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Cancer Letters 216 (2004) 9–20

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Ganoderma lucidum extracts inhibit growth and induce actin


polymerization in bladder cancer cells in vitro
Qing-Yi Lua, Yu-Sheng Jinb, Qifeng Zhanga, Zuofeng Zhangc, David Hebera,
Vay Liang W. Goa, Frederick P. Lid, Jian Yu Raob,*
a
Center for Human Nutrition, Department of Medicine, David Geffen School of Medicine, University of California, Los Angeles, CA 90095, USA
b
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, USA
d
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 human
urothelial cell (HUC) model consisted of HUC-PC cells and MTC-11 cells. Ethanol and water extracts of fruiting bodies and
spores of the G. lucidum were used to examine growth inhibition, actin polymerization status, and impact of actin remodeling
on 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 G2/M arrest. At non-cytotoxic concentrations
(40–80 mg/ml), these extracts induced actin polymerization, which in turn inhibited carcinogen 4-aminobiphenyl induced
migration in both cell lines. The increased actin polymerization was associated with increased stress fibers and focal adhesion
complex formation, however, expression of matrix metalloproteinase-2 and focal adhesion kinase (total and phospholated) were
unchanged, 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 ‘Lingzhi’. Its fruiting bodies have been used for their
medicinal properties in traditional Chinese medicine
Ganoderma lucidum (Fr.) Karst. (Polyporaceae) is for over 2000 years. This mushroom is described in
a medicinal mushroom known to the Chinese as detail in the Chinese Materia Medica classics, Shen
Nung Ben Cao Jin (dated 206 BC–8 AD), and the
Compendium of Materia Medica. G. lucidum has been
* Corresponding author. Address: Department of Pathology used in traditional Chinese medicine for promotion of
and Laboratory Medicine, UCLA School of Medicine, Box 951732,
Los Angeles, CA 90095-1732, USA. Tel.: C1-310-794-1567;
vitality and longevity, and more recently used in
fax: C1-310-206-5178 the treatment of debility and weakness, insomnia,
E-mail address: jrao@mednet.ucla.edu (J.Y. Rao). 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
10 Q.-Y. Lu et al. / Cancer Letters 216 (2004) 9–20

sickness, cardiovascular disease, AIDS, and cancer the polymeric filaments and monomeric globules,
[1–3]. In addition to the fruiting bodies, the spores and respectively, reflects cellular differentiation versus
cultivated mycelia of Ganoderma have been recently dedifferentiation status [12]. In general, cell differen-
consumed as health food and herbal medicines. tiation is associated with an increased F/G-actin ratio,
Chemical investigations on the fruiting bodies, spores, whereas dedifferentiation and malignant transform-
and mycelia of G. lucidum reveal that they contain ation is associated with a decreased F/G-actin ratio
various bioactive substances. Active constituents of [13,14]. Thus, the ratio of F-actin to G-actin functions
G. lucidum include polysaccharides, proteins, nucleo- as a surrogate marker for cellular differentiation and
sides, fatty acids, terpenoids, sterols, and cerebrosides dedifferentiation [13–15]. Since, actin is a major
[4]. Water or alcohol extracts of G. lucidum have been cytoskeletal protein involved in migration, the func-
used to investigate biological activities, as these tional signification of actin polymerization on cell
extracts contain compounds mainly responsible for motility was examined by ‘wound scratching assay’.
the immunological and anti-inflammatory properties. Mechanism for growth inhibition was investigated by
Alcohol extracts contain biological compounds that Laser Scanning Cytometry (LCS) based cell cycle
lower blood cholesterol and glucose levels, blood analysis and mechanism for migration inhibition was
pressure, and inhibit histamine release, while the studied by immunofluorescence analysis of adhesion
liver-protective, antiviral and anti-tumor effects complex, immunoblot analysis of matrix metallopro-
can be attributed to both the water-soluble polysac- teinase-2 (MMP-2) expression, and focal adhesion
charides and alcohol-soluble triterpenes [3,4]. Of kinase (FAK) activities.
particular interest among the reported biological/
pharmacological properties of G. lucidum are their
anti-tumor activities, including the effects on cell 2. Materials and methods
cycle arrest, apoptosis induction, motility inhibition,
antiangiogenic, and antimutagenic activities [3,5–10]. 2.1. Materials
The primary objective of this study was to evaluate
the chemopreventive effects of G. lucidum extracts G. lucidum spore powder was obtained from
using our unique in vitro human bladder cancer model Zhongke Capsule (Nanjing, China), and G. lucidum
consisting of two cell lines, both derived from the fruiting body powder was provided by Pharmanex
same human urothelial clone immortalized by SV-40. Inc. (Provo, UT). All solvents used for extraction are
In this model, HUC-PC is an untransformed cell line HPLC grade and were purchased from Fisher
that does not form tumor nodules when inoculated into Scientific. 4-Aminobiphenyl (4-ABP) was purchased
nude mice, and MTC-11 is a transformed low-grade from Sigma (St Louis, MO).
tumor that forms tumor nodules when inoculated
into nude mice [11]. After exposure to carcinogen 2.2. Extract preparation
4-aminobiphenyl (4-ABP), the HUC-PC cells can be
transformed to malignant and the MTC-11 cells Extraction was performed on both spores and
induced to form a high-grade tumor [11]. This cell fruiting bodies. Briefly, powder from spores or
culture system provides a unique model to rapidly test fruiting bodies was first extracted with 95% ethanol
the effect of chemopreventive agents on bladder and then with water by successive sonication at room
cancer in association with 4-ABP, a major carcino- temperature for 30 min. Extracts were filtered and
genic component found in cigarette smoke. In this filtrate was concentrated under reduced pressure to
study, the growth inhibition effect of various Gano- yield dark brown powder (water extracts) and oily
derma extracts on both cell lines was analyzed by [3H] residue (alcohol extracts).
thymidine incorporation assay and calorimetric tetra-
zolium (MTT) assay. In addition, dose–response 2.3. Cell culture
effects on actin polymerization were determined by
DNase I inhibition assay analysis. The quantitative Both HUC-PC and MCT-11 cells were grown
relationship between cytoplasmic F- and G-actin, in 90% F-12 nutrient mixture (Ham) medium
Q.-Y. Lu et al. / Cancer Letters 216 (2004) 9–20 11

(GIBCO BRL Island, NY) with 1% penicillin and The plates were then read on a Microplate Reader
streptomycin (10,000 mg/ml penicillin, 10 mg/ml Bio-Rad 550. Cell survival was calculated as the
streptomycin) and 10% FBS. Cultures were main- percentage of MTT inhibition, using the following
tained at 37 8C in 5% CO2 and 95% air, and medium formula:
changed two times per week. G. lucidum extracts were  
meanexperimentalabsorbance
dissolved in either dd H2O or ethanol to make a stock %survivalZ !100:
meancontrolabsorbance
solution of 10 mg/ml. 4-ABP was dissolved in 100%
dimethyl sulfoxide (DMSO) to make a stock solution
2.6. Determination of cellular F/G-actin ratio
of 100 mmol/ml. Logarithmically growing HUC-PC
and MTC-11 cells were harvested and seeded at an
Cells at various time points and conditions, as
initial density of 2!105 cells in 20 ml of fresh
described above, were harvested and F/G-actin ratio
medium in 60-mm petri dishes.
was determined by a biochemical DNase I inhibition
assay. This assay has been described extensively
2.4. [3H] thymidine incorporation assay
previously [12].
Cells were plated into 48-well culture plates (1! 2.7. Analysis of focal adhesion complex by triple-label
104 cells/well) for 24 h to allow for attachment. immunofluorescence
Subsequently, fresh medium (400 ml/well) containing
different concentrations of mushroom extracts was In this assay, cells were cultured directly on 1 cm
added and incubated at 37 8C with 5% CO2 for 24 h. diameter cover glass (Fisher Scientific, Pittsburgh,
[3H] thymidine (DuPont NEN, Boston, MA) at a final PA) placed on a 24-well flat-bottom plate. At the end
concentration of 1 mCi/ml was added 4 h prior to of the treatment, culture medium was aspirated and
termination of the experiment. The cells were rinsed cells on the cover glass were washed twice with PBS
twice with PBS and fixed with 10% trichloroacetic and then fixed with 3.7% formaldehyde solution in
acid at room temperature (200 ml/well) for 15 min, PBS for 20 min at room temperature. After fixation,
and again rinsed twice with 100% EtOH. Cells were cells on the cover glass were incubated sequentially
then lysed with 0.4 N NaOH 200 ml/well and heated with following reagents: 1% BSA for 30 min, 1:100
at 65 8C for 30 min. After the plate was cooled, monoclonal anti-Paxillin (clone 5H11, Upstate Bio-
100 ml/well glacial acetic acid was added and mixed technology) for 1 h, 1:150 Cy3-conjugated AffiniPure
in a shaker. Scintillation vials were prepared with Goat Anti-Mouse IgG (HCL) (Jackson ImmunoR-
4 ml scintillation fluid/bottle, and 100 ml of cell esearch lab Inc.) for 30 min, 1:40 Bodipy phallacidin
lysate was added to each vial. The vial was vortexed (for F-actin) (Molecular Probes Inc., Eugene, OR) for
well until the volume was clear, and the radioactivity 30 min, and 1:1000 dilution of 4 0 ,6-diamidino-2-
was determined. phenylindole (DAPI) (10 mg/ml Molecular Probes
Inc., Eugene, OR) for 5 min. Between each incubation
2.5. MTT assay step, the cover glass was rinsed with PBS three times.
The stained cover glass was then transferred onto a
The cytotoxicity of each extract was determined by regular microscopic slide, which was then mounted in
a calorimetric tetrazolium (MTT) assay. Briefly, MTT 100 mM n-propyl gallate (Sigma Chemical Co., St
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazo- Louis, MO) in spectranalyzed glycerol (Fisher
lium bromide; Sigma, MI) was dissolved in PBS at Scientifc), pH 6.5, for fluorescence microscopic
5 mg/ml and filtered. Serial triplicate dilutions of examination and cell cycle analysis by LCS.
the extract at 50 ml in volume were added to
1.5!104 cells/ml to 96-well flat-bottomed plates. 2.8. Cell cycle analysis by laser scanning
Plates were incubated for 24 h at 37 8C, pulsed with cytometry (LCS)
10 ml MTT (5 mg/ml) and incubated for 4 h at 37 8C.
DMSO (100 ml) was then added to all wells and The cell cycle was analyzed using laser scanning
mixed thoroughly for 30 min at room temperature. cytometer (CompuCyte, Cambridge, MA) by
12 Q.-Y. Lu et al. / Cancer Letters 216 (2004) 9–20

measuring the DAPI fluorescence and the peak 2.11. Statistical analysis
intensity of fluorescence in the cell nuclei. Fluor-
escence excitation was provided by a 488 nm laser Descriptive statistics, such as mean and standard
line. The blue fluorescence was measured using error, were used to summarize the results.
standard settings of LCS dichroic mirrors and The Student’s t-test and ANOVA test were used for
emission filters. Multicycle software (Phoenix univariate analysis. Statistical significance was
Flow System) and the software provided with the defined by a two-tailed P-value of 0.05.
LSC were used to deconvolute histograms to
estimate proportions of cells in particular phases
of the cycle. 3. Results

3.1. Growth inhibition and cell cycle analysis


2.9. Immunoblot analysis for MMP-2 and FAK
of Ganoderma lucidum extracts on HUC-PC
and MTC-11 cells
Cells that were washed twice in cold PBS were
scraped from culture dishes in lysis buffer (50 mM
To examine the growth inhibition of G. lucidum
Tris–HCl (pH 7.4), 150 mM NaCl, 2 mM EGTA,
extracts, HUC-PC and MTC-11 cells were exposed
MgCl2 2 mM, 1% (v/v) Triton X-100, 10% glycerol,
to various concentrations of the extracts for 24 h and
10 mM DTT, 1 mM phenylmethylsulfonyl fluoride,
then subjected to [3H] thymidine incorporation assay
10 mg/ml leupeptin, 10 mg/ml aprotinin, 5 mg/ml
(Fig. 1a). Both cells were exposed to incremental
pepstatin A, 50 mM NaF, and 1 mM NaVO4).
concentrations of four extracts (15–1000 mg/ml) and
Lysates were centrifuged at 12,000!g and 4 8C
incubated for 24 h. Cell survival was calculated as
for 10 min. Protein concentrations of lysates were
the percentage of [3H] thymidine incorporation assay
determined by Bio-Rad Protein Assay (Bio-Rad
(% surviva1Zmean experimental absorbance/mean
Laboratories, Hercules, CA). For IB analyses, the
control absorbance!100). Overall, the growth inhi-
same concentrations of proteins (30 or 50 mg) were
bition effects of these extracts were similar for both
subjected to 8% SDS-PAGE and were electrotrans-
cell lines, and alcohol extracts appear to be more
ferred to nitrocellulose membranes using electroblot
potent than water extract for both cell lines. Fruiting
buffers. Membranes were blocked in PBS containing
body ethanol extract was most potent in inhibiting
5% non-fat dry milk for 30 min. Reactions with the
untransformed HUC-PC cell line with its concen-
primary antibodies in TBS buffer containing 3% dry
tration producing 50% inhibition (IC50) being
milk were carried out at 4 8C overnight. After
124 mg/ml, followed by spore ethanol extract
extensive washing, membranes were placed on a
(IC50Z280 mg/ml), spore water extract (IC50Z
shaker with biotinylated secondary IgG for 1 h.
500 mg/ml), and fruiting body water extract (IC50Z
Upon further washing, membranes were reacted
1000 mg/ml). Similarly, fruiting body ethanol extract
with streptavidin-horseradish peroxidase for 45 min
was most potent (IC50Z113 mg/ml) in inhibiting
and ECL detection reagents immediately prior to
transformed MTC-11 cell line, followed by spore
autoradiography.
ethanol extract (IC50Z234 mg/ml), spore water
extract (IC50Z465 mg/ml), and fruiting body water
2.10. Wound-scratch assay for cellular migration extract (IC50Z990 mg/ml).
The cytotoxicity effects of G. lucidum extracts
A wound-scratch assay was performed to were further examined by MTT assay. The IC50 of
examine the cellular migration. In this assay, a HUC-PC cells by fruiting body ethanol extract,
uniform cell-free area was created by scratching spore ethanol extract, spore water extract, and
confluent monolayers with a plastic pipet tip, and fruiting body water extract were 325, 521, 362,
the wound area was inspected at different time and 1000 mg/ml, respectively. The IC50 of MTC-11
intervals to determine the distance migrated by the cells by fruiting body ethanol extract, spore ethanol
cells. extract, spore water extract, and fruiting body water
Q.-Y. Lu et al. / Cancer Letters 216 (2004) 9–20 13

Fig. 1. Effects of G. lucidum extracts on the proliferation (A) and cell cycle (B) of HUC-PC and MTC-11 cells, using [3H] thymidine
incorporation 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 [3H] 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 each
Ganoderma extract for 24 h on cover glass underwent triple-label immunofluorescence including paxillin, F-actin, and DNA, as detailed in
Section 2. Cell cycle analysis was performed using Laser Scanning Cytometry. GLfb, Ganoderma lucidum fruiting bodies; GLs, Ganoderma
lucidum spores. For A, —%—, Glfb–H2O; —:—, Glfb–ETOH; —&—, Gls– H2O; —!—, Gls–ETOH.

extract were 129.3, 274.7, 365.4, and 509 mg/ml, extracts. As shown in Fig. 1b, G. lucidum extracts at
respectively, which were similar to the above results this non-cytotoxic concentration induced G2/M arrest
obtained by [3H] thymidine incorporation assay. The in both HUC-PC and MTC-11 cells. In HUC-PC cells,
IC50 of ethanol extracts in HUC-PC cells was higher the percentage of cells in the G2/M phase increased
than that of MTC11 cells, suggesting the untrans- from 25% in untreated control cells to 31–35% in cells
formed HUC-PC cells was more resistant to the treated with various G. lucidum extracts. In MTC-11
cytotoxicity of ethanol extracts than the MTC-11 cells, the percentage of cells in the G2/M phase
cells. increased from 15% in untreated control cells to
Cell cycle analysis using LCS was performed 25–27% in cells treated with ethanol extracts of
on cells treated with 40 mg/ml (about or less than G. lucidum, but only to 17–18% in cells treated with
one-third of IC50) for 24 h with various G. lucidum water extracts. These findings were consistent with
14 Q.-Y. Lu et al. / Cancer Letters 216 (2004) 9–20

Table 1 3.3. Effects of Ganoderma lucidum on cellular


Dose–response effect of Ganoderma lucidum extracts on the migration
increment of F/G-ratio in MTC-11 and HUC-PC cells

Control Concentration HUC-PC MTC-11 We previously observed that in the human


(mg/ml) 1.02G0.03 0.84G0.10 urothelial cell (HUC) cells (including HUC-PC and
GLs–H2O 40 1.30G0.18 0.93G0.12 MTC11 cells), increased actin polymerization
80 1.80G0.12** 1.20G0.15** enhances cell attachment and adhesions, thereby
GLs–ETOH 40 1.20G0.23 0.83G0.23
decreasing cell motility and spread (unpublished
80 1.02G0.18 0.96G0.20
GLfb–H2O 40 1.11G0.15 0.87G0.00 data). To determine the functional impact of altered
80 1.20G0.11* 1.08G0.22* actin polymerization of G. lucidum extracts on cell
GLfb–ETOH 40 0.81G0.30 0.81G0.20 motility, a scratch wound assay was performed. In this
80 0.99G0.25 0.99G0.02 assay, a uniform cell-free area was created by
MTC-11 and HUC-PC cells were treated with various extracts at scratching confluent monolayers with a sterile plastic
concentrations of 40, 80 mg/ml. F/G-ratios were determined by pipet tip. HUC-PC and MTC-11 cells treated with
DNase I inhibition assay, as described in Section 2. The increment 200 mM 4-ABP were incubated for 24 h in the
of F/G-ratio was calculated using the percentage of increase of F/G- presence of each extract at doses of 40 or 80 mg/ml.
ratio in the treated sample versus the parallel-untreated control
sample (i.e. (sample F/G-ratioKcontrol F/G-ratio)/control F/G-
4-ABP, a known bladder cancer carcinogen, induces
ratio!100%). Values represent meanGSD of three independent cell migration. Fig. 2a and b show the results of HUC-
experiments. GLfb, Ganoderma lucidum fruiting bodies; GLs, PC cells and MTC11 cell migration, respectively. The
Ganoderma lucidum spores. E, ethanol extract; H, water extract. distance cells migrated was inspected at 12-h time
*P! 0.05 and **P!0.01 by ANOVA test. intervals. Compared to untreated control, 4-ABP
enhanced the migration capability of both cell
the observation that ethanol extract had stronger lines. However, the increased cell migration was
growth inhibition effect on MTC-11 cells. suppressed when each of the Ganoderma extracts (40
or 80 mg/ml) were added to 4-ABP treated cells. A
3.2. Effects of Ganoderma lucidum extracts clear dose–response effect was not observed, how-
on actin polymerization ever, in either cell line. At 24 h intervals spore water
extract at 80 mg/ml completely counteracted the cell
F/G-actin ratio reflects the actin polymerization migration effect exerted by 4-ABP in both cell lines.
status in a cell. In general, a high F/G-actin ratio
represents a more differentiated cellular status. 3.4. Effects of Ganoderma lucidum on expressions
Table 1 shows the effects of various Ganoderma of MMP-2 and FAK (total and phospholated)
extracts on actin polymerization, as reflected by the and formations of focal adhesion complex
F/G-actin ratio measured by DNase I inhibition
assay on HUC-PC and MTC-11 cells. Two inde- To elucidate the potential mechanisms involved in
pendent experiments were performed. Cells were cell migration inhibition by G. lucidum extracts, we
treated with each extract at non-cytotoxic concen- first examined how these extracts modulate focal
trations (40–80 mg/ml) for 24 h. As expected, in the adhesion complex formation. Cells with or without
untreated controls, the F/G-actin ratio in untrans- the treatment of 80 mg/ml of various extracts for 24 h
formed HUC-PC cells (1.02G0.03) was slightly were fixed, followed by triple-immunofluorescence
higher than the F/G-actin ratio in transformed MTC- labeling for F-actin, paxillin, and DNA. Fig. 3 shows
11 cells (0.84G0.10) (P!0.05 by Student’s t-test). the results obtained from MTC-11 cells, similar
A dose–response increase of the F/G-actin ratio was findings were obtained from HUC-PC cells (results
observed on both cell lines when treated with water not presented). As shown in Fig. 3a, cells treated
extracts, but ethanol extracts caused less of an with various G. lucidum extracts demonstrated
increase. Generally, the effect appeared more increased stress fiber formation (green fluorescence)
prominent in the transformed MTC-11 cells than and increased focal adhesion complex formation
in HUC-PC cells. (red fluorescence) at the periphery of cells in
Q.-Y. Lu et al. / Cancer Letters 216 (2004) 9–20 15

Fig. 2. Dose- and time- effect of migration inhibition by G. lucidum on HUC-PC (a) and MTC-11 (b) cells. Confluent monolayers of HUC-PC
and MTC-11 cells were scratch wounded with a sterile plastic pipet tip (tZ0 h) and then cultured in medium in the presence of 4-ABP and
Ganoderma 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 is
the distance cells migrated (mm). GLfb, Ganoderma lucidum fruiting bodies; GLs, Ganoderma lucidum spores; E, ethanol extract; H, water
extract.

a circumferential manner. However, immunoblot respectively. Our results show that G. lucidum inhibits
analysis revealed that the levels of phospho-FAK, cell growth, and at non-cytotoxic concentrations it
total FAK, and MMP2, were unchanged in untreated inhibits 4-ABP-induced migration and induces actin
control cells versus cells treated with various extracts polymerization as well as focal adhesion complex
at a concentration of 80 mg/ml for 24 h. Interestingly, formation. Both fruiting body aqueous and alcohol
MMP2, an important enzyme involved in tumor cell extracts inhibit the growth and induced G2/M arrest;
invasion [16], was almost undetectable in the MTC-11 however, slightly higher inhibitory activity was
cells. These findings suggested that other mechanisms observed in cells treated with alcohol extracts.
might be involved in actin remodeling of G. lucidum Variations of the effects on actin remodeling were
extracts in our model system. observed in different forms of extracts, which in
general correlated with their activities on motility
inhibition. These observations suggest that this
4. Discussion natural product may mediate its chemopreventive
effects via multiple components with multiple mech-
In this study we characterized the cell growth, anisms of action.
migration and actin polymerization effects of medic- Many studies on extracts and substances from
inal plant G. lucidum aqueous and alcohol extracts on G. lucidum have demonstrated their anti-tumor
an in vitro urothelial cell model. The purified, dietary activities. Chemical studies on the alcohol-soluble
supplements of both fruiting bodies and spores were extracts and their ethyl acetate fractions of the fruiting
extracted with ethanol and water to obtain triter- bodies, mycelia and spores of G. lucidum resulted in
penoid- and polysaccharide-enriched fractions, the isolation and structure determination of more than
16 Q.-Y. Lu et al. / Cancer Letters 216 (2004) 9–20

Fig. 3. Effects of Ganoderma lucidum on MMP-2 expression, FAK activity, and focal adhesion complex formation. (A) MTC-11 cells in
monolayer culture were either with or without (control) 80 mg/ml of various Ganoderma lucidum extracts for 24 h, and stained for F-actin
(stress fibers, green fluorescence), paxillin (adhesion complex, red fluorescence), and DNA (nuclei, blue fluorescence). Except for GLs–
H2O, other three types of Ganoderma lucidum extracts induced an increased formation of stress fiber and adhesion complex at around the
entire 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. MMP2
expression was almost undetectable in MTC-11 cells.

100 highly oxygenated lanostanoid-type triperenes. that the active ethyl acetate fraction obtained from
Some 20 of these G. lucidum triterpenes, of a diverse the alcohol extract altered the cell cycle and cellular
chemical nature, were shown to be cytotoxic against a signal transduction by modulating the calcium trans-
panel of human and murine tumor cell lines such as port system. They suggested that the decrease in
Hep G2, P388, Hepatoma, S180, T47D, LLC, Meth-A intracellular calcium was one of the reasons for the G1
[17]. In investigating the mechanism of the cytotox- arrest of the HeLa cell cycle, which resulted in cell
icity of the G. lucidum spores, Zhu et al. [18] showed growth inhibition in the presence of the G. lucidum
Q.-Y. Lu et al. / Cancer Letters 216 (2004) 9–20 17

spore extracts. Hu et al. [5] recently reported that including HL-60 [32], U937 [31,32], and K562 [33].
alcohol extract of G. lucidum fruiting bodies inhibited Thus, anti-tumor polysaccharides of G. lucidum are
breast cancer cell MCF-7 proliferation through up- a group of biological response modifiers (BRM) or
regulation of p21 and down-regulation of cyclin D1, immunopotentiators. A recent study demonstrated
which induced apoptosis through overexpression of that G. lucidum spore and fruiting body aqueous
Bax in human breast cancer cells. Recently, inhibitory extract inhibited the cell migration of highly invasive
triterpenoids for farnesyl protein transferase-cata- breast MDA-MB-231 and prostate PC-3 cancer cells
lyzed post-translational farnesylation of Ras protein by suppressing the transcription factors AP-1 and NF-
were isolated from fruiting bodies of G. lucidum [19] kB, the expression of urokinase-type plasminogen
and identified as ganoderic acid A and ganoderic acid activitor uPA, uPA receptor uPAR, and the secretion
C. Farnesyl protein transferase inhibitors also inhib- of uPA [6].
ited Ras-dependent cell transformation. Our finding Previous studies have showed that upon exposure
that extracts of G. lucidum directly modulate actin to carcinogen 4-ABP, untransformed PC cells could
polymerization suggests a novel mechanism of action be induced to transformation, whereas the trans-
for the anti-tumor effects of this mushroom. formed MTC-11 cells could progress into a high-
Ikekawa et al. [20] first reported in 1968 that hot grade form [11,12]. Thus, these cell lines provide a
water extracts obtained from G. lucidum fruiting useful in vitro model to examine the efficacy of
bodies showed a marked anti-tumor activity against chemopreventive agents on bladder carcinogenic
transplanted sarcoma 180 in mice. Since then, a processes and progression. Our results demonstrate
number of polysaccharides demonstrating anti-tumor that various Ganoderma extracts have an antagonistic
activity have been isolated from the G. lucidum
effect on 4-ABP-induced bladder carcinogenic pro-
fruiting bodies [21–26], cultured mycelia [27] and
cess in cell migration. The inhibition of migration is
G. lucidum spores [28,29]. Multiple studies sub-
associated with actin remodeling and redistribution of
sequently confirmed that the anti-tumor polysacchar-
focal adhesion complex [34]. It should be noted that
ides do not have direct cytotoxicity [26,27,30,31]
cellular motility is a process that is closely coordi-
against tumor cells, but showed profound immuno-
nated by actin remodeling and cell/substratum
modulatory effects on the host-immune system
adhesive interaction with maximum speed occurring
[28,29,32]. Using an in vitro culture system, Wang
et al. [32] performed a detailed analysis of the effects at an intermediate adhesiveness [34]. At higher
of a G. lucidum polysaccharide (PS-G) on the adhesiveness, as observed in this study in cells treated
functions of macrophages and T-lymphocytes, and with Ganoderma extracts, cells are unable to break
on the growth of leukemic cells. They demonstrated attachment, which leaves them extended. This sup-
that (1) PS-G had a strong stimulatory effect on both ports our observation that motility inhibition is
macrophages and T-cells in promoting the synthesis associated with increased actin polymerization and
and release of various cytokines (IL-1b, IL-6, IFNg, stress fiber formation.
TNFa) which are important in mounting an effective Bladder cancer, like other malignant cancers,
cell-mediated anti-tumor response; (2) PG-S itself and develops through multiple genetic and epigenetic
normal mononuclear cell culture medium (MNC-CM) alterations that lead to altered growth, differentiation,
had no tumoricidal activity, but conditioned media and apoptosis control [35,36]. The aggressiveness of
from PSG-activated mononuclear cells (PSG-MNC- cancer is characterized by tumor invasion and
CM) strongly inhibited leukemic cell growth; and metastasis, which is at least partially associated with
(3) IFNg and TNFa neutralizing antibodies greatly an increased ability of cells to migrate among others.
reduced PSG-MNC-CM-induced growth inhibition of Growth, migration, and differentiation are controlled,
the tumor cells. These results confirm that the anti- at least in part, by endocrine/paracrine mechanisms
tumor activity of PSG-MNC-CM was derived mainly involving external signals, usually peptides, that bind
from the elevated cytokines, especially IFNg to cell surface receptors linked to proteins in the inner
and TNFa, which induced apoptosis and differen- cell membrane that, in turn, generate internal second
tiation in the PSG-treated leukemic cell lines, messenger substances [35]. These second messengers
18 Q.-Y. Lu et al. / Cancer Letters 216 (2004) 9–20

induce changes in both the cytoplasm and the nucleus constituents, and may implicate its use as a potential
that lead to cell division. effective chemopreventive or therapeutic agent.
A number of studies have demonstrated that Further studies are required to define the operative
monitoring the change of these proteins, e.g. actin signaling pathways within the context of microeco-
polymerization status, as measured by an increase in systems of bladder cancer patients.
F/G-actin ratio (increased F-actin or decreased
G-actin), is a marker of cellular differentiation [37].
Active studies are underway to develop actin-targeting Acknowledgements
chemopreventive/chemotherapeutic agents. Examples
of actin polymerization chemicals include cytochala- Research supported by a research grant from the
sins (B, D and E), an actin depolymerization agent, and UCLA Center for Dietary Supplement Research in
Jasplakinolide (Jas), an actin over-polymerization Botanicals (NIH Grant No. U01 Ca96116 and
agent [38–40]. Our current study shows that Gano- AT00151), UCLA Lung Cancer SPORE (NIH Grant
derma extracts have dramatic effects in stimulating No. P50 CA90833) and the Starr Foundation.
growth inhibition, G2/M arrest, and actin polymeriz- Frederick P. Li, MD is a Harry and Elsa Jiler
ation as well as stress fiber formation. Since actin American Cancer Society Clinical Research Pro-
polymerization is a cell cycle-related event, in that fessor. We are grateful to Pharmanex Inc. for
actin polymerization is increased in late G1 and G2/M providing us with the source of fruiting bodies.
phases of the cell cycle [13], one may assume that
the observed growth inhibition and G2/M may be
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