ONCOLOGY REPORTS 16: , 2006

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1 ONCOLOGY REPORTS 16: , 2006 Inhibitory effect of wter-soluble extrct from the culture medium of Gnoderm lucidum (Rei-shi) myceli on the development of pulmonry denocrcinom induced by N-nitrosobis (2-hydroxypropyl) mine in Wistr rts NAOKI KASHIMOTO 1,2, MINORU HAYAMA 2, KENJI KAMIYA 1 nd HIROMITSU WATANABE 1 1 Deprtment of Experimentl Oncology, Reserch Institute for Rdition Biology nd Medicine, Hiroshim University, Ksumi 1-2-3, Minmi-ku, Hiroshim ; 2 Helth Cre Institute Wkung Phrmceuticl Co Ltd., 1624 Shimokoutchi, Kod-cho, Akitkt, Hiroshim , Jpn Received July 15, 2006; Accepted August 28, 2006 Abstrct. A wter-soluble extrct from the culture medium of Gnoderm lucidum (Rei-shi) myceli () hs been shown to exert potent chemopreventive effect. The present study ws designed to investigte the effects of dietry supplementtion on the development of lung tumors initited by N-nitrosobis (2-hydroxypropyl) mine (BHP) in mle Slc:Wistr rts. A totl of 77 nimls, 6 weeks of ge, were divided into 5 groups nd given BHP (2,000 ppm) in their drinking wter for 10 weeks. The norml controls were not supplied with BHP. After tretment with the crcinogen, the rts were fed norml control MF solid diet, or the sme diet contining (1.25%, 2.5% or 5%) for 12 weeks. Mcroscopiclly, ll the doses of reduced the number of nodules, nd the effect of 5% ws found to be especilly significnt. Microscopiclly, n increse in the number of proliferting cell nucler ntigen (PCNA)-negtive tumors nd decrese in the number of tumors strongly positive for PCNA were observed in the tissue sections from the rts tht hd received ll the doses of. The present results thus indicte tht dietry supplementtion with inhibits the Correspondence to: Dr Hiromitsu Wtnbe, Deprtment of Experimentl Oncology, Reserch Institute for Rdition Biology nd Medicine, Hiroshim University, Ksumi 1-2-3, Minmi-ku, Hiroshim , Jpn E-mil: tonko@hiroshim-u.c.jp Abbrevitions: BHP, N-nitrosobis (2-hydroxypropyl) mine; PCNA, proliferting cell nucler ntigen; PBS, phosphte-buffered sline; BSA, bovine serum lbumin; IgG, immunoglobulin G; COX, cyclooxygense Key words: lung tumor inhibition, N-nitrosobis (2-hydroxypropyl) mine, Gnoderm lucidum, Wistr rts development of lung tumors, suggesting tht my be potent chemopreventive gent ginst lung crcinogenesis. Introduction Gnoderm lucidum (Fr.) Krst. (Polyporcee) is medicinl mushroom known to the Jpnese s Rei-shi or Mnnentke, or to the Chinese s Lingzhi. Its fruiting bodies hve been used for their medicinl properties in trditionl Chinese medicine for over 2000 yers. This mushroom is described in detil in the Chinese clssics, Shen Nung Ben Co Jin (dted 206 BC - 8 AD), nd the Compendium of Gnoderm lucidum hs been used in trditionl Chinese medicine for the promotion of vitlity nd longevity, nd within the present decde for the tretment of debility nd wekness, hypertension, crdiovsculr disese, bronchitis, rthritis, neurstheni, insomni, heptopthy, chronic heptitis, nephritis, gstric ulcers, sthm, dibetes, ltitude sickness, AIDS, nd cncer (1-3), in Chin nd other orientl countries. The myceli of Gnoderm is consumed s helth food nd herbl medicines. Chemicl investigtions of the fruiting bodies, spores, nd the myceli of Gnoderm lucidum hve shown tht they contin vrious bioctive substnces, including polyscchrides, proteins, nucleosides, ftty cids, terpenoids, sterols, nd cerebrosides (4). Wter or lcohol extrcts of Gnoderm lucidum hve been used to investigte biologicl ctivities, s these extrcts contin compounds with immunologicl nd nti-inflmmtory properties. Of prticulr interest mong the reported biologicl/phrmcologicl properties of Gnoderm lucidum re their nti-tumor ctivities, including cell cycle rrest, poptosis induction, motility inhibition, ntingiogenesis, nd ntimutgenesis (3,5-10). A wter-soluble extrct from the culture medium of Gnoderm lucidum (Rei-shi) myceli () contins vrious kinds of high-moleculr-weight constituents, such s polyscchrides with protein or wter-soluble lignin, nd low-moleculr-weight constituents, such s triterpenes. We

2 1182 KASHIMOTO et l: INHIBITION BY Rei-shi OF LUNG TUMORS INDUCED IN RATS hve reported previously tht prevented the development of zoxymethne-induced berrnt crypt foci nd colon tumors in F344 rts (11,12), nd N,N'-dimethyl-hydrzineinduced colon tumors in ICR mice (13). To determine whether my exert chemopreventive effect on the crcinogenesis of orgns other thn the colon, the present study ws conducted ssessing the effects of on the development of rt lung tumors fter initition with BHP. Mterils nd methods Animls. Seventy-seven 6-week-old mle Slc:Wistr rts (SLC Jpn Inc., Hmmtsu, Jpn) were used in the present study. They were housed three or four to polycrbonte cge nd kept under constnt temperture conditions (24±2 C) nd reltive humidity (55±10%) with 12 h light/ drk cycle. The nimls were mintined in ccordnce with the Guidelines for the Cre nd Use of Lbortory Animls estblished by Hiroshim University nd fed commercil MF diet (Orientl Yest. Co. Ltd., Tokyo, Jpn) lone or with 1.25%, 2.5% or 5% supplement of. Norml tp wter ws lso provided d libitum. BHP. The crcinogen ws purchsed from Nkri Tesque Co. Inc., Kyoto, Jpn, nd dissolved in distilled wter t concentrtion of 2,000 mg/l just before use. This solution ws supplied to the rts d libitum for 10 weeks from lightopque bottles exchnged t 2- or 3-dy intervls.. A wter-soluble extrct from the culture medium of Gnoderm lucidum myceli (designted s ) ws prepred by Nod Shokkin-Kogyo Co., Ltd. (Chib, Jpn). In brief, Gnoderm lucidum (Rei-shi or Mnnnentke) myceli were cultured in solid medium composed minly of sugr-cne bgsse for three months, then the whole medium contining the myceli ws extrcted with hot wter. The extrct ws filtered nd spry-dried to obtin. Pthology. The nimls were scrificed nd necropsied if they becme moribund, nd ll the remining rts were scrificed 22 weeks fter the BHP tretment. The lungs were removed nd the loction nd size of the individul tumors ws recorded. All the tissues were fixed in 10% neutrl formlin. Lesions in the lung were clssified into four types: Hyperplsi, denom, denocrcinom, nd squmous cell crcinom. Dt indicte both the totl number of tumors nd the verge dimeter of the tumors mesured in ech section of five lobules. Immunohistochemistry. Prffin-embedded sections were deprffinized in xylene, nd rehydrted through grded ethnol series. A 0.05 M PBS buffer ws used to prepre the solutions nd lso for the wshes between the vrious steps. The incubtions were performed in humidified chmber. Three-micrometer-thick sections were treted for 30 min t room temperture with 2% BSA nd incubted with primry ntibodies ginst ß-ctenin (diluted 1:100; Trnsduction Lbortories, BD , KY, USA), monoclonl mouse nti-proliferting cell nucler ntigen ntibody (Dko-PCNA, PC 10, code No. M 879), monoclonl mouse nti-cyclooxygense-1 (diluted 1:50; Snt Cruz Biotechnology, sc-1754) nd monoclonl mouse nti-cyclooxygense-2 (diluted 1:100; Snt Cruz Biotechnology, sc-1746) for 1 h t room temperture. For ech cse, negtive controls were performed on seril sections whereby incubtion with the primry ntibody ws omitted. All the slides were then exposed to the secondry ntibody, biotinylted horse nti-universl-monkey IgG (Vectstin Universl Quick Kit, Vector Lbortories, CA, USA, Ctlog No PK-8800) nd peroxidse-conjugted streptvidin complex. Peroxidse ctivity ws visulized by tretment with H 2 O 2 nd diminobenzidine for 5 min. At the finl step, the sections were counterstined with hemtoxylin for 1 min. ß-ctenin-positive nuclei were counted nd divided by the totl epithelil cells in the tumors to give n index for nucler ccumultion. The PCNA index ws clssified into four types: -, Negtive (0%); ±, slightly positive (<50%); +, positive (<80%); ++, strongly positive (>80%). The PCNA index ws clculted from the intensity of PCNA stining of 100 rndomly selected tumors in sections of five lobules. Sttisticl significnce. Sttisticl significnce ws determined using Dunnett's method for multiple comprisons, the 2 nd the Student's t-test. Results The intke of drinking wter in the BHP tretment groups (6.7 to 7.1 ml/dy/niml) ws significntly decresed s compred with the norml controls (18.5 ml/dy/niml) but there ws no significnt difference mong the BHP tretment groups. The diet intke lso showed no significnt difference mong the experimentl groups fter BHP tretment (dt not shown). All of the nimls survived until the scheduled time of scrifice. Body weight in the BHP lone group ws significntly decresed s compred with the norml vlue (P<0.01) (Tble I). In the BHP lone group, thyroid weight ws significntly incresed reltive to the control (P<0.01), nd the weight of the liver, testis nd drenl glnd ws significntly decresed. The reltive weight of the thyroid ws significntly incresed s compred with the norml control vlue, wheres tht of the lung nd testis ws significntly decresed (Tble I). Mcroscopiclly, BHP-treted nimls hd mny lung nodules. Both the number of nodules in the nimls given BHP+5% nd the men dimeter of nodules in ll the groups were significntly decresed in comprison with the corresponding vlues in the BHP lone group (P<0.01) (Tble II). Microscopiclly, s shown in Fig. 1, lesions in the lung were clssified into four types: Hyperplsi, denom, denocrcinom, nd squmous cell crcinom. BHP-treted nimls hd pulmonry hyperplsi, denom nd denocrcinom, nd lso thyroid tumors. Squmous cell crcinoms were lso evident in 13%, 6%, 20% nd 0% of the nimls in the BHP, BHP+1.25%, BHP+2.5% nd the BHP+5% groups, respectively (Tble III). The numbers of pulmonry tumors nd denoms in ll the groups nd denocrcinoms in the BHP+2.5% nd BHP+5%

3 ONCOLOGY REPORTS 16: , Tble I. Body weight nd orgn weight (reltive weight). Body Hert (g) Thyroid (mg) Lung (g) Liver (g) Kidney (g) Testis (g) Adrenl Spleen (g) weight (g) glnd (mg) Norml 498.1± ± ± ± ± ± ± ± ±0.33 (0.27±0.01) (79.9±3.6) (0.32±0.01) (2.53±0.04) (0.51±0.01) (0.64±0.03) (89.4±3.3) (184.3±62.3) BHP 429.4± ± ± ± ± ± ± ± ±0.01 (0.30±0.02) (226.4±24.2 ) (0.40±0.02 ) (2.65±0.17) (0.56±0.03) (0.27±0.02 ) (85.1±5.0) (133.1±8) BHP+1.25% 448.9± ± ± ± ± ± ± ± ±0.02 (0.28±0.01) (180.3±18.6) (0.39±0.01) (2.59±0.03) (0.55±0.02) (0.26±0.01) (84.1±2.9) (127.5±5.6) BHP+2.5% 460.7± ± ± ± ± ± ± ± ±0.14 (0.28±0.01) (262.7±69.7) (0.47±0.09) (2.63±0.07) (0.59±0.06) (0.27±0.03) (89.1±8.9) (181.4±44.8) BHP+5% 456.8± ± ± ± ± ± ± ± ±0.03 (0.28±0.01) (212.7±17.2) (0.40±0.01) (2.68±0.04) (0.57±0.01) (0.26±0.01) (80.4±2.6) (137.1±5.3) Vlues re men ±SE, significntly different from the norml vlue (P<0.01). Tble II. Number nd size of the lung nodules. Group Number Men dimeter of nodules of nodules BHP 58.7± ±0.07 BHP+1.25% 44.4± ±0.06 BHP+2.5% 48.1±6 3.15±0.06 BHP+5% 35.4± ±0.07 Vlues re men ±SE, significntly different from the BHP vlue (P<0.01). groups were significntly decresed in comprison with the BHP lone group (Tble IV). The totl size of pulmonry tumors nd denoms in the BHP+5% group ws lso significntly lower thn tht in the BHP lone group (P<0.01) (Tble V). As shown in Fig. 2, the PCNA index ws clssified into four types: -, Negtive (0%); ±, slightly positive (<50%); +, positive (<80%); ++, strongly positive (>80%). The PCNA indices in the pulmonry tumors re shown in Tble VI. The numbers of PCNA-negtive tumors in the BHP+2.5% nd BHP+5% groups were significntly incresed. In the BHP+5% group, the numbers of both positive tumors nd strongly positive tumors were significntly decresed in comprison with the BHP lone group. None of the lung tumors showed ny nucler or cytoplsmic stining for ß-ctenin, COX-1 or COX-2. Discussion In the present study, did not ffect the incidence of tumors, but significntly reduced the number nd size of tumors in dose-dependent mnner. There re two interesting reports of studies crried out using similr niml model of lung crcinogenesis. One involved long-fermented miso, nturl product, which did not ffect the incidence or size of tumors, but significntly reduced the number of tumors (14). The other report describes tht combintion of n ntibiotic, erythromycin, or n nti-inflmmtory drug, mpicillin, with nturl product, sho-siko-to, decresed the incidence, size nd number of tumors (15). However, s in the cse of nd/or miso, the nturl product lone did not ffect tumor incidence. Drugs used for cncer chemotherpy re known to cuse undesirble side-effects. In contrst however, even though nturl products possess less potent nti-tumor effect thn chemicl compounds, they re superior in terms of sfety., being nturl product, could be useful when used in combintion with chemicl compound to slow the progression of tumor growth. However, further studies on the ppliction of nturl products re wrrnted. Yun et l reported tht totl extrct of the whole fruiting body of Gnoderm lucidum inhibited the incidence of pulmonry denom induced by benzo()pyrene (BP) in newborn mice (16,17). The nticncer ctivity of polyscchrides isolted from Gnoderm lucidum ws originlly tested in vivo using srcom 180 cells trnsplnted subcutneously into mice (18). The nticncer effects of Gnoderm lucidum were ttributed to the ctivity of ß-glucns (19), which hve lso been isolted from other mushrooms nd demonstrte potent immunostimultory ctivity (20). Therefore, the stimultion of immune effector cells leds to the subsequent production of cytokines, nd contributes to the nticncer ctivity of Gnoderm lucidum (19). Similr to the results obtined using the mouse srcom 180 cell model, polyscchride extrct of Gnoderm lucidum hs lso been shown to inhibit the growth of Lewis lung crcinom cells in mice (19,21). The sulfte derivtives of D-glucns demonstrte significntly higher ntitumor ctivity ginst Ehrlich scites crcinom (22). Furthermore, wter-soluble extrcts from the culture medium of Gnoderm lucidum prevent zoxymethn-induced berrnt crypt foci nd colon cncer in rts nd the development of N,N'- dimethylhydrzine-induced colon tumors in ICR mice (11-13). Finlly, Gnoderm lucidum polyscchride

4 1184 KASHIMOTO et l: INHIBITION BY Rei-shi OF LUNG TUMORS INDUCED IN RATS Figure 1. H&E stining of the lung lesions (x100). (A) Hyperplsi; (B) denom; (C) denocrcinom; (D) squmous cell crcinom. Tble III. Tumor incidence. Number of Pulmonry Squmous Thyroid nimls tumors cell tumor crcinom Norml BHP (100) b 2 (13) 15 (100) BHP+1.25% (100) 1 (6) 16 (100) BHP+2.5% (100) 3 (20) 15 (100) BHP+5% (100) 0 15 (100) Number of nimls with tumors, b incidence rte. Tble IV. Number of pulmonry tumors (per niml). Group Totl pulmonry Adenom Crcinom tumors b BHP 42.71± ± ±1.27 BHP+1.25% 28.80±2.14 c 20.27±1.38 c 11.33±1.31 BHP+2.5% 24.80±1.72 c 23±1.90 c 9.20±1.20 c BHP+5% 24.50±2.42 c 19.20±2.50 c 8.29±1.18 c Dt indicte the totl number of tumors mesured in ech section of 5 lobules; b denom + denocrcinom; vlues re men ±SE; c significntly different from the BHP vlue (P<0.01). extrct (Gnopoly) reportedly stimultes immune function in ptients with dvnced cncers, including those of the lung, colon, brest, liver, prostte, bldder, nd the brin (8). Twelve weeks of tretment with Gnopoly resulted in significnt increse in the plsm concentrtions of IL-2, IL-6 nd IFN-Á nd enhnced NK ctivity, wheres the levels of IL-1 nd TNF- were significntly decresed (8). In the present study, by clssifying the PCNA index into four types - negtive, slightly positive, positive or strongly positive - by microscopic observtion of 100 rndom tumors from five lobules of the lung, ws found to decrese

5 ONCOLOGY REPORTS 16: , Tble V. Size of the pulmonry tumors (mm). Group Totl pulmonry Adenom Crcinom tumor BHP 1.07± ± ±0.06 BHP+1.25% 1.11± ± ±0.08 BHP+2.5% 1.06± ± ±0.07 BHP+5.0% 0.93±0.03 b 0.85±0.03 b 1.14±0.07 Dt indicte the verge dimeter of the tumors mesured in ech section of five lobules; vlues re men ±SE; b significntly different from the BHP vlue (P<0.01). the number of strongly positive tumors nd increse the number of negtive tumors in dose-dependent mnner. In the present study, ttenuted the up-regulted cell cycle ctivity during crcinogenesis fter exposure to the crcinogen. As similr effect hs lso been reported in n niml model of colon crcinogenesis (11-13), is thought to function in this wy to chieve its nti-tumor effect. did not influence the cell cycle of norml tissue close to crcinom lesions (dt not shown), nd this hs lso been reported in the colon crcinogenesis niml model. Accordingly, is thought to hve no effect on the prolifertion of norml epithelil cells. In ddition, mcroscopic observtion reveled tht reduced the number nd size of tumors induced by the crcinogen, nd this reflected the decrese in the number of PCNA-positive crcinoms. A recent study hs shown tht for the prevention of tumor formtion it is importnt to rrest the poptosis guidnce bility of p53 nd the destbiliztion of the genome, nd the destbilized prevention of genome my be ttined by the guidnce of cell cycle stop (23). Therefore, it is shown tht countercts the up-regultion of the cell cycle in ssocition with proteins such s p53, or contributes to gene stbility. Further study is needed to shed more light on the function of. In ddition to in vivo biologicl ctivities other thn nticncer effects, the intrgstric ppliction of poly- Figure 2. H&E nd PCNA stining of the lung lesions (x100). (A) H&E stining on the 5% +BHP group rts; (B) PCNA stining on (A) s clssified to negtive; (C) H&E stining on the BHP group rts; (D) PCNA stining on (C) s clssified to strong positive.

6 1186 KASHIMOTO et l: INHIBITION BY Rei-shi OF LUNG TUMORS INDUCED IN RATS Tble VI. Immunochemistry study (PCNA). Group - ± + ++ BHP 4.85± ± ± ±9.97 BHP+1.25% 6.55± ± ± ±7.10 BHP+2.5% 13.81±10.02 b 55.26± ± ±3.57 b BHP+5.0% 25.08±12.91 b 56.14± ±19.46 b 0.93±1.84 b The PCNA index ws clculted from the intensity of the PCNA stining of 100 rndomly selected tumors in sections of five lobules; vlues re men±s.e.; b significntly different from the BHP vlue (P<0.05 nd 0.01); -, negtive; ±, slightly positive; +, positive; ++, strongly positive. scchride hs been shown to prevent indomethcin- nd cetic cid-induced gstric mucosl lesions in rts (24). The heptoprotective effects of the Gnoderm lucidum polyscchride hve lso been evluted in mouse infection model utilizing Bcillus Clmette-Guerin (25). Tretment with the polyscchride diminished the histologicl chnges ssocited with liver injury, such s hemorrhge nd necrosis in the heptic lobules nd the inflmmtory infiltrtion of lymphocytes nd Kupffer cells round the centrl vein (25). Glycns known s gnodern A nd B isolted from the fruiting bodies of Gnoderm lucidum hve been shown to possess hypoglycemic ctivity (26). Both gnoderns reduced the plsm glucose levels in norml mice nd mice with lloxn-induced hyperglycemi (26). Gnodern B incresed the level of insulin in the plsm but did not hve ny effect on insulin binding to isolted dipocytes (27). Gnodern B lso reduced the glycogen content of the liver but hd no influence on totl cholesterol nd triglyceride levels in the plsm nd liver (27). Polyscchrides isolted from the fruiting body demonstrte protective effects ginst lloxninduced dmge to insulin-producing pncretic ß cells, incresing their vibility nd protecting them from necrosis (28). A wter extrct of myceli hs lso been ssessed for crdiovsculr ctivity in rbbits nd rts, nd ws found to decrese systolic nd distolic blood pressure (29). Co nd Lin observed tht Gnoderm lucidum polyscchrides nd peptide inhibited in vivo ngiogenesis using the chick choriollntoic membrne ssy (9,30). In n in vivo experiment, orl dministrtion of the triterpenoid frction of the fruiting body of Gnoderm lucidum inhibited the growth of intrsplenic implnts of Lewis lung crcinom in mice (31). In ddition, the triterpenoid frction prevented the invsion of metsttic tumor cells into the white pulp of the spleen nd heptic prenchym (31). Furthermore, mtrigel-induced neovsculriztion ws lso inhibited, nd the biologiclly ctive compound responsible for the suppression of ngiogenesis ws identified s gnodric cid F (31). Combined tretment with lovsttin nd n oxygented triterpenoid frction from Gnoderm lucidum inhibited the growth of humn heptom Hep 3B cells (3). Moreover, lovsttin nd the triterpenoid frction reduced the growth of tumors in nude mice with orthotopiclly inoculted humn heptom Hep 3B/T2 cells (25). These dt suggest tht triterpenoids from Gnoderm lucidum my hve potentil s n djuvnt therpy for the tretment of cncer. All of the bove studies clerly show tht Gnoderm lucidum possesses ntitumor nd immunomodultory potentil. In conclusion, the results of the present study indicte tht Gnoderm lucidum my be useful chemopreventive gent, nd tht further study is wrrnted to identify the ctive component involved in its effect. Studies re now in progress in our lbortory to further clrify the ctive compounds in the wter-soluble extrct nd investigte their phrmceuticl effects. References 1. Lin ZB: Phrmcologicl functions of Gnoderm lucidum In: Modern Reserch of Gnoderm lucidum. Z.B. Lin (ed). 2nd edition. 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