Carcinogenesis, Vol. 25, No. 7, 1177-1183,
July 2004
Carcinogenesis vol.25 no.7 © Oxford University Press 2004; all rights reserved.
Medicinal mushroom extracts inhibit ras-induced cell transformation and the inhibitory effect requires the presence of normal cells
W. L. Wendy Hsiao1,2,4,
You Quan Li2,
Tin Lap Lee2,
Ning Li2,
Marilyn M. You3 and
Shu-ting Chang3
1 Biomedical Science, School of Chinese Medicine, Hong Kong Baptist University, Hong Kong, 2 Department of Biology, Hong Kong University of Science and Technology, Hong Kong and 3 Department of Biology, Chinese University of Hong Kong, Hong Kong, China
4 To whom correspondence should be addressed Email: bowhsiao{at}hkbu.edu.hk
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Abstract
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Previously, we developed a simple Rat 6 (R6) cell system by
which the inhibitory effects of non-cytotoxic chemicals can
be assessed by focus formation assay upon transfection of
ras oncogene to the host cells. Using this system, two well studied
medicinal mushrooms
Ganoderma lucidum and
Tricholoma lobayense with anticancer potential were examined for their possible advert
effects on cell transformation induced by
ras oncogene. Results
indicated that both species of mushrooms yielded strong inhibitory
effects on
ras-induced cell transformation. Further study on
T.lobayense indicated that the DEAE-column-bound, polysaccharides
(PS)-peptide enriched, but not the unbound fraction, showed
strong inhibition in a dosage-dependent manner. Subsequent time
course study revealed that the continued presence of the extract
in the transfected cultures was required for a maximum inhibitory
effect. At the same time, we also observed that significant
levels of inhibition occurred even when the application of the
extract was delayed until day 12 after transfection. Using a
stable transformed cell line, R6/GFP-Ras expressing green fluorescent
protein-
ras fusion protein in a co-culture assay with normal
R6 cells, we demonstrated that R6/GFP-Ras cells grew into green
fluorescent foci with striking transforming morphology in the
absence of extracts. However, in the presence of extracts, R6/GFP-Ras
cells, in most cases, remained as small colonies compiled with
only a few green fluorescent cells. Moreover, the inhibitory
effect requires the presence of R6 cells. In our study, mushroom
extracts have no effect on the growth of individually cultured
normal and transformed R6 cells. It is noteworthy that the extracts
do not affect the level, or the subcellular localization of
the Ras protein. Collectively, the data strongly suggest that
the inhibitory effect of the mushroom extracts is not due to
a direct killing of the transformed cells, rather, it may be
mediated through the surrounding normal R6. While the general
understanding of the antitumor effect of PS and PSPC is mediated
through the cytokines released by activated marcrophages and
T-lymphocytes, our data may provide a novel alternative mechanism
that the mushroom PS peptides may exert anticancer effect by
targeting the
ras-mediated signaling pathway.
Abbreviations: DMEM, Dulbecco's modified Eagle medium; D10CS, Dulbecco's modified Eagle medium supplemented with 10% calf serum; GFP, green fluorescent protein; PS, polysaccharides; PSPC, polysaccharide–peptide complexes; T24, activated human c-H-ras oncogene
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Introduction
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The antitumor effect of mushrooms has long been observed in
Asia, especially in China and Japan. The analysis of various
species of mushrooms has resulted in the identification of a
family of high molecular weight, hot-water-soluble polysaccharides
(PS) and polysaccharide–peptide complexes (PSPC), which
have tested positive for antitumor activities in animal studies
(
1). NMR analysis reveals that the antitumor PS are composed
of a variety of linear and branched glucans. They appear in
various conformations, and some are in the gel state. As a result
of the complexity and heterogeneity of PS and PSPC and limited
suitable bioassay systems, the mechanism of action of these
PS remain obscure. Until now, research on the antitumor activities
is based mainly on the results derived from experiments with
implanted Sarcoma180 or chemically induced tumors in mice (
2–
6).
Antimitotic tests in tumor cell lines have been, so far, rather
inconsistent or negative (
1,
2,
4,
7–
9). Aside from their
antitumor effects, mushroom-derived PS and PSPC seem to function
as immunomodulators; this was observed in animals as well as
in cultured macrophages and T-lymphocytes. It has been postulated
that the antitumor effect of PS and PSPC may be mediated through
the cytokines released by activated macrophages and T-lymphocytes,
instead of through direct cytocidal effects on tumor cells (
4,
8–
11).
In order to gain a more comprehensive understanding, it is necessary
to investigate the antitumor activity of mushrooms in a wider
range of cell systems.
Previously, we have demonstrated that the established rodent cell line R6 is resistant to transformation induced by a potent c-H-ras (T24) oncogene in a focus formation assay. The transforming efficiency of T24, however, can be modulated by treatment with various tumor promoters and factors (12,13). Using this R6/ras assay system, we have assessed the inhibitory or enhancing effects of various chemicals on T24 induced-transformation (14–16). In the current study, we explored the antitumor activity of mushrooms using the focus formation assay built around the R6/ras model system.
In the study, we focused on two medicinal mushrooms: Ganoderma lucidum and Tricholoma lobayense. Both mushrooms exhibit antitumor activities, based mainly on animal studies (1–4,17). Tricholoma lobayense is a native Hong Kong species. Ganoderma lucidum is an important traditional medicine in China and Japan, used for promoting health and treatment of various diseases, including cancer. Ganoderma lucidum is also a better documented natural product in terms of its pharmacological and chemical properties. Our results showed that ras-induced transformed foci were effectively inhibited by the addition of extracts of G.lucidum and T.lobayense in dosage-dependent and time-dependent manners. Data also revealed that the PS fraction of T.lobayense would only exert an inhibitory effect on Ras-transformed cells when cells were co-cultivated with normal R6 cells, suggesting a novel mechanism in which the inhibitory effect of PS is mediated through the surrounding normal R6 cells.
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Materials and methods
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Preparations of mushroom samples
Fruiting bodies of
G.lucidum were homogenized and extracted
with boiling distilled water for 6 h to obtain the PS-enriched
preparations. After centrifugation to remove the insoluble portion,
the water-soluble extracts were lyophilized, then kept at room
temperature for later usage. Liquid mycelium cultures of
G.lucidum were also used to obtain PS-extract. Prior to extraction, the
cultures were filtered and precipitated with ethanol according
to Liu
et al. (
17). The precipitates were dissolved in distilled
water, centrifuged to remove the insoluble, then lyophilized
and designated as mycelium filtrate.
Tricholoma lobayense was
originally isolated and established in cultures by S.T.Chang's
Laboratory at the Chinese University of Hong Kong, Hong Kong.
Tricholoma lobayense was cultured in nutrient broth as described
(
17). Liquid cultures containing the secreted fungal PS were
prepared as above. The water-soluble, PS-enriched components
were lyophilized and designated as mycelium filtrate. Part of
the filtrate was further fractionated into the unbound Fraction
A1 and the salt-eluted bound fraction A2 using a DEAE-cellulose
ion exchange column chromatography (
17). Both fractions were
dialyzed against ddH
2O and lyophilized for later usage.
Cell cultures and plasmids
The Rat 6 (R6) cell line was a subclone of the Fisher rat embryo fibroblasts originating from Freeman's Laboratory (18). R6/T24 cell line is a clonal R6 cell line transformed by the activated human c-Ha-ras oncogene (12). The R6/green fluorescent protein GFP-Ras cell line is a transformed clonal cell line established from a transformed focus derived from R6 cultures transfected by a GFP-ras fusion vector in our lab. Cells were grown in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% calf serum (D10CS) (Invitrogen, Carlsbad, CA). Cultures were maintained in a humidified incubator at 37°C with 5% CO2 in air and fed twice a week with fresh medium. Plasmid pT24 contains a 6.4 kb BamHI fragment corresponding to the coding sequence of the human bladder c-Ha-ras oncogene. The plasmid pT24 was obtained from M.Wigler's Laboratory.
Focus formation assay
The standard focus formation assay and treatment of the cultures were performed as described earlier (12). In brief, 5 x 105 cells seeded in 90 mm plate were transfected with 1 mg T24 plasmid DNA and 20 µg R6 genomic DNA as carrier DNA by DNA-mediated transfection procedure based on Bacchetti and Graham (19) and Wigler et al. (20), with slight modifications (12,13). To determine the effects of mushroom extracts on ras-induced focus formation, transfected cultures were fed with DMEM plus 5% fetal calf serum (D5FCS) in the presence and absence of test sample on day 2 upon transfection, then continued feeding with each respective growth medium twice a week throughout the experiment. Lyophilized samples of mushroom preparations were weighted, dissolved in boiling ddH2O, then centrifuged at 10 000 r.p.m. for 20 min to remove the residues. The supernatant was sterile filtered, then added to the growth medium at designated concentrations. All experiments were performed in six replicate plates.
Cytotoxicity assay
The cytotoxic effect of each sample was performed on both R6 and R6/T24 cell lines. Cells were seeded in triplicate at 104/60 mm plate in D10CS. The next day, the test sample was added to the cultures and kept for 5 days. At the end of treatments, cells were trypsinized and counted using a Coulter Counter Fullerton, CA. Cytotoxicity was expressed as percent survival, i.e. cell counts of the treated cultures divided by cell counts obtained from the untreated cultures.
Colony formation and co-culture assays
For a better quantitative assessment of the inhibitory effect for drug testing, we designed a co-culture assay to simulate the focus formation assay. The assay was set up by seeding 500 ras-transformed R6 cells on a 90 mm plate in triplicate pre-seeded with 2.5 x 105 normal R6 cells 24 h earlier. A day after the seeding of the transformed R6 cells, the test sample was added to the co-cultures of normal and transformed R6 cells grown in DMEM plus 5% calf serum (D5CS). At the end of 2 weeks, culture plates were fixed with 10% formaldehyde, stained with Giemsa stain and photographed. In order to distinguish the transformed from the neighboring normal R6 cells, a transformed cell line, R6/GFP-Ras expressing green fluorescent GFP-Ras fusion protein was used in the co-culture assay. In a parallel experiment, the possible toxic effect of mushroom extracts on the growth of R6 and R6/GFP-Ras cells were tested by seeding 500 of each cell line separately in D10CS in the presence and absence of test chemical for the duration of 12–14 days. Cultures were stained and scored for total number of colonies per plate. All the experimental cultures were fed twice a week with or without the mushroom extract.
Western blot analysis
R6 or R6/GFP-Ras-transformed cells were seeded 2.5 x 105 per 90 mm plate in DMEM plus D10CS. The next day, cultures were fed with fresh medium in the presence and absence of Tricholoma filtrate A2 fraction (500 µg/ml) and fed twice a week. Cells were washed with cold PBS three times and lysed in 400 µl NET buffer (150 mM NaCl, 50 mM Tris–HCl, pH 7.4, 5 mM EDTA, pH 8.0, 1 mM APMSF, 1 µM E-64, 1 µM pepstatin, 100 mM NaVO5 and 10 µg/ml aprotinin) plus 1% NP-40 on ice according to Lu et al. (21). For western blotting analysis, 40 µg of protein extracts were loaded on a 12% SDS–PAGE. After separation, the proteins were transferred to a Hybond-C nylon membrane (Amersham, Piscataway, NJ), reacted to either anti-Ras (Santa Cruz) or anti-GFP antibodies (Clontech, Palo Alto, CA) and visualized by the ECL detection kit (Amersham) according to the manufacturer's manual. Blots were hybridized with anti-actin antibodies (Santa Cruz, Santa Cruz, CA) to normalize the gel loading.
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Results
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Effect of extracts of G.lucidum on ras-induced transformation
In the primary experiment, extracts of fruiting bodies and filtrate
of
G.lucidum were tested. While both the fruiting bodies and
mycelium filtrate were effective in inhibiting foci formation
induced by c-Ha-
ras oncogene, the latter seemed to be more effective
(
Table I). At 200 µg (dry weight)/ml and above (
Table I,B),
mycelium filtrate showed nearly 100% reduction of foci.
On the other hand, the fruiting body extract-treated cultures
exhibited 56% inhibition at 200 µg/ml and 80% at a dose
of 500 µg/ml (
Table I,A). Neither samples posted cytotoxicity
on either the host R6 or R6/T24 cells (
Table II). Thus, the
inhibitory effect of the mushroom extracts on
ras-transformation
is not due to a direct killing of cells transformed by
ras oncogene
in the focus formation assay.
Effect of Tricholoma filtrate on ras-induced transformation
Both the total, and the DEAE-column-bound fraction of
T.lobayense filtrate were tested in R6 cells upon transfection of the
ras oncogene. Data showed that the total filtrate and the DEAE-column-bound
fraction A2 of
T.lobayense markedly inhibited
ras-foci, while
the unbound fraction produced no effect (
Table III and
Figure 1).
The preparations had no or slightly toxic effect on either
normal or transformed R6 cells (
Table IV). The nutrient broth
used for the
T.lobayense cultures alone presented no inhibitory
effect.
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Fig. 1. Giemsa stain of transformed foci in R6 cell cultures transfected with pT24 plasmid DNA. Transfected cultures were grown in normal medium alone (A); normal medium with 100 µg/ml DEAE-column-bound fraction A2 (i.e. PSPC-enriched fraction) (B); or normal medium with 100 µg/ml DEAE-column-unbound fraction A1 (C) of Tricholoma filtrates. Experiments were carried out as described in Table III.
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Dosage and time course studies of Tricholoma filtrate on ras-induced transformation
The inhibitory effect of the
Tricholoma filtrate was further
explored with regard to dosage and duration of treatment. Results
revealed that the inhibitory effect of
Tricholoma filtrate was
dosage-dependent. Extracts in concentration as low as 1 µg/ml
exerted a 19% inhibitory effect on the formation of
ras-foci
(
Table V). Time course study indicated that the maximal effect
was obtained when transfected cultures were treated with
Tricholoma filtrate from days 4 to 20 after the transfection. Interestingly,
a 32% reduction in foci number was still obtained when the treatment
was delayed until day 12 after the transfection of the
ras oncogene
(
Figure 2).
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Fig. 2. Time course studies of the effect of PSPC-enriched fraction A2 of Tricholoma filtrate on the number of transformed foci in R6 cultures transfected (at day 0) with pT24 plasmid DNA. Where indicated, transfected cultures were treated with 100 µg/ml of A2 fraction of Tricholoma filtrate described in Table III. Experiments were terminated at day 20, and stained with Giemsa stain for scoring. Relative number of foci is the ratio of foci obtained in the presence of drug to that in the absence of drug (i.e. no treatment control).
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Effect of Tricholoma filtrate on R6/GFP-Ras cells co-cultivated with normal R6 cells
To further explore the nature of the inhibitory effect of
Tricholoma filtrate, we reconstituted the focus formation assay by seeding
500 R6/GFP-Ras cells on a 90 mm culture plate pre-seeded with
2.5
x 10
5 normal R6 cells 24 h earlier. The co-cultures were
then maintained in D5CS medium in the presence and absence of
Tricholoma filtrate. The effect of
Tricholoma on colony formations
of individual R6 and R6/GFP-Ras cell lines were conducted in
parallel with the co-culture assay. Results indicated that addition
of
Tricholoma did not affect the colony formation (
Figure 3A and B),
nor the morphology of R6 or R6/GFP-Ras cells (data not
shown). In the co-culture of R6 and R6/GFP-Ras (
Figure 3C),
the R6/GPF-Ras cells formed many dense transformed colonies
on the top of the monolayer of R6 cells in the absence of treatment,
resembling the formation of transformed foci shown in
Figure 1.
Addition of
Tricholoma filtrate (200 µg/ml) effectively
blocked the formation of the GFP-Ras transformed colonies. Indeed,
under the fluorescent microscope, the green fluorescent colonies,
representing the R6/GFP-Ras cells, were severely retarded in
the presence of the
Tricholoma filtrate, while grown to sizable
colonies in the absence of the drug treatment (
Figure 3). It
is worthy to note that treatment with
Tricholoma does not affect
the initial plating efficiency of R6/GFP-Ras cells as nearly
the same number of GFP-positive cells were observed on both
the treated and untreated plates under a fluorescent microscope,
24 h upon the addition of the filtrate. Additional evidence
came from the fact that early withdrawal of the treatment substantially
reduces the inhibitory effect of the filtrate, resembling what
we observed in the focus formation assay (data not shown).
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Fig. 3. Effect of PSPC-enriched fraction A2 of Tricholoma filtrate on colony formation of normal and transformed R6 cells. (A and B) 500 of R6 or R6/GFP-Ras-transformed cells per 90 mm plate were seeded in DMEM plus D10CS in the presence and absence of Ticholoma filtrate A2 fraction (200 µg/ml). (C) 500 R6/GFP-Ras cells were seeded on a lawn of 2.5 x 105 normal R6 cells plated in 90 mm plate 24 h earlier. The Tricholoma filtrate A2 fraction (200 µg/ml) was then added to the co-cultures of normal and transformed R6 cells grown in D5CS 24 h after the seeding of the transformed cells. At the end of 2 weeks, culture plates were fixed with 10% formaldehyde, stained with Giemsa stain and photographed. GFP-Ras-colonies derived from the co-cultures were viewed and photographed under a fluorescent microscope at 100 and 400x magnifications.
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Tricholoma filtrate posted no effect on the expression of GFP-Ras protein
To determine whether the retardation of R6/GFP-Ras colonies
is due to the suppression of Ras protein, protein extracts derived
from the treated and untreated R6 and R6/GFP-Ras cells were
examined for Ras protein expression using western blot analysis.
Result showed that addition of
Tricholoma filtrate to the cultures
did not reduce the level of GFP-Ras fusion protein expression
identified with either anti-Ras or anti-GFP antibody (
Figure 4).
The same blot was hybridized with anti-actin antibody as
a protein loading control. Aside from the protein level,
Tricholoma treatment did not alter the normal subcellular localization
of the GFP-tagged Ras protein, nor the transforming morphology
of GFP-Ras cells as it shows in
Figure 5. In that, the GFP-Ras
proteins are correctly localized in the inner surface of the
plasma membrane of the untreated (
Figure 5A–C) as well
as of the treated (
Figure 5D–E) R6/GFP-Ras cells co-cultivated
with R6 cells as described in the experiments shown in
Figure 3C.
No alteration of subcellular localization of GFP-Ras proteins
was seen when R6/GFP-Ras cells were grown alone and treated
with
Tricholoma filtrate (data not shown).
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Fig. 4. Effect of PSPC-enriched fraction A2 of Tricholoma filtrate on Ras protein expression in normal and transformed Rat 6 cells. 2.5 x 105 R6 or R6/GFP-Ras-transformed cells per 90 mm plate were seeded in DMEM plus D10CS. Next day, cultures were fed with fresh medium in the presence (+) and absence (–) of Tricholoma filtrate (A2) (200 µg/ml) and fed twice a week. At the end of 14 days, cells were washed with cold PBS, lysed in NET buffer plus protease inhibitors and collected for western blot analysis as described in the Materials and Methods. Protein extracts obtained from the treated and untreated R6 and R6/GFP-Ras cultures were loaded 40 µg/lane and separated on 12% PAGE gel by electrophoresis. The resulting blot was hybridized sequentially with anti-Ras (Santa Cutz), anti-GFP (Clontech) and anti-actin (Santa Cutz) antibodies and visualized using Amersham ECL Western Blotting Detection Kit according to the manufacture manual.
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Fig. 5. Effect of PSPC-enriched fraction A2 of Tricholoma filtrate on the subcellular localization of GFP-Ras protein in GFP-ras-transformed R6 cells co-cultivated with normal R6 cells. The co-cultures were prepared as described in Figure 3 legend, and treated (+) or untreated (–) with Tricholoma filtrate (200 µg/ml) for 7 days. (A–C) Fluorescent microscopic views of three individual R6/GFP-Ras colonies derived from the untreated co-cultures; (D–F) three individual colonies derived from the treated co-cultures.
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Discussion
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The antitumor effects of PS and PSPC of higher fungi have long
been investigated in tissue cultures, animal models and clinical
patients, yet the mechanism underlying the action of PS is unclear.
Considerable evidence suggests that the antitumor effect of
PS is mediated through the cytokines released by activated macrophages
and T-lymphocytes, instead of through direct cytocidal effects
on tumor cells (
4,
8–
11). In order to explore other possible
cell-mediated responses to PS, we employed a non-lymphocytic
in vitro cell system and tested the antitumor activity of PS
against cell transformation induced by a defined
ras oncogene.
The results showed that both the fruiting bodies and the cultured
mycelia of
G.lucidum, a medicinal mushroom well known for its
antitumor activity, markedly inhibited the formation of
ras-induced
transformed foci assessed by the focus formation assay in the
R6 embryo fibroblast cell line. Interestingly, none of the PS
preparations were toxic to either normal or
ras-transformed
R6 cells. Thus, the inhibitory effect of the mushroom extracts
is not due to a direct cell killing of the transformed cells
used in the study. This finding is consistent with the observations
made in lymphatic cell system in which no cytotoxicity was detected
under the treatment of
G.lucidum (
4,
9,
10).
The inhibitory effect was not restricted to G.lucidum. When we examined culture filtrate from Tricholoma, similar results were obtained. In the previous study, an antitumor component was identified in the culture filtrate of T.lobayense (17). The active component, based on tumorigenesis studies in animals, was found to reside in the DEAE-cellulose ion exchange column bound fraction, but not in the unbound fraction. Further characterization of the bound fraction showed that the fraction is a PSPC with a molecular weight of 154 kDa. It is intriguing that the levels of inhibitory activity of the DEAE-bound and -unbound fractions of Tricholoma assessed by focus formation assay were remarkably similar to those obtained by animal tumorigenesis test studied by Liu et al. (17) (Table VI). The inhibitory activity of the bound fraction appeared to be dosage-dependent. Based on the time course study, early withdrawal of the component impaired the full activity of the PSPC as shown in Figure 2B–D. On the other hand, a 32% inhibition was still observed when the addition of the chemical was delayed until day 12 (Figure 2G). In fact, our study shows that the duration, rather than the time of application, dictates the efficacy of the compound, as demonstrated by the relative number of foci of groups B and H, C and G and D and F. Each group of the pair received the same duration, but different time frame of treatment, yet each yielded a similar number of foci. We also observed that the relative foci decreased from 0.32 to 0.04 when the experiment was carried out for 24 (Table III), instead of 20 days (Figure 2). This result reiterates the tentative conclusion that it is the length, not the time frame, of treatment that is more critical in determining the extent of inhibition of transformed foci.
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Table VI. Comparisons of anticancer effects of filtrates of T.lobayense cultures assessed by animal test versus in vitro focus formation assay
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The mechanism underlying the inhibitory effect of PS or PSPC
against
ras-foci remains unclear. In this study, the
ras-transformed
cells in focus formation were effectively inhibited during the
early stage of transformation, and were equally inhibited when
the stably transformed cells were mixed with normal cells, then
treated with PS extract in the co-culture assay (
Figure 3C).
Three key findings in this study may shed light on the possible
mode of inhibition against
ras-foci. First, treatment with the
extracts posts no cytocidal effect on either normal or established
ras-transformed cell line assessed by the cell proliferation
test and colony formation assay (
Table II and
Figure 3A and B).
Secondly, mushroom extracts do not block the expression
(
Figure 4), nor alter the membranous localization and the transforming
activity of the
ras oncoprotein tagged with GFP displayed in
R6/GFP-
ras cells (
Figure 5). Thirdly, the inhibitory effect
of mushroom extracts against
ras-transformed cells requires
the presence of normal cells. The last point was well illustrated
in the co-culture experiment, in which the colony formation
of R6/GFP-
ras cells was only inhibited in the present, but not
in the absence of the co-cultured normal R6 cells (
Figure 3C).
As mentioned earlier, treatment with
Tricholoma does not affect
cell adhesion as the number of seeded R6/GFP-Ras cells found
in the treated cultures was similar to that found in the untreated
cultures. Thus, the mushroom extract seems to exert its opposing
effect on cell expansion, rather than on cell adhesion of the
transformed cells. An early report indicated that certain triterpenoids
from
G.lucidum inhibited farnesyltransferase activity of Ras
protein and retarded the growth of k-
ras transformed cells (
22).
In our case, based on the clear display of the membranous GFP-tagged
Ras protein under the treatment with
Tricholoma filtrate observed
in vivo, the PS extract does not seem to act as farnesyltransferase
inhibitor. Taking all these observations together, our data
suggest that the antitumor effect of PS or PSCP from
G.lucidum and
T.lobayense is very likely mediated through the normal Rat
6 host cells, by direct or indirect cell contact. Based on our
preliminary investigation, however, inhibition of the
Tricholoma filtrate on the growth of transformed cells was not apparent
when normal and transformed cells were each grown on an individual
chamber (upper and lower) separated by a microporous membrane
using a Transwell culture chamber system, suggesting that the
inhibitory effect of mushroom filtrate may require a direct
cell-to-cell contact (data not shown). However, determining
the precise target of the PS and PSPC requires further investigation.
Our previous works indicate that the transforming ability of
the activated
ras oncogene can be modulated by various factors
and compounds (
14–
16). Early works by others suggested
that the antitumor effect of fungal PS and PSPC is mediated
through the cytokine released from the host cells. Later, Wang
et al. presented evidence that treatment with
G.lucidum stimulated
macrophages and T lymphocytes to release TNF-
and IFN-
, both
of which were cytotoxic to HL-60 and U937 (
9,
23). Other related
studies that may shed light on the mechanism of mushroom extract
are the recent works on glucan, a natural PS product widely
distributed in fungi. Glucan has been reported to act as immunomodulator
and cell response modifier. Binding of glucan to its specific
glucan receptors can elicit a serial cellular response through
the modulating of activities of various factors including IgE,
cytokines, chemokines, transcriptional factors and growth factors
(
24–
26). Interestingly, the bioactive glucan receptors
are present in human fibroblasts (
26). Whether a similar mechanism
applies to the inhibitory effect of mushroom extracts in our
cell system warrants further investigation.
This study is the first to demonstrate that the PS- and PSCP-enriched mushroom extracts can inhibit cell transformation induced by a defined oncogene through a novel non-cytocidal route. Ras proteins play a pivotal role in regulating cell growth and the development of human cancer. The demonstration of the inhibitory effect of mushroom extracts on ras-induced transformation in this current study may have broad implications for cancer prevention and treatment and may provide a better understanding of the underlying mechanism of the cancer inhibitory effect of mushroom PS.
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Received November 25, 2002;
revised December 17, 2003;
accepted February 4, 2004.
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Abstract
Introduction