0510:16p pp. 20" Medicirwl Mushrooms. Vol I",..r"uliv"al Jmlrnal 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 RS follows: ( I) GL-B 50. 100. 200 I1g ml-1 inhibited the growth of implanted Sarcomu 180 in viJ..o significantly and do.-e dcpcndently. (2) GL-B diret:tly udded to the culture medium neither induced HL-6Q apoptoslS nor restr.sined its 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 increased tumor necrosisfactor-a ( TNF-a) and interferon-)' (IFN-y) releasein dose-dependent and lime-dependent instance-'i. (4) As unu'eated macrophage!; and T Iymphl>cytes produced little or no 'rNF-a and IFN-y. and mlICrophnge culture 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 seemed that 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-yo macrophage. polysaccharides. proliferilii Sarcoma 180. T lymphocyte. tumor necrosisfactor-a. Many investigators have demonstrated that the polysaccharides isolated from Ganoderma /u- cidum cou]d significantly inllibit the growth of' implanted Sarcoma 180 and Lewis lung-carci- noma in animal models in vitro (Sasaki, 1971; Miyazaki and Nishiyama, ]981; Sone and Okuda. ]985; Maziyama et al., 1989). Although its anti- tumor activity is beyond doubt, the mechanisms remain unclear. Some scholars have suggested that its antitumor activity may be mediated to Ganoderma lucidum (Curt.: Fr.)P. Karst. (Ling Zhi) has beenwidely usedas a medicine to promote health and longevity in China for thou- sands of years.A series of experimentsin our lab- oratory have demonstratedthat G. lucidum has various pharmacological effects, such as im- munomodulating ones(Xia and Lin, .1989; Ma et al.. 1991; Lei and Lin, 199.1. 1992). Recently. G. lucidum hasattracted great interest due to the an- titumor activity of its polysaccharide fraction. ABBREVIATIONS Con A: concanavalin A: CY: cyclophosphamide: ELISA: enzyme-linked immunoabsorbent assay; FCS: fetal calf serum; IFN-y. interferon-y: LPS: lipopolysaccharide; NBS: newborn bovine serum: PHS: phosphate-buffered saline: PEC: peritoneal exudatecells: PI: propidinmiodide; TNF-a: tumor necrosis factor-a. 207 1521-9437/991$5.00 @ 1999 by Begell House. In.:
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0510:16p
pp. 20"
Medicirwl
Mushrooms. VolI",..r"uliv"al Jmlrnal
The Antitumor Activity of Ganodermalucidum (Curt.:Fr.)P. Karst. (Ling Zhi)(Aphyllophoromycetideae) Polysaccharides IsRelated 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 present study. 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 RS follows:( I) GL-B 50. 100. 200 I1g ml-1 inhibited the growth of implanted Sarcomu 180 in viJ..o significantly and do.-edcpcndently. (2) GL-B diret:tly udded to the culture medium neither induced HL-6Q apoptoslS nor restr.sined itsproliferation ill vitro. (3) The macrophage or T lymphocyte culture medium treated with GL-B (GL-B-M-CMor GL-B- T-CM) 50, 100, and 2()() I1g ml-1 !iignificantly induced HL-60 apoptosis and inhibited it!i proliferdtion.GL-B significantly increased tumor necrosis factor-a ( TNF-a) and interferon-)' (IFN-y) release in dose-dependentand lime-dependent instance-'i. (4) As unu'eated macrophage!; and T Iymphl>cytes produced little or no 'rNF-a andIFN-y. and mlICrophnge culture 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 seemed that the antitumor ac-tivity of GL-B was related to apoptosis induced by TNF-a-relea!ie from macrophages and IFN-y-releilse frOlI1
Many investigators have demonstrated that thepolysaccharides isolated from Ganoderma /u-cidum cou]d significantly inllibit the growth of'implanted Sarcoma 180 and Lewis lung-carci-noma in animal models in vitro (Sasaki, 1971;Miyazaki and Nishiyama, ] 981; Sone and Okuda.]985; Maziyama et al., 1989). Although its anti-tumor activity is beyond doubt, the mechanismsremain unclear. Some scholars have suggestedthat its antitumor activity may be mediated to
Ganoderma lucidum (Curt.: Fr.)P. Karst.(Ling Zhi) has been widely used as a medicine topromote health and longevity in China for thou-sands of years. A series of experiments in our lab-oratory have demonstrated that G. lucidum hasvarious pharmacological effects, such as im-munomodulating ones (Xia and Lin, .1989; Ma etal.. 1991; Lei and Lin, 199.1. 1992). Recently. G.lucidum has attracted great interest due to the an-titumor activity of its polysaccharide fraction.
some extent by the activation of host immunefunctions. However, such a hypothesis requiressubstantial evidence. In thi~ article. we describeour investigation of the antitumor activity ofGL-B, a mixture of partially purified polysaccha-rides isolated from G. Lucidum. at a cellu]ar andmolecular level both in vn'o and in vitro.
were harvested by peritoneal lavage using coldHank's solution containing 5% fetal calf serum(FCS). PECs were washed twice and resuspendedin an RPMI 1640 medium containing 10% FC'S.Peritoneal macrophages were further isolatedfrom the PECs by incubating the PECs in a 24-wel.lplate at 37°C in a humidified atmo!.'Phere for 2--4h to allow for peritoneal macrophage adherence.After this time, the nonadherent cells were re-moved by washing three times with waTnl RPMJ1640 medium. More than 95% of the adherent ce.llpopulation was macrophages, as determined bymorphology and esterase staining.
MATERIALS AND METHODS
Animals
Inbred male and female 1-2-month-old (bodyweight 18-25g) BALB/c mice were purchasedfrom the Department of Experimental Animals.Beijing Medical University. Conditioned Media
To investigate the effect of GL-B on stimulat-ing cytokine production by T lymphocytes andmacrophages, and the effect ofGL-B conditionedmedia with T lymphocytes and macrophage", onpruliferdtion and apoptosis of tumor cells, a purepopu lation of macrophage.~ or T I ymphocytes wasincubated separately in an RPMI 1640 mediumcontaining 10% NBS with or without variousconcentrations of GL-B at 37°C tor 12-72. Theconditioned media, which were caJ]ed macro-phage culture medium with GL-B (GL-B-M-CM) and T lymphocyte culture medium withGL-B (GL-B-T-CM), were then collected, fil-tered, and stored at -70°C, respectively, for use.
Drug
Ganoderma [u(.'idum polysaccharide (GL-B)consists of seven fractions of polysaccharide iso-lated from G, lucidllm. It is a yellowish and water-soluble powder with molecular weights of 7,000to 9,000 provided by the Department of Phyto-chemistry, College of Phannacy, Beijing MedicalUniversity (Li and He, 1991).
Cell Lines
HL-60 and Sarcoma 180 were obtained fromBeijing Tumor I.nsritute. L929 cells were providedby the Departmenl of Immunology, Beijing Med-ical University. RPMI. 1640 powder was fromGibco BRL. MTT, lipopolysaccharide (LPS), alldconcanavalin A (Con A) were from Sigma.
Assay of Cytokines
Conditioned media from GL-B stimulatedmacrophages or T lymphocytes were assayed foractivity of tumor necrosis factor (TNF-a) and in-terferon-y (IFN-y). TNF-a was a.~sayed by bioas-~y methods using L929 cells and IFN-y by solid-phase enzyme-linked immunoabsorbent assay(ELISA) a... described by the available kit.
Preparation of T Lymphocytes andPeritoneal Macrophages
Mice were killed and the spleens were choppedwith two ~Iides and filtered over a fine nylon mesh.Cells were washed three times in Hank's balancedsalt solutjon containing 5% heat-inacrivated new-born bovine serum (NBS). Cells were finallysuspended in RPMI-I640 supplemented with100 IV ml-1 penicillin, 100 ~g.ml-1 streptomycin,1 mmol.I-J sodium pyruvate, 2 mmol.I-1 L-gluta-mine, 3.4 x 10-3 mmol.I-1 2-mercaptoethanol, and10% NBS. Rat peritoneal exudate cells (PECs)
Treatment of HL-60 Cells
HL-60 I.;ells, maintained in an RPMI 1640medium containing 10% FCS. were cultured at allinitiaJ concentration of Ixl05.mI-1 in the pres~ence or absence of 20% (voVvol) of GL-B stimu-lating conditioned media or normal media. Fordetecting the direct effect of GL-B on HL-60
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growth. the culture was treated with GL-B at dif-ferent concentrations. Cultures were then incu-bated at 37°C in a humidified atmosphere for 3days.
Statistical Analysis
Results were expressed as X ::!:: SD and ana-lyzed by I-test to compare the difference betweenthe groups with the Statistica software for Win-dows@ (4.5. Statsoft Inc., 1993).Antitumor Experiment in
Tumor-Bearing Mice
Sarcoma 180 cells were injected subderrnal.lyinto the axillary fossa of the right foreleg. Themice were divided into several groups randomly.Different doses of drugs were administered bystomach tube. once a day. The mice were killed 10days later and the tumors were cut and weighed.
RESULTS
Antitumor Effect of GL-B on LocallyImplanted Sarcoma 180 In Vivo
Table I shows the antitumor effect of GL-Bon Sarcoma 180 in BALB/c mice in vivo. GL-B50, 100, and 200.mg kg-l inhibited the growth ofSarcoma 180 in a dose-dependent instance. Theinhibitory rates are 27.70%, 55.83%, and 66.70%,respectively. The inhibitory rate of the GL-Bgroup treated with cyclopho!iphamide (CY) washigher than that of the GL-B or the CY group.Their inhibitory rates were 80.98%,45.45%, and75.95%, respectively (Table 2).
Analysis of Apoptosis
HL-60 cells were separated from the mediumby centrifugation and washed with phosphate-buffered saline (PBS) after incubation. Hy-podiploid DNA was analyzed using the method ofpropidiumiodide (PI) labeling and flow cytome-try. After washing, samples were resuspended andplaced in the dark at 4"C. PI fluorescence of indi-vidual nuclei was analyzed using a FACScan flowcytometer (Becton Dickinson). Effect of GL-B on Proliferation of HL-60
and Sarcoma 180 Cells In VitroDetermination of Cells Proliferation
On the basis of the above results, we furtheradded GL-B directly to the in vitro HL-60 andSarcoma 180 culture media. We unexpectedlyfound that GL-B had no effect on proliferation ofeither HL-60 or Sarcoma 180 cells (Tables 3 and4). Table 3 shows that GL-B 400 ~g'ml-J slightlystimulated HL-60 cell proliferation.
MTT (20 1.11.5 mg I-I every well) was addedto the cell culture 4 h before the end of incuba-tion. After incubation, gave away supernatant, and150 III of isopropanol was added and OD wasassessed using the Enzyme Labeling lru;trument
(Bio-Rad).
TABLE 1Antitumor Effect of GL-B on Sarcoma 180 in BALB/c Mice (n = 10, x:l: SO)
Dose
(mgokg--1)
Tumor weight
(mg)
Inhibitory rate
(%)Groups
23.61 :t 1.4024.05:t 1.7523.90 r 1.6223.52:t 1.4823.84:t 1.43
Effects of GL-B-M-CM and GL-B- T-CM onProliferation of HL-60 Cells In VitroTABLE 4
Effect of GL-B on Proliferation of Sarcoma180 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 significantlyinhibited proliferation of HL-60 cells in "ilro.Compared with RPMI 1640 and N-M-CM or N-T-CM groups,p < 0.05 orO.OI.
Concentration(J1Q"mt-1)Groups CD 570 nm
RPMI1640 -GL.B 50
100200400
y~"--~'p< 0.01 VS. RPMt 1640.
0.369 j:0.337 j:0.405 :t0.411 :t0.442 :t0.244 :t Effect of GL-B-M-CM and GL-B- T-CM on
Inducing Apoptosis of HL-60 Cells In Vitro
TABLE 5Effect of GL-B on Apoptosis of HL-60 CellsAfter 72 h of In Vitro Incubation~n = 3, x:t: SD)
Concentration Apoptotic cellsGroups (~g.ml-1) (%)
Table 8 shows that 50. 100. and 200 Jlg.ml-1of GL-B-M-CM significantly induced HL-60cells apoptosis; the proportions of apt>ptotic cellswere 18.81 :i: 0.93%. 20.98 :i: 1.57%. 23.00 :i:0.56%. respectively. compared with the N-M-CMgroup. 7.44:1: 1.07%. P<O.Ol.
Table 9 shows that 50, ]00. and 200 Jlg.ml-Jof GL-B- T -CM significantly induced HL-60 cellapoptosis; the proportions of apoptotic cells were19.39:t 1.13%.. 2i,94:t0.84%, 22.85:t 1.49%, re-spectively, compared with the N- T-CM group,7.75 :t 1.14%,p<0.OI.
RPMI1640GL-B
1.94:t 0.410.87 t 0.240.65 :t 0.310.63 :t 0.45
67.70:t 4.04'.
0!10
2.111.731.421.36
1.87:t 0.781.02 t 0.51.M0.45:t O.48*'d0.36:!: 0.17**
0.1080.0450.0520.0570.093
0.037
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TABLE 6Effect of GL-B-M-CM on Proliferation ofHL-60 Cells In Vitro (n = 8, x:t SO)
TABLE 7Effect of GL-B- T-CM on Proliferation ofHL~60 Cells In Vitro (n = 8, x:I: SO)
Concentrationmgoml-1
Concentrationmgoml-1Groups OD 570nm Groups OD 570 nm
FIGURE 1. Effect of GL-B on apoptosis of HL-60 cellsincubated for 72 h in vitro.(A) Control: (8) GL-B 50 ~g.ml-1: (C) GL-B 100~g.mr 1; (D) GL-B 200 ~g.mt1; (E) Vp-16 80 ~g.ml-l.
;lll
FIGURE 2. Effect of Gl-B-M-CM on apoptosis ofHl-60 cells incubated for 72 h in vitro-(A) Control; (8) Gl-B 50 ~g-ml-l; (C) Gl-B 100~g-ml-1; (0) Gl-B 200 1J9-ml-1; (E) Vp-16 80 J.19-m.l.
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level during 24-48 h, and subsided after 72 h. TheTNF-a level in the supernatant of 12.5-400Jlg.ml-1 GL-B cultured with macrophages rose dur-ing 24 h as the dose increased (Fig. 5).
Effect of GL-B on IFN-y Productionof T Lymphocytes
Figure 6 shows that the IFN-y level in the su-pernatant of 200 ~g'ml-1 GL-B cultured with Tlymphocytes rose in 24 h and subsided after 48 h.The IFN-y level in the supernatant of 12.5-2()()~g.mI-1 GL-B cultured with T lymphocytes roseduring 24 h as the dose was increased to 400~g'mi-i. At that level it started to subside (Fig. 7).
DISCUSSION
FIGURE 3. Effect of GL-B- T-CM on apoptosis ofHL-60 cells incubated for 72 h in vitro.(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 present study, we investigated the antitu-mor activity of GL-B, a purified polysaccharideisolated from Ganodenna lucidum. The resultsshowed thal GL-B inhibited the growth of im-planted Sarcoma 180 significantly and in a dose-dependent, combination immunotherapy, GL-Bwith a chemotherapy such as cyclophosphamideachieved the best effect. On the ba is of the pre-ceding in vivo results, we added GL-B directly to
Effect of GL-B on MacrophageTNF-a Production
f'lgure 4 shows that the TNF-a level in the su-pernalant of 200 ~g'mJ-1 GL-B cultured withmacrophages rose in 24 h, remained at the highest
u zu 60 ISU40Time (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 ofincubation (n = 4, x:t SD).
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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()cells. Unexpected results showed that directlyadding GL-B to the in vitro tumor cell cultures can-not inhibit their proliferation. even at the very highconcentration of 400 Jlg.ml-l. GL-B also has no ef-fect on inducing tumor cell apoptosis measured by
using a FACScan flow cytometer. It :\eems (hatGL-B alone has no direct effect on proliferation oftumor cells.
In a further study, GL-B was first added to theculture medium of macrophages and T lymph()-cytes, then the conditioned GL-B-M-CM and GL-
-80
FIGURE 6. Effect of 200 Jig.ml-1 GL-B in IFN.'Y InOuCtlon in the supernatant ormurine spleen cells cultured with ConA after 3-72 h of incubation.
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growth of implanted Sarcoma 180 ill vi~'o signifi-cantly and dose-dependently. Second. GL-B di-rectly added to a culture medium neither inducedHL-60 apoptosis nor restrained its proliferation in~'itro. Third. the macrophage or T lymphocyte cul-ture medium treated with GL-B significantly in-duced HL-60 apoptosis and inhibited its prolifer-ation. GL-B significantly increased TNF-(X andIFN-y release dose- and time-dependently. TNF-(X and IFN-y were known to play imp<)rtant rolesin suppressing tumor cell growth and inducingapoptosis of rumor ceJIs. Finally. as untreatedmacrophages aJ1d T lymphocytes produced littleor no TNF-(X and IPN-y. and N-M-CM or N- T -CMdid not inhibit HL-60 proliferation or induce itsapoptosis, it seemed that the antitumor activity ofGL-B was related to promoting TNF-(X releasefrom macrophages and IFN -y release from T lym-
phocytes.
REFERENCES
B- T -CM media were added to the in vitro H.l.-60culture medium as a drug. The results showed thatboth GL-B-M-CM and GL-B-T-CM significantlyinhibited proliferation of HL-60; the FACScanflow cytometer also found that both of them sig-nificantly induced HL-6() cell apoptosis.
To reveal further the mechanism of the antitu-mor effect of GL-B-M-CM and GL-B- T-CM. theleve.J ofTNF-a in GL-B-M-CM and .lFN-yin GL-B- T -CM were ac;sayed by bioassay and ELISA. re-spective.Jy. TNF-a and IFN-y were known to playimportant roles in suppressing tumor cell growthand inducing apoptosis of many different kinds oftumor cells (Thomson, 1991; Malorni andRainaldi, 1996). Many experiments showed thatTNF-a and lFN-y cooperated with each other ininducing tumor cell apoptosis (Volm and Mattern,1995; Rawadi et aJ.. 1996: Sveinbjinsson andRushteldt; 1997). Our experimental resultsshowed that the levels ofTNF-a in GL-B-M-CMand IFN-y in GL-B-T-CM rose significantly in ado5e-dependent mode. Moreover. our l"e!;ultsshowed that there was a positive correlation be-tween the level of TNF-a in GL-B-M-CM andlFN-y in GL-B- T -CM and the antitumor effect ofGL-B-M-CM and GL-B-T-CM.
In the present study. we investigated the anti-tumor activity and mechanism of GL-B. The re-sults were a.'i follows: first, GL-B did inhibit the
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