Life Sciences 77 (2005) 400 – 413

www.elsevier.com/locate/lifescie

A novel polysaccharide of black soybean promotes myelopoiesis

and reconstitutes bone marrow after 5-flurouracil- and
irradiation-induced myelosuppression
Hui-Fen Liaoa,b,c,d,T, Yu-Jen Chena,c, Yuh-Cheng Yanga
a
Department of Medical Research, Mackay Memorial Hospital, Taipei 251, Taiwan
b
Department of Medical Research and Education, Taipei Veterans General Hospital, Taipei 112, Taiwan
c
Graduate Institute of Sport Coaching Science, Chinese Culture University, Taipei 111, Taiwan
d
Department of Molecular Biology and Biochemistry, National Chiayi University, Chiayi 600, Taiwan
Received 4 June 2004; accepted 22 October 2004

Abstract

The aim of this study was to investigate the promotion of myelopoiesis by an active polysaccharide of black
soybean (PSBS). Murine spleen cells were collected from ICR mice and conditioned media (SCM) was prepared
by incubating these cells without PSBS (normal-SCM) or with PSBS in concentrations ranging from 12.5 to 100
Ag/ml (PSBS-SCM). Murine bone marrow cells were treated with PSBS alone or SCM to induce the formation
of colonies, including CFU-GM, CFU-GEMM, BFU-E and HPP-CFC. The concentrations of six hematopoietic
growth factors contained in SCM were measured using enzyme-linked immunoassay. In the live animal
experiment, PSBS was administered orally to total body-irradiated (TBI) and 5-fluorouracil (5-FU)-treated mice
to assess the reconstitution of bone marrow after myelosuppression. PSBS-SCM stimulated CFU-GM, CFU-
GEMM, BFU-E and HPP-CFC colony formation with 45.0, 5.0, 6.2 and 6.6-fold increases, respectively.
However, neither PSBS alone nor normal-SCM had such a colony-stimulating effect. In PSBS-SCM, the levels
of IL-6, IL-17, G-CSF and GM-CSF were markedly increased, but not those of IL-3 and SCF. Oral
administration of PSBS in mice not only restored the leukocyte counts reduced by TBI and 5-FU treatment but
also enhanced CFU-GM colony formation of bone marrow cells without a significant change in body weight.

T Corresponding author. Department of Molecular Biology and Biochemistry, National Chiayi University, 300 University
Road, Chiayi 600, Taiwan. Tel.: +886 5 2817781; fax: +886 5 2817780.
E-mail address: liaohf@seed.net.tw (H.-F. Liao).

0024-3205/$ - see front matter D 2005 Elsevier Inc. All rights reserved.
doi:10.1016/j.lfs.2004.10.080
 H.-F. Liao et al. / Life Sciences 77 (2005) 400–413 401

We conclude that PSBS promotes myelopoiesis activity in the bone marrow, stimulates production of various
hematopoietic growth factors from spleen cells, and reconstitutes bone marrow that has been myelosuppressed
by irradiation and 5-FU.
D 2005 Elsevier Inc. All rights reserved.

Keywords: Polysaccharide of black soybean (PSBS); Myelopoiesis; Spleen cell-conditioned medium (SCM); Colony forming
unit (CFU); 5-Flurouracil; Irradiation

Introduction

Black soybean [Glycine max (L.) Merr.] is a soybean cultivar with a black seed coat. In traditional
Chinese medicine, black soybean has been used for detoxification, as an anti-inflammatory, and to
improve the blood. Soybean has been reported to possess many properties, including immune
modulation (Gladysheva et al., 2001), inhibition of carcinogenesis (Suzuki et al., 2002), antioxidation
(Minemoto et al., 2002), and a cholesterol lowering effect (Kerckhoffs et al., 2002). However, there are
fewer reports specifically concerning black soybean. It has been proven that black soybean possesses a
higher antioxidative activity than soybean (Yang et al., 1999). Wang (1992) reported that treatment of 38
leukopenic patients with black soybean-containing herbal prescriptions increased the number of
circulating white blood cells (Wang, 1992). In a previous study, we found that a high-molecular weight
polysaccharide of black-soybean (PSBS) with an a-linked glucan structure is the major component with
providing immune regulation and anti-tumor effects (Liao et al., 2001). Other investigations have also
demonstrated many biological functions of polysaccharides from natural products. For examples, a-
linked glucan in rice bran has been proven to stimulate anti-tumor immunity (Katyama et al., 2002;
Ghoneum and Jewett, 2000), and h-linked glucan isolated from medicinal fungi is responsible for their
immunomodulating activities (Bao et al., 2002; Chen et al., 1997; Weng et al., 2002).
Myelosuppression after radiotherapy or chemotherapy is a major limiting factor in the clinical
treatment of cancers. Concurrent chemoradiation is now a standard treatment for locally advanced
malignancies originating from the esophagus, stomach, pancreas, colorectal, anus, uteri cervix, etc
(Henning et al., 2000; Mitchell et al., 2001), and, the results are promising, the severity of
myelosuppression is greater than that of single treatment modality. Therefore, promotion of
hematopoiesis remains an extremely important challenge in clinical cancer treatment.
Hematopoietic stem and progenitor cells in the bone marrow have the ability to self-replicate and
differentiate into mature blood cells. Various hematopoietic growth factors (HGFs) stimulate these
processes, including granulocyte-macrophage (GM-CSF), granulocyte (G-CSF), and macrophage (M-
CSF) colony stimulating factor; interleukin (IL)-3; IL-6; and stem cell factor (SCF) (Chen et al., 1993;
Wang et al., 1991b). Several potential drugs, such as norcantharidin (NCTD) and Astragalus
polysaccharide (APS), have been tested for their ability to activate cell-mediated immunity. These
drugs not only inhibit tumor growth (Chen et al., 2002; Pickering et al., 2003), but also promote
myelopoiesis (Wang, 1989; Zhu and Zhu, 2001).
In the present study, we attempted to clarify whether PSBS stimulates stem and progenitor cell
proliferation directly or activates spleen cells to secrete HGFs to promote myelopoiesis by in vitro assay.
The effects of PSBS on reconstitution of bone marrow in myeloablated mice were also examined.
402 H.-F. Liao et al. / Life Sciences 77 (2005) 400–413

Materials and methods

Materials and animals

PSBS was purified from an extract of black soybean through a series of separation procedures as
described in our previous report (Liao et al., 2001). In brief, lipid-soluble and low-molecule weight
components in black soybean were removed using dichloromethane and ethanol and then extracted by
water at 708C. The water-soluble extract was collected and deproteinized with proteinase K. Further
separation of the non-protein water-soluble fraction was performed by liquid chromatography and gel
filtration, and the active PSBS, with a high molecular weight was isolated.
Male ICR mice (10 to 12 weeks of age, 35 F 5 g), purchased from the National Laboratory Animal
Breeding and Research Center (Taipei, Taiwan, ROC), were kept under specific pathogen-free
conditions. All experiments were performed in accordance with guidelines in the NIH Guide for the
Care and Use of Laboratory Animals (DHHS publication No. NIH 85-23, revised 1996).

Preparation of splenocyte-conditioned medium (SCM)

The spleens were removed from the mice, homogenated into single cells, and dispersed into
suspension using a 1-mm metal sieve. The spleen cells (1  107 cells/ml) were cultured in 10% fetal calf
serum (FCS) containing RPMI 1640 medium without (normal SCM) and with PSBS at concentrations of
12.5, 25, 50, and 100 Ag/ml (PSBS-SCM) at 378C in a fully humidified atmosphere containing 5% CO2.
After 72 hours, the supernatant was collected to serve as splenocyte-conditioned media (SCM), sterilized
by filtration, and stored as 1-ml aliquots at 708C until use.

Preparation of PHA-SCM and lung-conditioned medium (LCM) as positive control

Phytohemagglutinin P (PHA, 10 Ag/ml; Difco Lab., Detroit, MI) was used to prepare SCM (PHA-
SCM) as above described that served as a positive control. Lung-conditioned medium (LCM) was
prepared as previous investigation with modification (Fedorocko et al., 2002). The lung was removed
from the mice, clipped into pieces, and cultured in 10% fetal calf serum (FCS) containing RPMI 1640
medium at 378C incubator. After 5 days, the supernatant was collected to serve as lung-conditioned
medium (LCM), sterilized by filtration, and stored as 1-ml aliquots at 708C until use.

Assay for granulocyte-macrophage (CFU-GM) colony-forming cells

Colony-forming unit-granulocyte/macrophage (CFU-GM) assay using a soft-agar culture method

was performed for analyzing the granulocyte-macrophage colony-forming potential of bone marrow
stem cells as previously described (Wang et al., 1996). Bone marrow cells collected from ICR mice
were resuspended in RPMI 1640 medium supplemented with 10% FCS and cultured for 90 minutes at
378C in a humidified 5% CO2 incubator. Then, discard the adherent cells, and the hematopoietic stem
cells exist in the non-adherent layer. The non-adherent bone marrow cells (1  105 cells/ml) were then
plated in a 1-ml layer of 0.3% agar (Sigma) in McCoys’ 5A medium supplemented with 15% FCS,
essential and non-essential amino acids, vitamin C and sodium pyruvate. Each SCM and PSBS alone,
all at 20% (v/v), was added while plating the cells, with each plate having the same final cell
 H.-F. Liao et al. / Life Sciences 77 (2005) 400–413 403

concentration. After 7 days of incubation, the total number of colonies was counted using an inverted
microscope (Olympus, Melville, NY). The morphology of the colony was determined in situ after
fixation with 5% glutaraldehyde, dehydration with methanol, and staining with HarrisT hematoxylin
(Wang et al., 1992).

Assay for multipotential (CFU-GEMM) and erythroid (BFU-E) colony-forming cells

Colony assay for multipotential (colony-forming unit-granulocyte/erythrocyte/macrophage/ mega-

karyocyte, denoted as CFU-GEMM) and erythroid (burst-forming unit-erythrocyte, denoted as BFU-E)
colony-forming cells were carried out according to procedures previously described (Sun et al., 2003;
Liu et al., 2003b). The non-adherent bone marrow cells (5  104), rich of hematopoietic stem cells, were
plated in 35-mm culture dishes containing a 1 ml mixture of 1% methylcellulose in Iscove’s modified
Dulbecco’s medium (StemCell Technologies, Vancouver, BC, Canada), 30% FCS, 1% bovine serum
albumin, 0.1 mM 2-mercaptoethanol, 2 mM L-glutamine, 1 unit erythropoietin (EPO; Connaught
Laboratories Ltd,Willowdale, Ontario, Canada), and 20% SCM. The dishes were incubated at 378C in a
humidified atmosphere flushed with 5% CO2 for 7 days. Colonies were identified and scored with an
inverted microscope (100X). CFU-GEMM had mixed colonies containing granulocytes (G),
erythrocytes (E), monocyte/macrophages (M) and megakaryocytes (M), while BFU-E had multicentric
(burst) erythroid colonies with a red or pink color.

Assay for high proliferative potential colony-forming cells (HPP-CFC)

ICR mice were single-bolus injected intraperitoneally (i.p.) with 150 mg/kg body weight of 5-
flurouracil (5-FU; F. Hoffmann-La Roche Ltd., Basel, Switzerland) and sacrificed 30 hours later (Liu et
al., 2003a). Only the rested stem cells, named HPP-CFC, survived in bone marrow after mice treated
with high dose 5-FU. Bone marrow cells recovered from the mice were counted and assayed for cell
cycle by flow cytometry to estimate the percentage of cells arrested in G0/G1 phase. After which, 105
cells were plated in agarose cultures with or without an addition of 10% (v/v) mouse lung-conditioned
medium (LCM), similar to the CFU-GM colony-forming assay (Wang et al., 1991a; Timeus et al., 2003).
The proliferative activity of these 5-FU-resistant early stage stem and progenitor cells (HPP-CFC) was
expressed as the number of colonies at day 14.

Measurement of cytokine levels

Enzyme-linked immuno-sorbent assay (ELISA) kits were purchased from R&D Systems (Minneap-
olis, MN) for assay of cytokines. The concentrations of IL-3, 6, 17, SCF, G-CSF and GM-CSF in SCM
collected after 3 days’ incubation with various concentrations of PSBS were measured. Normal SCM
was used as a control.

Total body irradiation and PSBS administration in vivo

To examine the effect of PSBS on mice with radiation-induced myelosuppression, the ICR mice
were divided into four groups (6 mice per group): (1) untreated controls, (2) PSBS alone, (3) total
body irradiation (TBI) alone, (4) TBI plus PSBS administration. For TBI, the entire body was
404 H.-F. Liao et al. / Life Sciences 77 (2005) 400–413

irradiated in a single fraction with 8 Gy (6 MeV photon beam) using a linear accelerator (ClinacR
1800, Varian Associates, Inc., CA, USA, dose rate 2.4 Gy/min) on day 0. Oral administration of PSBS
(400 mg/kg body weight/day) was started 6 hours after TBI and continued from day 0 to 4. In the
control group, mice received normal saline orally. Peripheral blood was collected from the orbital
sinus on days 0, 2, 4, 6, 8, 10, 12, 14 and 16, and leukocytes were counted using a Coulter counter. In
addition, each mouse’s weight was measured every other day during the experimental period. On day
17, the mice were sacrificed and bone marrow cells were removed for an ex vivo CFU-GM colony-
forming assay.

Reconstitution effect of PSBS on myelosuppression induced by 5-FU

5-FU was injected i.p. at a single dose of 150 mg/kg into ICR mice (6 mice per group). Six
hours later, 400 mg/kg body weight/day of PSBS was given orally and continued for 5 consecutive
days. Body weight and number of leukocytes (obtained as described above) were examined every
two days, and bone marrow cells were collected for colony-forming assay when the mice were
sacrificed on day 11.

Statistical analysis

Results are expressed as the mean F standard error (SE) from at least three separate experiments.
Statistical comparisons were performed by using Student’s t-test or one-way analysis of variance
(ANOVA) as indicated. Differences were considered significant at a p b 0.05.

Results

Effect of PSBS on granulocyte-macrophage (CFU-GM) colony formation

As shown in Table 1, PSBS-SCM treatment significantly increased the colony formation of cultured
bone marrow cells as compared with normal SCM. The optimal dose of PSBS to achieve a maximal

Table 1
Effect of PSBS and PSBS-SCM on CFU-GM colony formation of bone marrow cells
Concentration of PSBS (Ag/ml) No. of CFU-GM/105 bone marrow cellsa
PSBS-SCM PSBS alone
0 1F1 1F 1
12.5 20 F 2T 2F 1
25 29 F 4T 4F 2
50 45 F 6T 3F 1
100 41 F 8T 5F 2
Spleen cells (107) were cultured with or without various concentrations of PSBS (0–100 Ag/ml) for 72 hours and the SCM
collected for clonogenic assay. Data from six separate experiments are expressed as mean F SE.
a
The colony-stimulating effects of positive control PHA-SCM and LCM are 29 F 7 and 38 F 6 colonies, respectively.
T P b 0.05, significant difference compared with control group.
 H.-F. Liao et al. / Life Sciences 77 (2005) 400–413 405

effect was 50 Ag/ml, which is much higher than the positive control PHA-SCM and LCM. However,
even at a high dose of 100 Ag/ml, PSBS was not toxic to spleen cells (Data not shown). PSBS alone,
even up to 100 Ag/ml, had no significant promoting activity (p N 0.05). Morphologic analysis of the
colonies induced by PSBS-SCM shows four cell types, including granulocytes, monocytes, macro-
phages, and eosinophils (Fig. 1A–D).

Effect of PSBS on mixed (CFU-GEMM) and erythroid (BFU-E) colony formation

To test the effect of PSBS on the activity of putative pluripotent hematopoietic stem cells (CFU-
GEMM) and early erythroid progenitors (BFU-E), PSBS-SCM was added to methylcellulose cultures
containing erythropoietin (EPO) for GEMM-mixed colonies and erythroid burst formation. PSBS-
SCM augmented EPO to induce CFU-GEMM and BFU-E colony formation in a dose-dependent
manner. PSBS (100 Ag/ml)-SCM increases 5.0-fold and 6.2-fold of CFU-GEMM and BFU-E,
respectively (Fig. 2A). The colony-stimulating results of positive control are 38 F 5 CFU-GEMM
colonies and 19 F 2 BFU-E colonies for PHA-SCM, and 48 F 3 CFU-GEMM colonies and 21 F 3
BFU-E colonies for LCM. Morphologic analysis of CFU-GEMM colonies treated with PSBS-SCM

Fig. 1. Types of CFU-GM colony that formed by PSBS-SCM-treated bone marrow cells. 105 bone marrow cells were culture in
a semi-solid medium with the stimulation of PSBS (50 Ag/ml)-SCM for 7 days, and the colony was determined in situ staining
with Harris’ hematoxylin. (A) Morphology of eosinophil colony; (B) morphology of granulocyte colony; (C) morphology of
granulocyte/macrophage colony; (D) morphology of monocyte and macrophage colony. All colonies were observed under a
microscope (100X).
406 H.-F. Liao et al. / Life Sciences 77 (2005) 400–413

Fig. 2. Effect of PSBS-SCM on multipotential (CFU-GEMM) and erythroid (BFU-E) colony formation. (A) Non-adherent
bone marrow cells were cultured for 7 days in semi-solid mylocellulose in the addition of 20% (v/v) PSBS-SCM and 1 unit
EPO for CFU-GEMM and BFU-E colony-forming activity; (B) morphology of BFU-E; (C) morphology of CFU-GEMM. All
colonies were observed under a microscope (100X). The colony-stimulating effects of positive control are 38 F 5 CFU-
GEMM colonies and 19 F 2 BFU-E colonies for PHA-SCM, and 48 F 3 CFU-GEMM colonies and 21 F 3 BFU-E
colonies for LCM. Data from four separate experiments are expressed as mean F SE. * P b 0.05, significant difference
compared with control group.

showed large-mixed colonies, while BFU-E colonies were red or pink color similar to that of
erythrocytes (Fig. 2B, C).

Effect of PSBS on HPP-CFC colony formation

5-FU is a cell cycle-specific agent sparing only G0 cells. We injected 5-FU into mice to recover a
5-FU-resistant subset of non-cycling cells in the bone marrow, as shown in Fig. 3A (before 5-FU
treatment) and Fig. 3B (after 5-FU treatment). A total of 105 surviving bone marrow cells with a low
percentage in the S phase (HPP-CFC) were recovered, and they had no colony-forming activity in the
presence of LCM or PSBS-SCM alone. However, a combination of PSBS-SCM and LCM activate the
 H.-F. Liao et al. / Life Sciences 77 (2005) 400–413 407

A B

400
G0/G1: 70.4% G0/G1: 93.3%
2000
S: 24.2% S: 4.7%

3200
G2/M: 5.4% G2/M: 2.0%
1500

1600 2400
Number

Number
1000 500

800
0
0

0 50 100 150 200 25 0 50 100 150 200

Channels Channels

C
40
Without LCM *
No. of colonies/105 bone marrow cells

With LCM
*
30
*
*
20

*
10 *
*
0
0 12.5 25 50 100
Concentration of PSBS (µg/ml)

Fig. 3. Effect of PSBS-SCM on HPP-CFC colony formation with or without LCM stimulation. (A) Cell cycle analysis of bone
marrow cells in mice without 5-FU (Control); (B) cell cycle analysis of bone marrow cells in 5-FU-treated mice (HPP-CFC);
(C) 105 HPP-CFC were removed from 5-FU-treated mice and cultured for 14 days in semi-solid agar in the addition of 20% (v/
v) PSBS-SCM with or without 10% (v/v) LCM for colony formation assay. The colony-stimulating effects of positive control
PHA-SCM are 4 F 1 colonies (without LCM) and 20 F 3 colonies (with LCM), respectively. Data from three separate
experiments are expressed as mean F SE. * P b 0.05, significant difference compared with control (without PSBS-SCM
treatment) group by one-way ANOVA.

HPP-CFC and enhances colony generation more than 3-fold (Fig. 3C). The colony-stimulating results
of positive control PHA-SCM are 4 F 1 (without LCM) and 20 F 3 (with LCM) colonies,
respectively.

HGFs in PSBS-SCM

Compared with normal SCM, PSBS-SCM dramatically increased the amounts of the following HGF
after 3 days of stimulation: IL-17 (230.9 pg/ml), GM-CSF (72.4 pg/ml), G-CSF (2260.2 pg/ml), and IL-6
(2126.3 pg/ml) (Fig. 4A and B). However, PSBS-SCM did not significantly increase the levels of SCF
and IL-3.
408 H.-F. Liao et al. / Life Sciences 77 (2005) 400–413

A B
300 IL-17 2500 G-CSF * *
GM-CSF
SCF * IL-6
IL-3
*
Level of cytokines (pg/ml) 250

Level of cytokines (pg/ml)

* * 2000 *
*
200 * * *
1500
150
1000 *
100 *
*
50 500
*
0 0
0 12.5 25 50 100 0 12.5 25 50 100
Concentration of PSBS (µg/ml) Concentration of PSBS (µg/ml)

Fig. 4. Levels of hematopoietic growth factors contained in PSBS-SCM. 107 Spleen cells (107) were cultured with PSBS (0–100
Ag/ml) for 72 hours and the resulting SCM collected for measurement of hematopoietic growth factors by ELISA. (A) Level of
IL-17, GM-CSF and SCF; (B) level of G-CSF, IL-6 and IL-3. Data from six separate experiments are expressed as mean F SE.
* P b 0.05, significant difference compared with control group.

Bone marrow reconstitution effect of PSBS in TBI and 5-FU-treated mice

In these two in vivo models, the baseline leukocyte counts of ICR mice in untreated control and
PSBS-treated groups were similar. Both TBI and 5-FU caused apparent myelosuppression. As
shown in Fig. 5, the number of leukocytes markedly decreased after TBI and 5-FU treatment with a
nadir value about 1.5 F 0.3  103 cells/mm3 and 2.1 F 0.5  103 cells/mm3, respectively (14.7%
and 19.6% of untreated controls). All the leukocyte counts slowly increased thereafter but remained

A B
Control
PSBS Control
14 TBI+PSBS PSBS
14 5-FU+PSBS
TBI
5-FU
12
Leukocyte count (x103/mm3)

12
Leukocyte count (x103/mm3)

10 10

8 * 8
*
6 6 *
4 4

2 2

0 2 4 6 8 10 12 14 16 0 2 4 6 8 10
Day Day

Fig. 5. Effect of PSBS on the leukocytes in irradiated and 5-FU-treated mice. Mice underwent total body irradiation in a single
fraction with 8 Gy or were injected i.p. with a single dose of 5-FU (150 mg/kg body weight). After 6 hours, PSBS, 400 mg/kg
body weight/day, was begun and given orally for 5 consecutive days, and leukocytes were counted every 2 days. (A) Total body
irradiation (TBI)-treated groups; (B) 5-FU-treated groups. Results represent means F SE of six mice. * P b 0.05, significant
difference compared with TBI (A) and 5-FU (B) group by ANOVA with repeated measurement followed by Bonferroni’s test.
 H.-F. Liao et al. / Life Sciences 77 (2005) 400–413 409

A B
# # # #
100 100
No. of CFU-GM / 105 BMCs

No. of CFU-GM/105 BMCs

80 80 *#
*#
60 60
*
40
* 40

20 20

0 0
Control PSBS TBI+PSBS TBI Control PSBS 5-FU+PSBS 5-FU

Fig. 6. Effect of PSBS on CFU-GM colony formation of bone marrow cells in irradiated and 5-FU-treated mice. Mice were
treated with total body irradiation (8 Gy) or 5-FU (150 mg/kg, i.p.), then PSBS (400 mg/kg) was given for 5 consecutive days,
and bone marrow cells were collected for colony-forming assay when the mice were sacrificed on day 11. (A) TBI-treated
groups; (B) 5-FU-treated groups. Results represent means F SE of six mice. * P b 0.05, significant difference compared with
control group; #P b 0.05, significant difference compared with TBI or 5-FU group.

below baseline until the mice were killed. Continuous administration of PSBS for 5 days not only
reduced the degree of leukopenia caused by TBI and 5-FU but also shortened the recovery time of
leukocytes counts. Normal mouse bone marrow cells (1  105) formed 97 F 5 granulocyte-
macrophage (GM) colonies (Fig. 6). After TBI and 5-FU, the CFU-GM numbers were greatly
diminished (33 F 6 and 41 F 5 colonies, respectively). Administration of PSBS reconstituted
myelopoiesis, with the number of CFU-GM increasing about 2.0-fold in TBI-treated and 1.8-fold 5-
FU-treated mice, as compared with the TBI and 5-FU alone group. None of the PSBS-treated mice
died, nor did their body weights change significantly (p N 0.05) during the experimental period
(Fig. 7).

A B
55 55
Control Control
PSBS PSBS
50 TBI+PSBS 50 5-FU+PSBS
TBI 5-FU
45 45
Body weight (g)

Body weight (g)

40 40

35 35

30 30

25 25

20 20
0 2 4 6 8 10 12 14 16 0 2 4 6 8 10
Day Day

Fig. 7. Effect of PSBS on change in body weight in irradiated and 5-FU-treated mice. The mice were irradiated with 8 Gy or
injected with 5-FU (150 mg/kg); 6 hours later, 400 mg/kg PSBS was given orally for 5 consecutive days and body weight was
checked every 2 days. (A) TBI-treated groups; (B) 5-FU-treated groups. Results represent means F SE of six mice.
410 H.-F. Liao et al. / Life Sciences 77 (2005) 400–413

Discussion

In the present study, we found that PSBS, a novel polysaccharide purified from black-soybean,
promotes myelopoiesis in vitro, stimulates secretion of various hematopoietic growth factors from spleen
cells, and reconstitutes the bone marrow in a myelosuppressed animal model. We also compared the
myelopoietic promotion effect of PSBS with positive control PHA-SCM and LCM. PHA is a kind of
lectin which stimulates splenic mononuclear cells to secrete cytokines including several hematopoietic
growth factors (Mecucci et al., 1986). LCM is the source of colony stimulating factors, especially GM-
CSF, that stimulate myelopoiesis of stem cells (Fedorocko et al., 2002). Our results demonstrated that
PSBS promotes CFU-GM, CFU-GEMM, BFU-E and HPP-CFC colony formation through an indirect
effect of hematopoietic growth factors production.
There are two pathways by which a natural product can promote hematopoiesis. The first is direct
stimulation of proliferation and differentiation of myeloid progenitor cells, an effect that can be
monitored by assessing CFU-GM (Wang et al., 1991b). The second pathway is stimulation of HGF-
producing cells, including T-lymphocytes, macrophages, fibroblasts, and endothelial cells, to secrete
HGF (Chen et al., 1993). Our results demonstrate that PSBS promotes myelopoiesis primarily by
stimulation of HGF production rather than the stimulation of progenitor cell proliferation. In addition,
PSBS-SCM induced maturation of a broad spectrum of blood cells including CFU-GM, CFU-GEMM,
BFU-E and HPP-CFC.
HGF content in PSBS-SCM included two groups of molecules: stimulating factors and synergizing
factors. We found that PSBS stimulated the secretion of IL-6, IL-17, G-CSF and GM-CSF. This indicates
that both stimulating and synergizing effects are involved in the PSBS-induced myelopoiesis. IL-3, G-,
M-and GM-CSF are mainly hematoopoietic growth factors that stimulate myeloid colony formation
directly. IL-6 and SCF directly support proliferation and differentiation of only a small number of murine
hematopoietic cells (Varnum-Finney et al., 2003; Wadhwa et al., 2003), but they act synergistically with
G-, M-and GM-CSF to enhance the total number of colonies (Muench et al., 1992; Caracciolo et al.,
1989). IL-17, a novel cytokine produced by activated ahTCR+CD4 CD8 T cells, induces stromal cells
to secrete cytokines involved in pro-inflammation and hematopoiesis (Schwarzenberger and Kolls,
2002). Several articles indicated that IL-17 increased granulopoiesis in leukocyte adhesion molecule-
deficient mice (Forlow et al., 2001) and regulated CFU-GM and BFU-E progenitors in normal and post-
irradiated murine bone marrow (Jovcic et al., 2001). The signaling mechanism of IL-17 involves
tyrosine phosphorylation of several members of the JAK and STAT proteins (Subramaniam et al., 1999).
Furthermore, Meth-A cells transfected with the hIL-17 gene can induce tumor-specific antitumor
immunity by augmenting the expression of MHC class I and II antigens (Hirahara et al., 2001). We are
going to test each mAb against IL-17, IL-6, G-CSF and GM-CSF, and various mAb combinations to
clarify which HGF is predominant.
The reason PSBS failed to stimulate the production of SCF and IL-3 remains to be elucidated. One
possibility is the lack of receptors for PSBS on cells that produce SCF or IL-3. Another possibility is the
presence of PSBS-induced inhibitors of SCF and IL-3 production. Normal cells, such as mononuclear
cells, CD34+ cells, myeloblasts, etc, secrete numerous growth factors, cytokines and chemokines that
form the basis of intercellular cross-talk networks and regulate the various stages of hematopoiesis
(Majka et al., 2001). Our study showed that HGF contained in PSBS-SCM acted with EPO and LCM to
form BFU-E and HPP-CFC colonies. This suggests that myelopoiesis is in fact a complex process
involving a variety of regulatory molecules, at least some of which are induced by PSBS.
 H.-F. Liao et al. / Life Sciences 77 (2005) 400–413 411

Several clinical trials have shown that injection of G-CSF or GM-CSF significantly increases the
number of neutrophils and monocytes in patients with hematopoietic hypofunction, such as that seen in
aplastic anemia (Ginopoulos et al., 2002), idiopathic neutropenia (Sultana et al., 2003), and especially in
cancer patients following treatment with cytotoxic drugs (Sanchez et al., 2002; Cesaro et al., 2003). The
fact that PSBS strongly promotes myelopoiesis but has low cytotoxicity makes PSBS it an attractive
candidate for clinical testing, especially in cancer patients who often develop severe neutropenia after
high-dose chemotherapy (Kimura and Okuda, 1999; Varveris et al., 2003) or radiotherapy (Calderoni et
al., 2002). PSBS may also potentiate G-CSF-induced mobilization of stem cells and thus be useful in
peripheral blood stem cell transplantation. The promotion of myelopoiesis by PSBS is quite different
from that of recombinant G-CSF. PSBS activates immunocompetent cells to secrete endogenous HGF.
G-CSF is only one arm of the HGF system, but PSBS appears to stimulate the entire system.
Additionally, while recombinant G-CSF is associated with adverse effects, including muscle and bone
pain, headache, fever, fatigue and nausea, PSBS is much less toxic.
PSBS thus is a natural product that with high safety and stability that promotes myelopoiesis both in
vitro and in vivo (in an animal model). We are engaged in a further in vivo animal study using a tumor
implantation model to observe the effects of PSBS on inhibiting tumor growth and promoting
myelopoiesis after radiation-and chemotherapy-induced bone marrow suppression.

Acknowledgments

This works was supported by a research grant NSC 92-2311-B-195-002 from the National Science
Council of the Republic of China and a grant VGH 377-1 from the Veterans General Hospital, Taipei,
Taiwan. The authors would like to thank Prof. Sheng-Yuan Wang (Department of Medical Research and
Education, Veterans General Hospital, Taipei, Taiwan) for helpful advice and critical revisal of the
manuscript.

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