antitumor activity ofP. heterophylla may be related, at least in part, to its capacity to induce TNF production in vivo. The strongest TNF eliciting and antitumor activity was found in the PH-I Cb fraction which is an acidic polysaccharide or proteoglycan with molecular weight of approximately 5000.

Tumor necrosis factor (TNF) was first discovered in 1975 by Carswell and her colleagues (Carswell, Old, Kassel, Green, Fiore & Williamson, 1975) in the serum of mice infected with Bacillus Camette- Guerin (BCG) and challenged subsequently with lipopolysaccharide (LPS). TNF is capable of inducing necrosis of certain tumors in experimental animals (Carswell et al., 1975) and exerted cytotoxic or cytostatic effects on many tumor cell lines in vitro (Watanabe, Nfitsu, Neda, Sone & Yamauchi, 1985; Haranaka & Satomi, 1981). However, when recombinant TNF was injected into human patients, many side effects including fever and cachexia were observed (Spriggs, Sherman, Ill & Kufe, 1987). Therefore, antitumor therapy by the induction of endogenous TNF is a possible alternative.

Previous studies showed that endogenous TNF could be induced in zymosan-primed mice bearing EAT cells with subsequent challenge with lipopoly- saccharide (LPS) (Wong, Fung, Lee & Choy, 1992a). The in vivo production of TNF was found to suppress the proliferation of EAT cells simultane- ously. Another study showed that TNF could be induced in the solid MH134 hepatoma by the systemic administration of an antitumor polysaccha- ride, MGA, in mice (Takahashi, Watanuki, Yamazaki & Abe, 1988a). Therefore, the endo- genous induction of TNF was proved to be an

effective method to suppress the growth of certain tumors in mice. Previous work from this laboratory have shown that a water soluble fraction (PH-I) extracted from the roots of Pseudostellaria hetero- phylla possesses potent mitogenic and antitumor activities. In addition, PH-I was shown to act as a priming agent for TNF release in vivo (Wong, Leung, Fung, Pang & Choy, 1992b). In the present study, PH-I was further fractionated, characterized and the antitumor activities of various bioactive fractions were investigated.

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 1 1 of water at 4°C overnight. One litre 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 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 (2.67% w/v) and slowly added into an equal volume of 95% ethanol with

271

272

3.0

2.5

E.0

o ,¢

¢~ 1.5 O

1.0

0 .5

0 .0

P H -

PH-I A PH-I B

0 5 i 0 15 20 25 30 35 45

C. K, WONG et al.

v

4O

FRACTION NUMBER

Fig. 1. Gel filtration of PH-I. PH-I (400 mg) was dissolved in 4 ml distilled water and applied onto a Sephadex G-100 column (2.5 × 90 cm). Fractions were eluted with water. Fraction of 12 ml were obtained at a flow rate 12 ml/h. Fractions 11-19 were collected as fraction PH-I A. Fractions 2 0 - 3 0 were collected as PH-I B. Fractions 3 1 - 3 8 were collected as fraction PH-I C. The relative amount of carbohydrate in each fraction was determined by

phenol sulphuric acid method (O.D.490).

con t inuous stirring. The precipi tate was then cent r i fuged down. The resul t ing superna tan t was slowly added into an equal volume of 95% ethanol . The precipi tate was then centr i fuged down and was designated as the PH- I f ract ion.

PH-I was applied on to a Sephadex G-100 (Pharmac ia ) co lumn (2.5 × 90 cm) and eluted with water to ob ta in f ract ions PH-I A, PH- I B and PH- I C fract ions. Frac t ions of 12 ml were collected at a flow rate of 12 m l / h . PH-I C was then applied on to a D E A E anion-exchange Sephadex co lumn (2.5 × 6 cm). PH-I Ca was eluted with 10 m M NaCl phospha te buf fe r (pH 8.0) and PH-I Cb was eluted with 500 m M NaC1 phospha t e buf fe r (pH 8,0). Frac t ions of 10 ml were collected at a flow rate of 1 0 0 m l / h . The f ract ions were dialysed against distilled water with benzola ted dialysis tub ing (Sigma) having a cut of f value of 1200 to remove salts. C a r b o h y d r a t e con ten t of the isolated f ract ions was de termined by the phenol sulphuric acid me thod (Dubois , Gilles, Hami l t on , Rebers & Smith, 1956), while prote in conten t was de termined by the Lowry m e t h o d (Lowry, Rosebrough , Farr & Randal l , 1951) and uronic acid con ten t was de te rmined by the carbazole me thod (Bitter & Muir , 1962). The puri ty and the molecular weight of the f rac t ion PH-I Cb

3 5

~.0

2 5

Z 2 0

~03 1 5

1.0

0 5

O0

P H - I Ca

VV v ~ .

,5 10 15 20 E5 30

FRACTION NUMBERS Fig. 2. Anion-exchange chromatography of PH-I C. PH-I C (150 mg) was dissolved in 1.5 ml 10 mM NaC1 phosphate buffer (pH 8.0) and applied onto a DEAE Sephadex column (2.5 × 6 cm). PH-I Ca was eluted with 10 mM NaCI phosphate buffer (pH 8.0) and PH-I Cb was eluted with 500 mM NaCI phosphate buffer (pH 8.0). Fractions of 10 ml were collected at a flow rate of 100ml/h. Fractions 1 - 7 were collected as PH-I Ca. Fractions 14 - 20 were collected as PH-I Cb. The fractions were then dialysed against distilled water with benzolated dialysis tubing having a cut off value of 1200 to remove salts. The relative carbohydrate and protein contents of each fraction were determined by phenol sulphuric acid method (O.D.49o) and Lowry method (O.D.6s0), respectively.

were de termined by analyt ical H P L C (Bio-Gel TSK-20 column, Bio-Rad) at flow rate 1 m l / m i n and compared with s tandards .

Determination o f the toxicity o f samples

The toxicity of different f ract ions was determined by the br ine shr imp assay (Meyer, Ferrigni, P u t n a m , Jacobsen , Nichols & Mclaughl in , 1982; Anderson , Goetz & Mclaughl in , 1991). Individual f ract ions at 1000, 100, 10 ~g /ml were mixed with the brine shr imp (Artemia salina L E A C H ) . The n u m b e r of shr imps being killed was counted two days later and the toxicity (LC;o) was de termined. LCso is def ined as the concen t ra t ion of sample (~g/ml) which causes 50°70 of the to ta l shr imps dea th in the br ine medium. Berber ine chloride and hippur ic acid were used as posit ive controls .

In vitro stimulation o f TNF release f rom macro- phages

An individual f rac t ion (PH-I A, PH-I B, PH-I C, PH-I Ca and PH-I Cb) f rom P. heterophylla was

TNF from P.

Table 1. Protein, carbohydrate and uronic acid contents in different fractions

Protein Carbohydrate Uronic acid (%) (%) (%)

PH-I 7.60 56.80 4.45 PH-I A 5.20 53.15 6.37 PH-I B 4.90 40.00 3.49 PH-I C 9.07 43.00 9.43 PH-I Ca 2.50 50.21 18.50 PH-I Cb 4.77 61.46" 21.75"

Bovine serum albumin (BSA), dextran (M.W. 72,200) and a-D-galacturonic acid were used as standards for the assays of protein, carbohydrate and uronic acid, respectively. *In the case of PH-I Cb, heparin containing glucuronic acid was used as a standard for carbohydrate and uronic acid determinations.

heterophylla 273

on day 0. On day 6, each mouse was injected i.v. with 2 mg P. heterophylla fractions in 0.2 ml PBS. Three to four mice from each group were bled f rom subclavian vessels to obtain the serum and the ascitic fluid was collected f rom the peritoneal cavity 2 h after the injection. T N F titers in the pooled sera and ascitic fluids were determined by the mouse TNF-a ELISA test kit (Genzyme) using recombinant murine TNF-a as the standard. The remaining 6 - 7 mice in each group were sacrificed on day 7, E A T cells in each mouse were harvested from the peritoneal cavity and counted. Pooled cells were washed once with half-isotonic solution to remove the erythro- cytes. The final cell suspension was prepared in PBS. Cells were counted with a haemacytometer using 0.3% trypan blue.

Table 2. Determination of the toxicity (LCso) of different fractions by brine shrimp assay

LCs0 (~g/ml)

Berberine chloride 50 Hippuric acid 310 PH-I >1000.0 PH-I A >1000.0 PH-I B >1000.0 PH-I C >1000.0

P. heterophylla fractions at 1000, 100, 10/ag/ml were mixed with the brine shrimp (Artemia salina LEACH). The number of shrimps being killed was counted two days later and the toxicity (LCs0) was determined. Berberine chloride and hippuric acid were used as positive controls. LCs0 is defined as the concentration of sample (tag/ml) which causes 50% of the total shrimps death in the brine medium.

incubated with resident peritoneal macrophages f rom ICR mice (1 × 106/ml) at various concentra- tions for 24 h at 37°C in a humidified atmosphere of 5% CO2 in air. The supernatants were harvested and their T N F titers were determined by a mouse TNF-a ELISA test kit (Genzyme) using recombinant murine TNF-a as the standard.

Effects o f P. heterophylla polysaccharides on the TNF production and E A T growth in vivo

Male ICR mice, about 3 0 - 3 5 g, were used in all experiments. Ehrlich ascites tumor (EAT), Ny Klein cell type, was maintained by weekly intraperi toneal (i.p.) implantat ion in mice. Mice in groups of 10 were inoculated i.p. with 1 × 105 E A T cells harvested f rom seven-day-old tumors in 0.2 ml PBS

RESULTS

The roots of P. heterophylla were extracted by hot water (80°C) and then subjected to stepwise alcohol precipitation. The yield of fraction PH-I was found to be 0.20%. In Fig. 1, PH-I was size fractionated into PH-I A (15.0%), PH-I B (31.0%) and PH-I C (36.3%) by Sephadex G-100 chromatography. In Fig. 2, PH-I C was further purified into PH-I Ca (40.1%) and PH-I Cb (30.4%) by D E A E anion exchange Sephadex chromatography.

The carbohydrate, protein and uronic acid contents of various fractions are shown in Table 1. All fractions were found to contain mainly carbohy- drate, a small amount of protein and some uronic acid. F rom the results, it seems obvious that the bioactive fractions are polysaccharides or proteo- glycans.

PH-I A, PH-I B and PH-I C were found to be non-toxic as determined in vivo by the Brine shrimp assay (Table 2). Table 3 shows that PH-I A, PH-I B, PH-I C, PH-I Ca and PH-I Cb could stimulate TNF release from peritoneal macrophages in vitro. Table 4 shows that endogenous TNF could be induced in the serum of EAT-bear ing mice by intravenous injection with PH-I A, PH-I B, PH-I C, PH-I Ca and PH-I Cb. Moreover , PH-I C, PH-I Ca and PH-I Cb could also induce TNF release in peritoneal ascitic fluid of EAT-bear ing mice. Since PH-I Cb was found to possess the most potent TNF- inducing and ant i tumor activities among all fractions being tested, it was further characterized by H P L C . In Fig. 3, PH-I Cb showed a single peak in analytical H P L C column (Bio-Gel TSK-20 column, Bio-Rad). Its molecular weight was found to be approximately

274 C. K. WONG et al.

Table 3. In vitro stimulation of TNF release from resident peritoneal macrophages

Sample Concentration of TNF (~g/ml) (ng/ml)

Medium 0.140 _+ 0.032

PH-I A

PH-I B

PH-I C

PH-I Ca

PH-I Cb

100 0.330 _+ 0.031 200 0.330 _+ 0.013 500 0.340 _+ 0.029

100 0.570 +_ 0.020 200 0.670 _+ 0.020 500 0.830 _+ 0.002

100 0.250 _+ 0.035 200 0.300 _+ 0.013 500 0.310 _+ 0.016

100 0.280 _+ 0.040 200 0.290 _+ 0.047 500 0.280 _+ 0.035

100 0.281 _+ 0.014 200 0.430 _+ 0.008 500 0.500 _+ 0.064

Different fractions (PH-I A, PH-I B, PH-I C, PH-I Ca and PH-I Cb) at various concentrations were incubated with peritoneal macrophages from ICR mice (1 x 106/ml) for 24 h. The supernatants were harvested and the TNF titers were determined by a mouse TNF-a ELISA test kit (Genzyme) using recombinant murine TNF-e as the standard. Results were expressed as mean +_ S.D. of three determinations.

5000 when compar ing with the dext ran s tandards . Fu r the rmore , the survival rate of PH-I Cb- t rea ted EAT-bea r ing mice was higher t han tha t of the un t rea ted EAT-bea r ing mice or zymosan-pr imed EAT-bea r ing mice af ter the in ject ion of LPS (Fig. 4). There were 40% EAT-bea r ing mice or zymosan-pr imed EAT-bea r ing mice which died on day 11 af ter LPS inject ion. However , only 10% PH-I Cb- t rea ted EAT-bea r ing mice died at the same time.

DISCUSSION

Al though the phase II clinical trials of r ecombinan t TNF had been carr ied out, the side effects of this t r ea tmen t were still a d rawback (Spriggs et al., 1987; Frei & Spriggs, 1989; Lenk, Tanneberger , Miiller, Eber t & Shiga, 1989; Fiedler, Zeller, Pe imann , Weh & Hossfeld, 1991). Therefore , the ac t iva t ion of the i m m u n e system to induce

endogenous TNF produc t ion so as to kill the tumor cells or to suppress the t u m o r growth may be a more safe and effective a l ternat ive for cancer t rea tment . Nowadays , many immunopo ten t i a t i ng polysaccha- rides extracted f rom na tura l products are found to possess a n t i t u m o r activities (Okutomi et al., 1989; Luett ig, Steinmuller , Gi f ford , Wagner & L o h m a n n - Mat thes , 1989; Shen, Zhai , Chen, Luo, Tu & Ou, 1991). One of the possible mechan isms of their a n t i t u m o r effect was related to the induct ion of TNF (Takahash i et al., 1988a, b; Luett ig et al., 1989; Mori , I toh, Y a m a d a & Tamaya , 1989; Usami et al., 1988). M G A , an a n t i t u m o r m a n n o g l u c a n prepared f rom Microe l lobospor ia grisea, was found to be one of the s t rong eliciting agents of TNF (Takahash i el al., 1988a, b). OK-432, a lyophilized p repara t ion of Strep tococcus pyogenes , could elicit endogenous TNF in mice by in t ravenous admin is t ra t ion (Sakagami & Takeda , 1992). OK-432 could also p romote an increase in the n u m b e r of pr imed per i toneal macrophages and the release of TNF from

TNF from P. heterophylla

Table 4. Effects of P. heterophylla polysaccharides on the TNF production and EAT growth in vivo

TNF titer (ng/ml) % Suppression of

Trea tment Serum Ascitic fluid EAT growth

PBS U.D. U.D. 0.00 + 13.70 PH-I U.D. U.D. 16.75 _+ 28.77 PH-I A 0.05 U.D. 50.94 _+ 47.53 PH-I B 0.97 U.D. 38.00 +_ 20.67* PH-I C 0.54 1.15 47.44 _+ 11.34 ~ PH-I Ca 0.29 0.45 49.30 _+ 19.58 t PH-I Cb 12.00 3.16 83.91 _+ 18.49 t

ICR mice in groups of 10 were inoculated i.p. with 1 × 105 EAT cells in 0.2 ml PBS on day 0. On day 6, each mouse was injected i.v. with 2 mg samples in 0.2 ml PBS. Three to four mice were bled from subclavian vessels to obtain the serum and the ascitic fluid was collected from the peritoneal cavity 2 h after the injection. TNF titers in the pooled sera and ascitic fluids were determined by the mouse TNF-a ELISA test kit (Genzyme) using recombinant murine TNF-a as the s tandard. The suppression of EAT growth was determined with the remaining mice (6 - 7 mice) on day 7. EAT cells in each mouse were harvested and counted. Results were expressed as mean _+ S.D. The differences between control and treatment groups were determined by the Student 's t-test. *P<0.01 ; tP<0.002. U.D. = undetectable.

275

P H - I C b

I N J E C T I O N D E X T R A N

( M . W . 5 , 0 0 0 )

I N J E C T I O N

I i i i i i i i I

0 4 8 1 2 1 6

T I M E ( m i n . )

Fig. 3. Analytical H P L C of PH-I Cb. PH-I Cb (1 mg in 20/A distilled water) was injected to a TSK-20 column (Bio- rad). The flow rate was 1 ml /min . The carbohydrate content was monitored by a refractive index detector. The molecular weight o f PH-I Cb was determined by comparing

with dextran standards.

t h e s e cells a f t e r i .p . a d m i n i s t r a t i o n in t he o v a r i a n

c a n c e r p a t i e n t s (Mor i et al., 1989). S o m e o f t he

C h i n e s e h e r b s ( H a r a n a k a , S a t o m i , S a k u r a i ,

H a r a n a k a , O k a d a & K o b a y a s h i , 1985) a n d f u n g i

1 0 0

8 0

v 6 0

> 4 0

O'3

2 0

r I I I ~ I

2 4 6 8 l O 12

D a y s a f t e r L P S i n j e c t i o n

Fig. 4. Effect of PH-I Cb on the survival rate of EAT- bearing mice. ICR mice in groups of 10 were inoculated with 2 × 105 EAT cells on day 0. In one group of mice, 5 mg zymosan was injected into each mouse i.p. on day 1 and on day 7 each mouse was injected i.v. with 25/ag LPS. In a second group of mice, each mouse was injected with 2 mg PH-I Cb i.v. on day 6. The percentage survival in these two groups of mice was monitored over a period of 18 days and compared with the control EAT-bearing mice. ( O ) Control, ( [] ) zymosan- and LPS-treated, ( • ) PH-I

Cb-treated.

276 C. K. WONG et al.

(Mori et al., 1987; Wong et al., 1992b) were found to be priming agents for TNF release in mice. However, eliciting agents of TNF isolated from higher plants and Chinese herbal medicine have seldom been reported.

The roots of P. heterophylla are commonly used in China as a mild tonic drug in the treatment of asthenia-syndrome cases which manifest as insufficiency of vital energy. PH-I, a mitogenic fraction separated from P. heterophylla, was previously shown to act as a priming agent for TNF release in vivo. It suppressed the growth of EAT cells in vivo but not in vitro (Wong et al., 1992b). Our present investigation showed that PH-I could be separated into several bioactive fractions, PH-I A, PH-I B and PH-I C. Among these three fractions, PH-I C exerted the most potent effect for the induction of endogenous TNF in vivo.

PH-I C was further fractionated into two bioactive fractions PH-I Ca and PH-I Cb by anion-exchange chromatography. It was found that the acidic fraction PH-I Cb exerted a strong effect on TNF release from macrophages in vitro (Table 3) and was the most potent inducer of endogenous TNF production in serum and tumor sites of mice bearing EAT cells (Table 4). PH-I Cb was found to be an acidic polysaccharide with molecular weight of approximately 5000. The EAT cell number was greatly suppressed after the i.v. injection of PH-I Cb into EAT-bearing mice. Preliminary results showed that i.v. injection of heparin but not galacturonic acid could also induce TNF production in the serum of normal and EAT-bearing mice as well as in the ascitic fluid of EAT-bearing mice (data not shown). It seems likely that the acidic properties of the polysaccharide or uronic acidic structure containing polymer may be related to its TNF eliciting and antitumor activities, but this has yet to be elucidated.

The endogenous induction of TNF by P. heterophylla fractions is probably one of the contributing factors for their suppressive effect on the growth of the EAT cells in vivo (Table 4). Supporting evidence is our observation that TNF coutd be found in the sera and peritoneal tumor sites

shortly after i.v. injection of the P. heterophylla fractions into EAT-bearing mice. Moreover, it was found that fractions PH-I, PH-I A, PH-I B, PH-I C, PH-I Ca and PH-I Cb did not exert any cytotoxic or cytostatic effect on the EAT cells in vitro (data not shown). Therefore, it seems likely that the antitumor activity of P. heterophylla polysaccharides is mediated through the activation of the host immune mechanisms rather than by exhibiting direct cytotoxicity on the tumor cells.

Although TNF could be induced in zymosan- primed EAT-bearing mice followed by the injection of LPS (Wong et al., 1992a), nevertheless, mice began to die gradually due to endotoxin shock 1 day after LPS injection (Fig. 4). In contrast, EAT-bearing mice injected with PH-I Cb did not die until eight days after the i.v. injection of PH-I Cb (Fig. 4). Therefore, the use of the PH-I Cb fraction to induce the endogenous TNF for the treatment of EAT-bearing mice might provide a novel, safe and effective method to combat cancer.

The use of natural products such as P. heterophylla to induce TNF for the treatment of cancer has some advantages. It can minimize the lethal side effects such as anorexia and cachexia caused by the direct injection of recombinant TNF (Frei & Spriggs, 1989; Lenk et al., 1989) as observed in the longer survival time when EAT-bearing mice were treated with PH-I Cb (Fig. 4). Since P. heterophylla was an immunostimulating agent (Wong et al., 1992b), it probably could modulate other known host antitumor immune mechanisms in vivo such as the activation of lymphokine activated killer cells, natural killer cells, tumor infiltrating lymphocytes, as well as their effects on the production of cytokines such as gamma-interferon, colony stimulating factor etc. The study on the immunostimulating effect of P. heterophylla is now in progress and will be reported.

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

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