0510:16p

I",..r"uliv"al Jmlrnal Mushrooms. Vol pp. 20"

The Antitumor Activity of Ganoderma

lucidum (Curt.:Fr.)P. Karst. (Ling Zhi)
(Aphyllophoromycetideae) Polysaccharides Is
Related to Tumor Necrosis Factor-a and Interferon-'Y
Dun-Hao Zhang and Zhi-Bin Un *
Department of Pharmacology, School of Basic Medical Sciences. Beijing Medical University, Beijing
100083, China

Iln all C(H-re"pUildellce should be addrcs!lCd

ABSTRACT: In the presentstudy. the antitumor activity I)f GL-B. a polysacchande isolated from Ganodenn"
/ucidum (Cun:Fr.)P. Karst. and its mechanism were stUdied in l'il'O and in vitro. The results were RSfollows:
( I) GL-B 50. 100. 200 I1g ml-1 inhibited the growth of implanted Sarcomu 180 in viJ..osignificantly and do.-e
dcpcndently. (2) GL-B diret:tly udded to the culture medium neither induced HL-6Q apoptoslSnor restr.sinedits
proliferation ill vitro. (3) The macrophageor T lymphocyte culture medium treated with GL-B (GL-B-M-CM
or GL-B- T-CM) 50, 100, and 2()()I1g ml-1 !iignificantly induced HL-60 apoptosis and inhibited it!i proliferdtion.
GL-B significantly increasedtumor necrosisfactor-a ( TNF-a) and interferon-)' (IFN-y) releasein dose-dependent
and lime-dependentinstance-'i.(4) As unu'eatedmacrophage!;and T Iymphl>cytesproduced little or no 'rNF-a and
IFN-y. and mlICrophngeculture medium with normal saline (N-M-CM) or T lymphocyte culture medium with nor-
mal saline (N- T-CM) did nllt inhibit HL-60 proliferation or induce its apoptosis, it seemedthat the antitumor ac-
tivity of GL-B was related to apoptosis induced by TNF-a-relea!ie from macrophages and IFN-y-releilse frOlI1
T lymphocytes.

KEY WORDS: Apoplosis. Ganodenn(llucidum. HL-60. interferon-yomacrophage.polysaccharides.proliferilii

Sarcoma 180. T lymphocyte. tumor necrosisfactor-a.

Ganoderma lucidum (Curt.: Fr.)P. Karst. Many investigators have demonstrated that the
(Ling Zhi) has beenwidely usedas a medicine to polysaccharides isolated from Ganoderma /u-
promote health and longevity in China for thou- cidum cou]d significantly inllibit the growth of'
sandsof years.A seriesof experimentsin our lab- implanted Sarcoma 180 and Lewis lung-carci-
oratory have demonstrated that G. lucidum has noma in animal models in vitro (Sasaki, 1971;
various pharmacological effects, such as im- Miyazaki and Nishiyama, ]981; Sone and Okuda.
munomodulatingones(Xia and Lin, .1989;Ma et ]985; Maziyama et al., 1989). Although its anti-
al.. 1991; Lei and Lin, 199.1.1992). Recently. G. tumor activity is beyond doubt, the mechanisms
lucidum hasattracted great interestdue to the an- remain unclear. Some scholars have suggested
titumor activity of its polysaccharide fraction. that its antitumor activity may be mediated to

ABBREVIATIONS
Con A: concanavalinA: CY: cyclophosphamide:ELISA: enzyme-linked immunoabsorbentassay;
FCS: fetal calf serum; IFN-y. interferon-y: LPS: lipopolysaccharide; NBS: newborn bovine serum:
PHS: phosphate-bufferedsaline: PEC: peritoneal exudatecells: PI: propidinmiodide; TNF-a: tumor
necrosisfactor-a.

1521-9437/991$5.00
@ 1999 by Begell House. In.: 207

Medicirwl
Jan 16 05 10:19p p.3

some extent by the activation of host immune were harvested by peritoneal lavage using cold
functions. However, such a hypothesis requires Hank's solution containing 5% fetal calf serum
substantialevidence. In thi~ article. we describe (FCS). PECs were washed twice and resuspended
our investigation of the antitumor activity of in an RPMI 1640 medium containing 10% FC'S.
GL-B, a mixture of partially purified polysaccha- Peritoneal macrophages were further isolated
rides isolated from G. Lucidum.at a cellu]ar and from the PECs by incubating the PECs in a 24-wel.l
molecular level both in vn'o and in vitro. plate at 37°C in a humidified atmo!.'Pherefor 2--4
h to allow for peritoneal macrophage adherence.
After this time, the nonadherent cells were re-
MATERIALS AND METHODS
moved by washing three times with waTnl RPMJ
Animals 1640 medium. More than 95% of the adherent ce.ll
population was macrophages, as determined by
Inbred male and female 1-2-month-old (body morphology and esterase staining.
weight 18-25g) BALB/c mice were purchased
from the Departmentof Experimental Animals.
Beijing Medical University. Conditioned Media

To investigate the effect of GL-B on stimulat-

Drug ing cytokine production by T lymphocytes and
macrophages, and the effect ofGL-B conditioned
Ganoderma [u(.'idum polysaccharide (GL-B) media with T lymphocytes and macrophage", on
consists of seven fractions of polysaccharide iso-
pruliferdtion and apoptosis of tumor cells, a pure
lated from G, lucidllm. It is a yellowish and water-
popu lation of macrophage.~or T Iymphocytes was
soluble powder with molecular weights of 7,000
incubated separately in an RPMI 1640 medium
to 9,000 provided by the Departmentof Phyto- containing 10% NBS with or without various
chemistry, College of Phannacy, Beijing Medical
concentrations of GL-B at 37°C tor 12-72. The
University (Li and He, 1991).
conditioned media, which were caJ]ed macro-
phage culture medium with GL-B (GL-B-
Cell Lines M-CM) and T lymphocyte culture medium with
GL-B (GL-B-T-CM), were then collected, fil-
HL-60 and Sarcoma 180were obtained from tered, and stored at -70°C, respectively, for use.
Beijing Tumor I.nsritute.L929 cells were provided
by the Departmenlof Immunology, Beijing Med- Assay of Cytokines
ical University. RPMI. 1640 powder was from
Gibco BRL. MTT, lipopolysaccharide(LPS), alld Conditioned media from GL-B stimulated
concanavalinA (Con A) were from Sigma. macrophagesor T lymphocytes were assayedfor
activity of tumor necrosis factor (TNF-a) and in-
Preparation of T Lymphocytes and terferon-y(IFN-y). TNF-a was a.~sayedby bioas-
Peritoneal Macrophages ~y methodsusingL929 cells and IFN-y by solid-
phase enzyme-linked immunoabsorbent assay
Mice were killed and the spleens were chopped (ELISA) a...described by the available kit.
with two ~Iides and filtered over a fine nylon mesh.
Cells were washed three times in Hank's balanced Treatment of HL-60 Cells
salt solutjon containing 5% heat-inacrivated new-
born bovine serum (NBS). Cells were finally HL-60 I.;ells,maintained in an RPMI 1640
suspended in RPMI-I640 supplemented with mediumcontaining 10%FCS. werecultured at all
100 IV ml-1 penicillin, 100~g.ml-1 streptomycin, initiaJ concentration of Ixl05.mI-1 in the pres~
1 mmol.I-J sodium pyruvate, 2 mmol.I-1 L-gluta- ence or absenceof 20% (voVvol) of GL-B stimu-
mine, 3.4 x 10-3 mmol.I-1 2-mercaptoethanol, and lating conditioned media or normal media. For
10% NBS. Rat peritoneal exudate cells (PECs) detecting the direct effect of GL-B on HL-60

208
Jan 16 05 10:23p p.4

growth. the culture was treated with GL-B at dif- Statistical Analysis
ferent concentrations. Cultures were then incu-
bated at 37°C in a humidified atmospherefor 3 Results were expressed as X ::!::SD and ana-
days. lyzed by I-test to compare the difference between
the groups with the Statistica software for Win-
dows@ (4.5. Statsoft Inc., 1993).
Antitumor Experiment in
Tumor-Bearing Mice

Sarcoma 180cells were injected subderrnal.ly RESULTS

into the axillary fossa of the right foreleg. The
mice were divided into severalgroups randomly.
Different doses of drugs were administered by Antitumor Effect of GL-B on Locally
stomachtube.oncea day.The mice were killed 10 Implanted Sarcoma 180 In Vivo
days later and the tumors were cut and weighed.
Table I shows the antitumor effect of GL-B
on Sarcoma 180 in BALB/c mice in vivo. GL-B
Analysis of Apoptosis 50, 100,and 200.mg kg-l inhibited the growth of
Sarcoma 180 in a dose-dependentinstance.The
HL-60 cells were separated from the medium inhibitory ratesare 27.70%, 55.83%,and 66.70%,
by centrifugation and washed with phosphate- respectively. The inhibitory rate of the GL-B
buffered saline (PBS) after incubation. Hy- group treated with cyclopho!iphamide(CY) was
podiploid DNA was analyzed using the method of higher than that of the GL-B or the CY group.
propidiumiodide (PI) labeling and flow cytome- Their inhibitory rateswere 80.98%,45.45%,and
try. After washing, samples were resuspended and 75.95%, respectively(Table 2).
placed in the dark at 4"C. PI fluorescence of indi-
vidual nuclei was analyzed using a FACScan flow
cytometer (Becton Dickinson). Effect of GL-B on Proliferation of HL-60
and Sarcoma 180 Cells In Vitro
Determination of Cells Proliferation
On the basis of the above results, we further
MTT (20 1.11.5 mg I-I every well) was added added GL-B directly to the in vitro HL-60 and
to the cell culture 4 h before the end of incuba- Sarcoma 180 culture media. We unexpectedly
tion. After incubation,gave away supernatant,and found that GL-B had no effect on proliferation of
150 III of isopropanol was added and OD was either HL-60 or Sarcoma 180cells (Tables 3 and
assessed using the Enzyme Labeling lru;trument 4). Table 3 shows that GL-B 400 ~g'ml-J slightly
(Bio-Rad). stimulated HL-60 cell proliferation.

TABLE 1
Antitumor Effect of GL-B on Sarcoma 180 in BALB/c Mice (n = 10, x:l: SO)
Dose Tumor weight Inhibitory rate
Groups (mgokg--1) (mg) (%)

23.61 :t 1.40 10.32 :!:2.60: 2.02:t 0.16

24.05:t 1.75 7.57:! 1.82" 1.46:t 0.61 27.70
23.90 r 1.62 6.33j :1.77"": 0.89 :t 0.45". 55.83
23.52:t 1.48 6.46 j 1.46".: 0.67 :t 0.47"" 66.70
23.84:t 1.43 5.84:1 1.68"" 0.44 j; 0.54"" 78.02

209
Jan 16 05 10:26p p.5

TABLE 2
Antitumor Effect of GL-B on Sarcoma 180 in BALB/c Mice (n = 10,.i:t. SD)

Dose Body weight (g) Tumor weight Inhibitory rate

Groups (mgokg-1) Origin difference (mg) (%)

NS -23.14:t 1.62 9.58 :t 2.11 1.87:t 0.78

GL-B 100 22.79:t 1.41 7.22 :t 1.73 1.02 t 0.51.M 45.45
CY 20 22.87 ;t 1.55 6.65 :t 1.42 0.45:t O.48*'d 75.95
Gl-B +CY 100+20 23.35:!: 1.34 6.70:1: 1.36 0.36:!: 0.17** 80.98
.p<O.05vs. N$; ..p< 0.01 va. NS
Ap < 0.05 va. OB + CY; Mp < 0.01 vs. OL-B + CY

TABLE 3 Effect of GL-B on Inducing HL-60 Cells

Effect of Gl-B on Proliferation of Hl-60 Cells Apoptosls In Vitro
In Vitro (n = 8, x :I: SD)

Concentration
Table 5 indicatesGL-B 50. .100.200 ~g mil
Groups (j1g'ml-1) 00570 nm cannot induce HL-60 cell apoptosis;the propor-
tions of apoptic cells were 0.87 :t 0.24%. 0.65 :t
APMI 1640 -0.346 :t 0.041 0.31 %, 0.63 :t 0.45%, respectively.The positive
GL-B 50 0.384:t 0.137 control drug Etopeside Vp-16 significantly in-
100 0.402 j: 0.44
ducedHL-60 cell apoptosis;theapoplic cells wa1'
200 0.418:t 0.063
400 0.465:t 0.139' 67.70 :t 4.04%.
Vp-16
- 80 0.241 j: 0.067"'
'p< 0.05 Ys. RPMI 1640; "p< 0.01 VS. RPMI1640

Effects of GL-B-M-CM and GL-B-T-CM on

TABLE 4 Proliferation of HL-60 Cells In Vitro
Effect of GL-B on Proliferation of Sarcoma
180 Cells In Vitro (n = 8, x f: SD) Tables 6 and 8 show that every concentration
of GL-B-M-CM and GL-B- T-CM significantly
Concentration
Groups (J1Q"mt-1) CD 570 nm inhibited proliferation of HL-60 cells in "ilro.
Compared with RPMI 1640and N-M-CM or N-
RPMI1640 - 0.369 j: 0.108 T-CM groups,p < 0.05 orO.OI.
GL.B 50 0.337 j: 0.045
100 0.405 :t 0.0520.0570.093
200 0.411 :t
400 0.442 :t
y~"--~ 0.244 :t 0.037 Effect of GL-B-M-CMand GL-B-T-CM on
'p< 0.01 VS. RPMt 1640. Inducing Apoptosis of HL-60 Cells In Vitro

TABLE 5 Table 8 shows that50. 100. and 200 Jlg.ml-1

Effect of GL-B on Apoptosis of HL-60 Cells of GL-B-M-CM significantly induced HL-60
After 72 h of In Vitro Incubation~n = 3, x:t: SD) cells apoptosis;the proportions of apt>ptoticcells
were 18.81 :i: 0.93%. 20.98 :i: 1.57%. 23.00 :i:
Concentration Apoptotic cells 0.56%. respectively.comparedwith the N-M-CM
Groups (~g.ml-1) (%) group. 7.44:1:1.07%. P<O.Ol.
RPMI1640 1.94:t 0.41
Table 9 shows that 50, ]00. and 200 Jlg.ml-J
GL-B 0.87 t 0.24 of GL-B- T-CM significantly induced HL-60 cell
0.65 :t 0.31 apoptosis;the proportions of apoptotic cells were
0.63 :t 0.45 19.39:t 1.13%..2i,94:t0.84%, 22.85:t 1.49%,re-
67.70:t 4.04'. spectively, compared with the N- T-CM group,
7.75 :t 1.14%,p<0.OI.

0!10
 lO:29p p.6

TABLE 6 TABLE 7
Effect of GL-B-M-CM on Proliferation of Effect of GL-B- T-CM on Proliferation of
HL-60 Cells In Vitro (n = 8, x:t SO) HL~60 Cells In Vitro (n = 8, x:I: SO)

Concentration Concentration
Groups mgoml-1 OD 570nm Groups mgoml-1 OD 570 nm
RPMI1640 RPMI1640 0.604 j; 0.130
N-M-CM N- T-CM - 0.577:t 0.141
GL-B-M-CM GL-B- T-CM 50 0.476:t 0.123',1.
100 0.439:t 0.162'M
200 0.266 i 0.1 02" ~
Vp-16 80 0.208:t 0.116" ~

'p< 0.05 YS RPMI1640; '.p<0.01 YS. RPMI1640:

4p < 0.05 Y5. N-S-CM; Mp < 0.01 YS. N-S-CM

TABLE 8 TABLE 9
Effect of GL-B-M-CM on Apoptosis of HL.60 Effect of GL-B-T-CM on Apoptosis of HL-60
Cells In Vitro (n = 3, x:t SD) Cells In Vitro (n = 3, x::I: SO)
Concentration Apoptotlc cells Concentration Apoptotic cells
Groups mg-ml-1 (%) Groups mg'ml-1 (%)
RPMI1640 -1.29:!: 0.46 RPMI1640
N-M-CM -7.44:t 1.07- N.T-CM
GL-B-M-CM 50 18.81 :t 0.93"-d GL-B-T-CM
100 20.98 :t 1.57"" M
200 23.00 :t 0.56-- M
yp-16- ~ 71.45:1:2.43"",1A
"p<O.05 YS. RPMI164Q;""p<0.O1 Y5. RPMI1640;
Ap < 0.05 Y5. N-M-CM; 6Ap < 0.01 Ys. N-M-CM.

FIGURE 1. Effect of GL-B on apoptosis of HL-60 cells FIGURE 2. Effect of Gl-B-M-CM on apoptosis of
incubated for 72 h in vitro. Hl-60 cells incubatedfor 72 h in vitro-
(A) Control: (8) GL-B 50 ~g.ml-1: (C) GL-B 100 (A) Control; (8) Gl-B 50 ~g-ml-l; (C) Gl-B 100
~g.mr 1; (D) GL-B 200 ~g.mt1; (E) Vp-16 80 ~g.ml-l. ~g-ml-1; (0) Gl-B 200 1J9-ml-1;(E) Vp-16 80 J.19-m.l.

;lll
 16 05 10:31p p.?

level during 24-48 h, and subsidedafter 72 h. The

TNF-a level in the supernatant of 12.5-400
Jlg.ml-1 GL-B cultured with macrophagesrosedur-
ing 24 h as the dose increased(Fig. 5).

Effect of GL-B on IFN-y Production

of T Lymphocytes

Figure 6 shows that the IFN-y level in the su-

pernatantof 200 ~g'ml-1 GL-B cultured with T
lymphocytesrose in 24 h and subsidedafter 48 h.
The IFN-y level in the supernatantof 12.5-2()()
~g.mI-1 GL-B cultured with T lymphocytes rose
during 24 h as the dose was increased to 400
~g'mi-i. At that level it started to subside(Fig. 7).

FIGURE 3. Effect of GL-B- T-CM on apoptosis of

HL-60 cells incubated for 72 h in vitro. DISCUSSION
(A) Control; (B) GL-B 50 J.1g.ml-l; (C) GL-B 100
~g.ml-1; (0) GL-B 200 J.1g.ml-1;(E) Vp-16 80 J.1g.ml-'.
In the presentstudy,we investigatedthe antitu-
mor activity of GL-B, a purified polysaccharide
isolated from Ganodenna lucidum. The results
Effect of GL-B on Macrophage showed thal GL-B inhibited the growth of im-
TNF-a Production planted Sarcoma 180 significantly and in a dose-
dependent, combination immunotherapy, GL-B
f'lgure 4 shows that the TNF-a level in the su- with a chemotherapysuch as cyclophosphamide
pernalant of 200 ~g'mJ-1 GL-B cultured with achieved the best effect. On the ba is of the pre-
macrophages rose in 24 h, remained at the highest ceding in vivo results, we added GL-B directly to

u zu 40 60 ISU
Time (hour)

t'IGURE: 4. E:ftect of 200 j.lg'ml-1 GL-B on TNF-a induction in the super-

natant of murine peritoneal macrophages cultured with lPS after 3-72 h of
incubation (n = 4, x:t SD).

~l~

Jan
JAn 16 05 10:34p p.B

FIGURE 5. Effect of different doses of GL-B on TNF-a induction in the super-

natant of murine peritoneal macrophages cultured with LP5 after 24 h of incuba-
tion (n = 4. x:!: SO).

the in vitro cultures of Sarcoma] 80 and HL-6() using a FACScan flow cytometer. It :\eems (hat
cells. Unexpected results showed that directly GL-B alone hasno direct effect on proliferation of
adding GL-B to the in vitro tumor cell culturescan- tumor cells.
not inhibit their proliferation. evenat the very high In a further study,GL-B was first addedto the
concentrationof 400 Jlg.ml-l. GL-B also hasno ef- culture medium of macrophagesand T lymph()-
fect on inducing tumor cell apoptosismeasuredby cytes, then the conditioned GL-B-M-CM and GL-

-
80

FIGURE 6. Effect of 200 Jig.ml-1 GL-B in IFN.'YInOuCtlonin the supernatant or

murine spleen cells cultured with ConA after 3-72 h of incubation.

;ll~
 lO:37p p.9

B- T-CM media were added to the in vitro H.l.-60 growth of implanted Sarcoma 180 ill vi~'o signifi-
culture medium as a drug. The results showed that cantly and dose-dependently. Second. GL-B di-
both GL-B-M-CM and GL-B-T-CM significantly rectly added to a culture medium neither induced
inhibited proliferation of HL-60; the FACScan HL-60 apoptosis nor restrained its proliferation in
flow cytometer also found that both of them sig- ~'itro.Third. the macrophage or T lymphocyte cul-
nificantly induced HL-6() cell apoptosis. ture medium treated with GL-B significantly in-
To reveal further the mechanism of the antitu- duced HL-60 apoptosis and inhibited its prolifer-
mor effect of GL-B-M-CM and GL-B- T-CM. the ation. GL-B significantly increased TNF-(X and
leve.JofTNF-a in GL-B-M-CM and .lFN-yin GL- IFN-y release dose- and time-dependently. TNF-
B- T -CM were ac;sayedby bioassay and ELISA. re- (Xand IFN-y were known to play imp<)rtant roles
spective.Jy.TNF-a and IFN-y were known to play in suppressing tumor cell growth and inducing
important roles in suppressing tumor cell growth apoptosis of rumor ceJIs. Finally. as untreated
and inducing apoptosis of many different kinds of macrophages aJ1dT lymphocytes produced little
tumor cells (Thomson, 1991; Malorni and or no TNF-(X and IPN-y. and N-M-CM or N- T-CM
Rainaldi, 1996). Many experiments showed that did not inhibit HL-60 proliferation or induce its
TNF-a and lFN-y cooperated with each other in apoptosis, it seemed that the antitumor activity of
inducing tumor cell apoptosis (Volm and Mattern, GL-B was related to promoting TNF-(X release
1995; Rawadi et aJ.. 1996: Sveinbjinsson and from macrophages and IFN -y release from T lym-
Rushteldt; 1997). Our experimental results phocytes.
showed that the levels ofTNF-a in GL-B-M-CM
and IFN-y in GL-B-T-CM rose significantly in a
do5e-dependent mode. Moreover. our l"e!;ults
showed that there was a positive correlation be-
tween the level of TNF-a in GL-B-M-CM and
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215