Ganoderma Lucidum Extracts Inhibit Growth and Induce Actin Polymerization in Bladder Cancer in Vitro

8/8/2019 Ganoderma Lucidum Extracts Inhibit Growth and Induce Actin Polymerization in Bladder Cancer in Vitro

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Ganoderma lucidum extracts inhibit growth and induce actinpolymerization in bladder cancer cells in vitro

Qing-Yi Lu a , Yu-Sheng Jin b , Qifeng Zhang a , Zuofeng Zhang c , David Heber a ,Vay Liang W. Go a , Frederick P. Li d , Jian Yu Rao b, *

aCenter for Human Nutrition, Department of Medicine, David Geffen Schoolof Medicine, University of California, Los Angeles, CA 90095, USAb Department of Pathology and Laboratory Medicine, David Geffen School of Medicine, University of California, Los Angeles, CA 90095, USA

c Department of Epidemiology, School of Public Health, University of California, Los Angeles, CA 90095, USAd Dana-Farber Cancer Institute, Boston, MA 02115, USA

Received 25 January 2004; received in revised form 1 June 2004; accepted 2 June 2004

Abstract

This study was conducted to investigate chemopreventive effects of Ganoderma lucidum using a unique in vitro humanurothelial cell (HUC) model consisted of HUC-PC cells and MTC-11 cells. Ethanol and water extracts of fruiting bodies andspores of the G. lucidum were used to examine growth inhibition, actin polymerization status, and impact of actin remodelingon cell migration and adhesion. Results showed that ethanol extracts had a stronger growth inhibition effect than water extracts.Cell cycle analysis showed that the growth inhibition effect was associated with G 2 /M arrest. At non-cytotoxic concentrations(40–80 mg/ml), these extracts induced actin polymerization, which in turn inhibited carcinogen 4-aminobiphenyl inducedmigration in both cell lines. The increased actin polymerization was associated with increased stress bers and focal adhesioncomplex formation, however, expression of matrix metalloproteinase-2 and focal adhesion kinase (total and phospholated) wereunchanged, which suggests that other mechanisms may be involved.q 2004 Elsevier Ireland Ltd. All rights reserved.

Keywords: Ganoderma lucidum ; Chemoprevention; Bladder cancer; Actin polymerization

1. Introduction

Ganoderma lucidum (Fr.) Karst. (Polyporaceae) isa medicinal mushroom known to the Chinese as

‘Lingzhi’. Its fruiting bodies have been used for theirmedicinal properties in traditional Chinese medicinefor over 2000 years. This mushroom is described indetail in the Chinese Materia Medica classics, ShenNung Ben Cao Jin (dated 206 BC–8 AD), and theCompendium of Materia Medica. G. lucidum has beenused in traditional Chinese medicine for promotion of vitality and longevity, and more recently used inthe treatment of debility and weakness, insomnia,hepatitis, bronchitis and asthma, diabetes, altitude

0304-3835/$ - see front matter q 2004 Elsevier Ireland Ltd. All rights reserved.doi:10.1016/j.canlet.2004.06.022

Cancer Letters 216 (2004) 9–20www.elsevier.com/locate/canlet

* Corresponding author. Address: Department of Pathologyand Laboratory Medicine, UCLA School of Medicine, Box 951732,Los Angeles, CA 90095-1732, USA. Tel.: C 1-310-794-1567;fax: C 1-310-206-5178

E-mail address: [email protected] (J.Y. Rao).

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sickness, cardiovascular disease, AIDS, and cancer[1–3] . In addition to the fruiting bodies, the spores andcultivated mycelia of Ganoderma have been recently

consumed as health food and herbal medicines.Chemical investigations on the fruiting bodies, spores,and mycelia of G. lucidum reveal that they containvarious bioactive substances. Active constituents of G. lucidum include polysaccharides, proteins, nucleo-sides, fatty acids, terpenoids, sterols, and cerebrosides[4]. Water or alcohol extracts of G. lucidum have beenused to investigate biological activities, as theseextracts contain compounds mainly responsible forthe immunological and anti-inammatory properties.Alcohol extracts contain biological compounds thatlower blood cholesterol and glucose levels, bloodpressure, and inhibit histamine release, while theliver-protective, antiviral and anti-tumor effectscan be attributed to both the water-soluble polysac-charides and alcohol-soluble triterpenes [3,4] . Of particular interest among the reported biological/ pharmacological properties of G. lucidum are theiranti-tumor activities, including the effects on cellcycle arrest, apoptosis induction, motility inhibition,antiangiogenic, and antimutagenic activities [3,5–10] .

The primary objective of this study was to evaluatethe chemopreventive effects of G. lucidum extracts

using our unique in vitro human bladder cancer modelconsisting of two cell lines, both derived from thesame human urothelial clone immortalized by SV-40.In this model, HUC-PC is an untransformed cell linethat does not form tumor nodules when inoculated intonude mice, and MTC-11 is a transformed low-gradetumor that forms tumor nodules when inoculatedinto nude mice [11] . After exposure to carcinogen4-aminobiphenyl (4-ABP), the HUC-PC cells can betransformed to malignant and the MTC-11 cellsinduced to form a high-grade tumor [11] . This cell

culture system provides a unique model to rapidly testthe effect of chemopreventive agents on bladdercancer in association with 4-ABP, a major carcino-genic component found in cigarette smoke. In thisstudy, the growth inhibition effect of various Gano-derma extracts on both cell lines was analyzed by [ 3 H]thymidine incorporation assay and calorimetric tetra-zolium (MTT) assay. In addition, dose–responseeffects on actin polymerization were determined byDNase I inhibition assay analysis. The quantitativerelationship between cytoplasmic F- and G-actin,

the polymeric laments and monomeric globules,respectively, reects cellular differentiation versusdedifferentiation status [12] . In general, cell differen-

tiation is associated with an increased F/G-actin ratio,whereas dedifferentiation and malignant transform-ation is associated with a decreased F/G-actin ratio[13,14] . Thus, the ratio of F-actin to G-actin functionsas a surrogate marker for cellular differentiation anddedifferentiation [13–15] . Since, actin is a majorcytoskeletal protein involved in migration, the func-tional signication of actin polymerization on cellmotility was examined by ‘wound scratching assay’.Mechanism for growth inhibition was investigated byLaser Scanning Cytometry (LCS) based cell cycleanalysis and mechanism for migration inhibition wasstudied by immunouorescence analysis of adhesioncomplex, immunoblot analysis of matrix metallopro-teinase-2 (MMP-2) expression, and focal adhesionkinase (FAK) activities.

2. Materials and methods

2.1. Materials

G. lucidum spore powder was obtained from

Zhongke Capsule (Nanjing, China), and G. lucidumfruiting body powder was provided by PharmanexInc. (Provo, UT). All solvents used for extraction areHPLC grade and were purchased from FisherScientic. 4-Aminobiphenyl (4-ABP) was purchasedfrom Sigma (St Louis, MO).

2.2. Extract preparation

Extraction was performed on both spores andfruiting bodies. Briey, powder from spores or

fruiting bodies was rst extracted with 95% ethanoland then with water by successive sonication at roomtemperature for 30 min. Extracts were ltered andltrate was concentrated under reduced pressure toyield dark brown powder (water extracts) and oilyresidue (alcohol extracts).

2.3. Cell culture

Both HUC-PC and MCT-11 cells were grownin 90% F-12 nutrient mixture (Ham) medium

Q.-Y. Lu et al. / Cancer Letters 216 (2004) 9–2010



(GIBCO BRL Island, NY) with 1% penicillin andstreptomycin (10,000 mg/ml penicillin, 10 mg/mlstreptomycin) and 10% FBS. Cultures were main-

tained at 378

C in 5% CO 2 and 95% air, and mediumchanged two times per week. G. lucidum extracts weredissolved in either dd H 2 O or ethanol to make a stock solution of 10 mg/ml. 4-ABP was dissolved in 100%dimethyl sulfoxide (DMSO) to make a stock solutionof 100 mmol/ml. Logarithmically growing HUC-PCand MTC-11 cells were harvested and seeded at aninitial density of 2 ! 105 cells in 20 ml of freshmedium in 60-mm petri dishes.

2.4. [ 3 H] thymidine incorporation assay

Cells were plated into 48-well culture plates (1 !

104 cells/well) for 24 h to allow for attachment.Subsequently, fresh medium (400 ml/well) containingdifferent concentrations of mushroom extracts wasadded and incubated at 37 8 C with 5% CO 2 for 24 h.[3 H] thymidine (DuPont NEN, Boston, MA) at a nalconcentration of 1 mCi/ml was added 4 h prior totermination of the experiment. The cells were rinsedtwice with PBS and xed with 10% trichloroaceticacid at room temperature (200 ml/well) for 15 min,and again rinsed twice with 100% EtOH. Cells were

then lysed with 0.4 N NaOH 200 ml/well and heatedat 65 8 C for 30 min. After the plate was cooled,100 ml/well glacial acetic acid was added and mixedin a shaker. Scintillation vials were prepared with4 ml scintillation uid/bottle, and 100 ml of celllysate was added to each vial. The vial was vortexedwell until the volume was clear, and the radioactivitywas determined.

2.5. MTT assay

The cytotoxicity of each extract was determined bya calorimetric tetrazolium (MTT) assay. Briey, MTT(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazo-lium bromide; Sigma, MI) was dissolved in PBS at5 mg/ml and ltered. Serial triplicate dilutions of the extract at 50 ml in volume were added to1.5 ! 104 cells/ml to 96-well at-bottomed plates.Plates were incubated for 24 h at 37 8 C, pulsed with10 ml MTT (5 mg/ml) and incubated for 4 h at 37 8 C.DMSO (100 ml) was then added to all wells andmixed thoroughly for 30 min at room temperature.

The plates were then read on a Microplate ReaderBio-Rad 550. Cell survival was calculated as thepercentage of MTT inhibition, using the following

formula:% survival Z

meanexperimentalabsorbancemeancontrolabsorbance ! 100 :

2.6. Determination of cellular F/G-actin ratio

Cells at various time points and conditions, asdescribed above, were harvested and F/G-actin ratiowas determined by a biochemical DNase I inhibitionassay. This assay has been described extensivelypreviously [12] .

2.7. Analysis of focal adhesion complex by triple-labelimmunouorescence

In this assay, cells were cultured directly on 1 cmdiameter cover glass (Fisher Scientic, Pittsburgh,PA) placed on a 24-well at-bottom plate. At the endof the treatment, culture medium was aspirated andcells on the cover glass were washed twice with PBSand then xed with 3.7% formaldehyde solution inPBS for 20 min at room temperature. After xation,cells on the cover glass were incubated sequentiallywith following reagents: 1% BSA for 30 min, 1:100monoclonal anti-Paxillin (clone 5H11, Upstate Bio-technology) for 1 h, 1:150 Cy3-conjugated AfniPureGoat Anti-Mouse IgG (H C L) (Jackson ImmunoR-esearch lab Inc.) for 30 min, 1:40 Bodipy phallacidin(for F-actin) (Molecular Probes Inc., Eugene, OR) for30 min, and 1:1000 dilution of 4 0,6-diamidino-2-phenylindole (DAPI) (10 mg/ml Molecular ProbesInc., Eugene, OR) for 5 min. Between each incubationstep, the cover glass was rinsed with PBS three times.The stained cover glass was then transferred onto a

regular microscopic slide, which was then mounted in100 mM n-propyl gallate (Sigma Chemical Co., StLouis, MO) in spectranalyzed glycerol (FisherScientifc), pH 6.5, for uorescence microscopicexamination and cell cycle analysis by LCS.

2.8. Cell cycle analysis by laser scanningcytometry (LCS)

The cell cycle was analyzed using laser scanningcytometer (CompuCyte, Cambridge, MA) by

Q.-Y. Lu et al. / Cancer Letters 216 (2004) 9–20 11



measuring the DAPI uorescence and the peak intensity of uorescence in the cell nuclei. Fluor-escence excitation was provided by a 488 nm laser

line. The blue uorescence was measured usingstandard settings of LCS dichroic mirrors andemission lters. Multicycle software (PhoenixFlow System) and the software provided with theLSC were used to deconvolute histograms toestimate proportions of cells in particular phasesof the cycle.

2.9. Immunoblot analysis for MMP-2 and FAK

Cells that were washed twice in cold PBS were

scraped from culture dishes in lysis buffer (50 mMTris–HCl (pH 7.4), 150 mM NaCl, 2 mM EGTA,MgCl 2 2 mM, 1% (v/v) Triton X-100, 10% glycerol,10 mM DTT, 1 mM phenylmethylsulfonyl uoride,10 mg/ml leupeptin, 10 mg/ml aprotinin, 5 mg/mlpepstatin A, 50 mM NaF, and 1 mM NaVO 4 ).Lysates were centrifuged at 12,000 ! g and 4 8 Cfor 10 min. Protein concentrations of lysates weredetermined by Bio-Rad Protein Assay (Bio-RadLaboratories, Hercules, CA). For IB analyses, thesame concentrations of proteins (30 or 50 mg) weresubjected to 8% SDS-PAGE and were electrotrans-ferred to nitrocellulose membranes using electroblotbuffers. Membranes were blocked in PBS containing5% non-fat dry milk for 30 min. Reactions with theprimary antibodies in TBS buffer containing 3% drymilk were carried out at 4 8 C overnight. Afterextensive washing, membranes were placed on ashaker with biotinylated secondary IgG for 1 h.Upon further washing, membranes were reactedwith streptavidin-horseradish peroxidase for 45 minand ECL detection reagents immediately prior toautoradiography.

2.10. Wound-scratch assay for cellular migration

A wound-scratch assay was performed toexamine the cellular migration. In this assay, auniform cell-free area was created by scratchingconuent monolayers with a plastic pipet tip, andthe wound area was inspected at different timeintervals to determine the distance migrated by thecells.

2.11. Statistical analysis

Descriptive statistics, such as mean and standard

error, were used to summarize the results.The Student’s t -test and ANOVA test were used forunivariate analysis. Statistical signicance wasdened by a two-tailed P -value of 0.05.

3. Results

3.1. Growth inhibition and cell cycle analysisof Ganoderma lucidum extracts on HUC-PC and MTC-11 cells

To examine the growth inhibition of G. lucidumextracts, HUC-PC and MTC-11 cells were exposedto various concentrations of the extracts for 24 h andthen subjected to [ 3 H] thymidine incorporation assay(Fig. 1a). Both cells were exposed to incrementalconcentrations of four extracts (15–1000 mg/ml) andincubated for 24 h. Cell survival was calculated asthe percentage of [ 3 H] thymidine incorporation assay(% surviva1 Z mean experimental absorbance/meancontrol absorbance ! 100). Overall, the growth inhi-bition effects of these extracts were similar for both

cell lines, and alcohol extracts appear to be morepotent than water extract for both cell lines. Fruitingbody ethanol extract was most potent in inhibitinguntransformed HUC-PC cell line with its concen-tration producing 50% inhibition (IC 50 ) being124 mg/ml, followed by spore ethanol extract(IC 50

Z 280 mg/ml), spore water extract (IC 50Z

500 mg/ml), and fruiting body water extract (IC 50Z

1000 mg/ml). Similarly, fruiting body ethanol extractwas most potent (IC 50

Z 113 mg/ml) in inhibitingtransformed MTC-11 cell line, followed by spore

ethanol extract (IC 50Z

234 mg/ml), spore waterextract (IC 50Z 465 mg/ml), and fruiting body water

extract (IC 50Z 990 mg/ml).

The cytotoxicity effects of G. lucidum extractswere further examined by MTT assay. The IC 50 of HUC-PC cells by fruiting body ethanol extract,spore ethanol extract, spore water extract, andfruiting body water extract were 325, 521, 362,and 1000 mg/ml, respectively. The IC 50 of MTC-11cells by fruiting body ethanol extract, spore ethanolextract, spore water extract, and fruiting body water




extract were 129.3, 274.7, 365.4, and 509 mg/ml,respectively, which were similar to the above resultsobtained by [ 3 H] thymidine incorporation assay. TheIC50 of ethanol extracts in HUC-PC cells was higherthan that of MTC11 cells, suggesting the untrans-formed HUC-PC cells was more resistant to thecytotoxicity of ethanol extracts than the MTC-11cells.

Cell cycle analysis using LCS was performedon cells treated with 40 mg/ml (about or less thanone-third of IC 50 ) for 24 h with various G. lucidum

extracts. As shown in Fig. 1b, G. lucidum extracts atthis non-cytotoxic concentration induced G 2 /M arrestin both HUC-PC and MTC-11 cells. In HUC-PC cells,the percentage of cells in the G 2 /M phase increasedfrom 25% in untreated control cells to 31–35% in cellstreated with various G. lucidum extracts. In MTC-11cells, the percentage of cells in the G 2 /M phaseincreased from 15% in untreated control cells to25–27% in cells treated with ethanol extracts of G. lucidum , but only to 17–18% in cells treated withwater extracts. These ndings were consistent with

Fig. 1. Effects of G. lucidum extracts on the proliferation (A) and cell cycle (B) of HUC-PC and MTC-11 cells, using [ 3 H] thymidineincorporation assay and Laser Scanning Cytometry analysis, respectively. For proliferation analysis, HUC-PC cells were plated at a density of 1! 105 cells/ml, and cultured for 24 h in a medium containing Ganoderma extract at various concentrations (15.6, 31.2, 62.5, 125, 250, 500,1000 mg/ml). Cell growth was determined by the [ 3 H] thymidine incorporation assay. The y-axis represents the percentage of inhibition of treated cells versus untreated culture, and the x-axis represents the concentration. For cell cycle analysis, cells treated with 40 mg/ml of eachGanoderma extract for 24 h on cover glass underwent triple-label immunouorescence including paxillin, F-actin, and DNA, as detailed inSection 2. Cell cycle analysis was performed using Laser Scanning Cytometry. GLfb, Ganoderma lucidum fruiting bodies; GLs, Ganodermalucidum spores. For A, — % —, Glfb–H 2 O; — : —, Glfb–ETOH; — & —, Gls– H 2 O; —! —, Gls–ETOH.




the observation that ethanol extract had strongergrowth inhibition effect on MTC-11 cells.

3.2. Effects of Ganoderma lucidum extracts

on actin polymerization

F/G-actin ratio reects the actin polymerizationstatus in a cell. In general, a high F/G-actin ratiorepresents a more differentiated cellular status.Table 1 shows the effects of various Ganodermaextracts on actin polymerization, as reected by theF/G-actin ratio measured by DNase I inhibitionassay on HUC-PC and MTC-11 cells. Two inde-pendent experiments were performed. Cells weretreated with each extract at non-cytotoxic concen-

trations (40–80 mg/ml) for 24 h. As expected, in theuntreated controls, the F/G-actin ratio in untrans-formed HUC-PC cells (1.02 G 0.03) was slightlyhigher than the F/G-actin ratio in transformed MTC-11 cells (0.84 G 0.10) (P ! 0.05 by Student’s t -test).A dose–response increase of the F/G-actin ratio wasobserved on both cell lines when treated with waterextracts, but ethanol extracts caused less of anincrease. Generally, the effect appeared moreprominent in the transformed MTC-11 cells thanin HUC-PC cells.

3.3. Effects of Ganoderma lucidum on cellular migration

We previously observed that in the humanurothelial cell (HUC) cells (including HUC-PC andMTC11 cells), increased actin polymerizationenhances cell attachment and adhesions, therebydecreasing cell motility and spread (unpublisheddata). To determine the functional impact of alteredactin polymerization of G. lucidum extracts on cellmotility, a scratch wound assay was performed. In thisassay, a uniform cell-free area was created byscratching conuent monolayers with a sterile plasticpipet tip. HUC-PC and MTC-11 cells treated with200 mM 4-ABP were incubated for 24 h in thepresence of each extract at doses of 40 or 80 mg/ml.4-ABP, a known bladder cancer carcinogen, inducescell migration. Fig. 2a and b show the results of HUC-PC cells and MTC11 cell migration, respectively. Thedistance cells migrated was inspected at 12-h timeintervals. Compared to untreated control, 4-ABPenhanced the migration capability of both celllines. However, the increased cell migration wassuppressed when each of the Ganoderma extracts (40or 80 mg/ml) were added to 4-ABP treated cells. Aclear dose–response effect was not observed, how-

ever, in either cell line. At 24 h intervals spore waterextract at 80 mg/ml completely counteracted the cellmigration effect exerted by 4-ABP in both cell lines.

3.4. Effects of Ganoderma lucidum on expressionsof MMP-2 and FAK (total and phospholated)and formations of focal adhesion complex

To elucidate the potential mechanisms involved incell migration inhibition by G. lucidum extracts, werst examined how these extracts modulate focal

adhesion complex formation. Cells with or withoutthe treatment of 80 mg/ml of various extracts for 24 hwere xed, followed by triple-immunouorescencelabeling for F-actin, paxillin, and DNA. Fig. 3 showsthe results obtained from MTC-11 cells, similarndings were obtained from HUC-PC cells (resultsnot presented). As shown in Fig. 3a, cells treatedwith various G. lucidum extracts demonstratedincreased stress ber formation (green uorescence)and increased focal adhesion complex formation(red uorescence) at the periphery of cells in

Table 1Dose–response effect of Ganoderma lucidum extracts on theincrement of F/G-ratio in MTC-11 and HUC-PC cells

Control Concentration(mg/ml) HUC-PC MTC-111.02G 0.03 0.84 G 0.10

GLs–H 2O 40 1.30G 0.18 0.93 G 0.1280 1.80G 0.12** 1.20G 0.15**

GLs–ETOH 40 1.20 G 0.23 0.83 G 0.2380 1.02G 0.18 0.96 G 0.20

GLfb–H 2 O 40 1.11G 0.15 0.87 G 0.0080 1.20G 0.11* 1.08 G 0.22*

GLfb–ETOH 40 0.81 G 0.30 0.81 G 0.2080 0.99G 0.25 0.99 G 0.02

MTC-11 and HUC-PC cells were treated with various extracts atconcentrations of 40, 80 mg/ml. F/G-ratios were determined byDNase I inhibition assay, as described in Section 2. The incrementof F/G-ratio was calculated using the percentage of increase of F/G-ratio in the treated sample versus the parallel-untreated controlsample (i.e. (sample F/G-ratio K control F/G-ratio)/control F/G-ratio ! 100%). Values represent mean G SD of three independentexperiments. GLfb, Ganoderma lucidum fruiting bodies; GLs,Ganoderma lucidum spores. E, ethanol extract; H, water extract.*P ! 0.05 and ** P ! 0.01 by ANOVA test.




a circumferential manner. However, immunoblotanalysis revealed that the levels of phospho-FAK,total FAK, and MMP2, were unchanged in untreatedcontrol cells versus cells treated with various extractsat a concentration of 80 mg/ml for 24 h. Interestingly,MMP2, an important enzyme involved in tumor cellinvasion [16] , was almost undetectable in the MTC-11cells. These ndings suggested that other mechanismsmight be involved in actin remodeling of G. lucidumextracts in our model system.

4. Discussion

In this study we characterized the cell growth,migration and actin polymerization effects of medic-inal plant G. lucidum aqueous and alcohol extracts onan in vitro urothelial cell model. The puried, dietarysupplements of both fruiting bodies and spores wereextracted with ethanol and water to obtain triter-penoid- and polysaccharide-enriched fractions,

respectively. Our results show that G. lucidum inhibitscell growth, and at non-cytotoxic concentrations itinhibits 4-ABP-induced migration and induces actinpolymerization as well as focal adhesion complexformation. Both fruiting body aqueous and alcoholextracts inhibit the growth and induced G 2 /M arrest;however, slightly higher inhibitory activity wasobserved in cells treated with alcohol extracts.Variations of the effects on actin remodeling wereobserved in different forms of extracts, which ingeneral correlated with their activities on motilityinhibition. These observations suggest that thisnatural product may mediate its chemopreventiveeffects via multiple components with multiple mech-anisms of action.

Many studies on extracts and substances fromG. lucidum have demonstrated their anti-tumoractivities. Chemical studies on the alcohol-solubleextracts and their ethyl acetate fractions of the fruitingbodies, mycelia and spores of G. lucidum resulted inthe isolation and structure determination of more than

Fig. 2. Dose- and time- effect of migration inhibition by G. lucidum on HUC-PC (a) and MTC-11 (b) cells. Conuent monolayers of HUC-PCand MTC-11 cells were scratch wounded with a sterile plastic pipet tip ( t Z 0 h) and then cultured in medium in the presence of 4-ABP andGanoderma extract (40 or 80 mg/ml). The wound area was inspected at 12 and 24 h intervals to determine distance cells migrated. The y-axis isthe distance cells migrated ( mm). GLfb, Ganoderma lucidum fruiting bodies; GLs, Ganoderma lucidum spores; E, ethanol extract; H, waterextract.




100 highly oxygenated lanostanoid-type triperenes.Some 20 of these G. lucidum triterpenes, of a diversechemical nature, were shown to be cytotoxic against apanel of human and murine tumor cell lines such asHep G2, P388, Hepatoma, S180, T47D, LLC, Meth-A[17] . In investigating the mechanism of the cytotox-icity of the G. lucidum spores, Zhu et al. [18] showed

that the active ethyl acetate fraction obtained fromthe alcohol extract altered the cell cycle and cellularsignal transduction by modulating the calcium trans-port system. They suggested that the decrease inintracellular calcium was one of the reasons for the G 1

arrest of the HeLa cell cycle, which resulted in cellgrowth inhibition in the presence of the G. lucidum

Fig. 3. Effects of Ganoderma lucidum on MMP-2 expression, FAK activity, and focal adhesion complex formation. (A) MTC-11 cells inmonolayer culture were either with or without (control) 80 mg/ml of various Ganoderma lucidum extracts for 24 h, and stained for F-actin(stress bers, green uorescence), paxillin (adhesion complex, red uorescence), and DNA (nuclei, blue uorescence). Except for GLs–H2 O, other three types of Ganoderma lucidum extracts induced an increased formation of stress ber and adhesion complex at around theentire cell periphery. (B) Immunblot analysis of phospho-FAK, total FAK, and MMP2. No difference in expression of phospho-FAK, total

FAK, or MMP2 between control and cells treated with 80 mg/ml of various Ganoderma lucidum extracts for 24 h were observed. MMP2expression was almost undetectable in MTC-11 cells.




spore extracts. Hu et al. [5] recently reported thatalcohol extract of G. lucidum fruiting bodies inhibitedbreast cancer cell MCF-7 proliferation through up-

regulation of p21 and down-regulation of cyclin D1,which induced apoptosis through overexpression of Bax in human breast cancer cells. Recently, inhibitorytriterpenoids for farnesyl protein transferase-cata-lyzed post-translational farnesylation of Ras proteinwere isolated from fruiting bodies of G. lucidum [19]and identied as ganoderic acid A and ganoderic acidC. Farnesyl protein transferase inhibitors also inhib-ited Ras-dependent cell transformation. Our ndingthat extracts of G. lucidum directly modulate actinpolymerization suggests a novel mechanism of actionfor the anti-tumor effects of this mushroom.

Ikekawa et al. [20] rst reported in 1968 that hotwater extracts obtained from G. lucidum fruitingbodies showed a marked anti-tumor activity againsttransplanted sarcoma 180 in mice. Since then, anumber of polysaccharides demonstrating anti-tumoractivity have been isolated from the G. lucidumfruiting bodies [21–26] , cultured mycelia [27] andG. lucidum spores [28,29] . Multiple studies sub-sequently conrmed that the anti-tumor polysacchar-ides do not have direct cytotoxicity [26,27,30,31]against tumor cells, but showed profound immuno-

modulatory effects on the host-immune system[28,29,32] . Using an in vitro culture system, Wanget al. [32] performed a detailed analysis of the effectsof a G. lucidum polysaccharide (PS-G) on thefunctions of macrophages and T-lymphocytes, andon the growth of leukemic cells. They demonstratedthat (1) PS-G had a strong stimulatory effect on bothmacrophages and T-cells in promoting the synthesisand release of various cytokines (IL-1 b , IL-6, IFN g ,TNFa ) which are important in mounting an effectivecell-mediated anti-tumor response; (2) PG-S itself and

normal mononuclear cell culture medium (MNC-CM)had no tumoricidal activity, but conditioned mediafrom PSG-activated mononuclear cells (PSG-MNC-CM) strongly inhibited leukemic cell growth; and(3) IFNg and TNF a neutralizing antibodies greatlyreduced PSG-MNC-CM-induced growth inhibition of the tumor cells. These results conrm that the anti-tumor activity of PSG-MNC-CM was derived mainlyfrom the elevated cytokines, especially IFN gand TNF a , which induced apoptosis and differen-tiation in the PSG-treated leukemic cell lines,

including HL-60 [32] , U937 [31,32] , and K562 [33].Thus, anti-tumor polysaccharides of G. lucidum area group of biological response modiers (BRM) or

immunopotentiators. A recent study demonstratedthat G. lucidum spore and fruiting body aqueousextract inhibited the cell migration of highly invasivebreast MDA-MB-231 and prostate PC-3 cancer cellsby suppressing the transcription factors AP-1 and NF-kB, the expression of urokinase-type plasminogenactivitor uPA, uPA receptor uPAR, and the secretionof uPA [6].

Previous studies have showed that upon exposureto carcinogen 4-ABP, untransformed PC cells couldbe induced to transformation, whereas the trans-

formed MTC-11 cells could progress into a high-grade form [11,12] . Thus, these cell lines provide auseful in vitro model to examine the efcacy of chemopreventive agents on bladder carcinogenicprocesses and progression. Our results demonstratethat various Ganoderma extracts have an antagonisticeffect on 4-ABP-induced bladder carcinogenic pro-cess in cell migration. The inhibition of migration isassociated with actin remodeling and redistribution of focal adhesion complex [34] . It should be noted thatcellular motility is a process that is closely coordi-

nated by actin remodeling and cell/substratumadhesive interaction with maximum speed occurringat an intermediate adhesiveness [34] . At higheradhesiveness, as observed in this study in cells treatedwith Ganoderma extracts, cells are unable to break attachment, which leaves them extended. This sup-ports our observation that motility inhibition isassociated with increased actin polymerization andstress ber formation.

Bladder cancer, like other malignant cancers,develops through multiple genetic and epigeneticalterations that lead to altered growth, differentiation,and apoptosis control [35,36] . The aggressiveness of cancer is characterized by tumor invasion andmetastasis, which is at least partially associated withan increased ability of cells to migrate among others.Growth, migration, and differentiation are controlled,at least in part, by endocrine/paracrine mechanismsinvolving external signals, usually peptides, that bindto cell surface receptors linked to proteins in the innercell membrane that, in turn, generate internal secondmessenger substances [35] . These second messengers




induce changes in both the cytoplasm and the nucleusthat lead to cell division.

A number of studies have demonstrated that

monitoring the change of these proteins, e.g. actinpolymerization status, as measured by an increase inF/G-actin ratio (increased F-actin or decreasedG-actin), is a marker of cellular differentiation [37] .Active studies are underway to develop actin-targetingchemopreventive/chemotherapeutic agents. Examplesof actin polymerization chemicals include cytochala-sins (B, D and E), an actin depolymerization agent, andJasplakinolide (Jas), an actin over-polymerizationagent [38–40] . Our current study shows that Gano-derma extracts have dramatic effects in stimulatinggrowth inhibition, G 2 /M arrest, and actin polymeriz-ation as well as stress ber formation. Since actinpolymerization is a cell cycle-related event, in thatactin polymerization is increased in late G 1 and G2 /Mphases of the cell cycle [13], one may assume thatthe observed growth inhibition and G 2 /M may beassociated with actin remodeling. However, the exactrelationships between actin remodeling, G 2 /M arrest,and growth inhibition remain to be elucidated.Since actin remodeling is regulated by a number of signal transduction pathways, the most notable one isRas superfamily GTPase such as Rac, Rho, and

CDC42, the exact upstream events in the observedeffect of Ganoderma exacts remain to be determinedas well.

Given that these mushrooms have been consumedfor generations by Chinese, a direct next step might beto study a small group of patients and monitor thedifferentiating effect of these chemicals using F/G-actin level as a surrogate marker, as has been reportedpreviously in patients treated with DMSO [41] . Since,multiple signaling pathways may be involved in theobserved effect of these mushroom extracts, in

particular regarding to actin remodeling, monitoringF/G-actin ration may be more advantageous thanexamining specic upstream molecular events of aparticular pathway to predict response in clinical trialsinvolving these chemicals.

The results of this study indicate that extracts of G. lucidum are capable of inhibiting human bladdercancer cell growth and migration in vitro. Multiplemechanisms may be responsible for the anti-tumoreffects of G. lucidum . The anti-tumor properties of G. lucidum may attribute to its chemically diversied

constituents, and may implicate its use as a potentialeffective chemopreventive or therapeutic agent.Further studies are required to dene the operative

signaling pathways within the context of microeco-systems of bladder cancer patients.

Acknowledgements

Research supported by a research grant from theUCLA Center for Dietary Supplement Research inBotanicals (NIH Grant No. U01 Ca96116 andAT00151), UCLA Lung Cancer SPORE (NIH GrantNo. P50 CA90833) and the Starr Foundation.Frederick P. Li, MD is a Harry and Elsa JilerAmerican Cancer Society Clinical Research Pro-fessor. We are grateful to Pharmanex Inc. forproviding us with the source of fruiting bodies.

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Ganoderma Lucidum Extracts Inhibit Growth and Induce Actin Polymerization in Bladder Cancer in Vitro