Extracts of Medicinal Mushrooms Agaricus bisporus and Phellinus linteus Induce Proapoptotic Effects in the Human Leukemia Cell Line K562

Abstract

We have studied the effects of Agaricus bisporus and Phellinus linteus ethanol extracts on transcriptional regulation of genes involved in cytokine release and apoptosis in the human leukemia cell line K562. In particular, we applied quantitative real-time PCR (Q-PCR) assays to monitor alterations of gene expression for the following genes: Bcl-2, Casp-9, NF-κB, TNF-α, IFN-γ, and IL-10. We found stronger proapoptotic activity for the Ph. linteus alcohol extract on K562 cells than for the A. bisporus extract: 4.4- and 2.2-fold increase of Bcl-2 and Casp-9 transcripts. Mushroom alcohol extracts are suggested to exert their effects on tumor cells via the induction of apoptosis. K562 leukemia cells were shown to be most responsive to the transcriptional induction of tumor necrosis factor TNF-α when stimulated with IFN-γ and then treated with Ph. linteus alcohol extract: up to a 4.5-fold increase. Treatment of K562 cells with A. bisporus extract promoted transcription of the cytokine gene IL-10

Full text

167
International Journal of Medicinal Mushrooms, 12(2):167–175 (2010)
1521-9437/10/$35.00
© 2010 by Begell House, Inc.
Extracts of Medicinal Mushrooms Agaricus bisporus
and Phellinus linteus Induce Proapoptotic Effects in
the Human Leukemia Cell Line K562
Alla V. Shnyreva,
1
Wei Song,
2
& Leo J. L. D. Van Griensven
2,
*
1
Department of Mycology and Algology, Moscow State University, Moscow 119991, Russia;
2
Plant
Research International, Wageningen UR, Wageningen 6700 AA, The Netherlands
* Address all correspondence to Leo J. L. D. Van Griensven,
Plant Research International, Wageningen UR, Wageningen 6700 AA,
The Netherlands; Leo van Griensven <leo.vangriensven@gmail.com>
ABSTRACT: We have studied the effects of Agaricus bisporus and Phellinus linteus ethanol extracts
on transcriptional regulation of genes involved in cytokine release and apoptosis in the human leukemia
cell line K562. In particular, we applied quantitative real-time PCR (Q-PCR)
assays to monitor altera-
tions of gene expression for the following genes: Bcl-2, Casp-9, NF-κB, TNF-α, IFN-γ, and IL-10.
We found stronger proapoptotic activity for the Ph. linteus alcohol extract on K562 cells than for the
A. bisporus extract: 4.4- and 2.2-fold increase of Bcl-2 and Casp-9 transcripts. Mushroom alcohol ex-
tracts are suggested to exert their effects on tumor cells via the induction of apoptosis. K562 leukemia
cells were shown to be most responsive to the transcriptional induction of tumor necrosis factor TNF-α
when stimulated with IFN-γ and then treated with Ph. linteus alcohol extract: up to a 4.5-fold increase.
Treatment of K562 cells with A. bisporus extract promoted transcription of the cytokine gene IL-10.
KEY WORDS: medicinal mushrooms, Agaricus bisporus, Phellinus linteus, mushroom ethanol ex-
tracts, proapoptotic and anticancer activities
ABBREVIATIONS
Bcl-2: B-cell lymphoma 2; BSA: bovine serum albumin; Casp-9: caspase 9; CML: chronic myeloid leukemia;
DEAE: diethyl diamino ethyl; FCS: fetal calf serum; HMW: high molecular weight; IFN-γ: interferon gamma;
IL-10: interleukin 10; K562: myelogenous erythroid leukemia cell line; NF-κB: nuclear factor-kappa B; Q-PCR:
quantitative real-time polymerase chain reaction; RT-PCR: reverse transcription polymerase chain reaction; ROS:
reactive oxygen species; RPMI 1640: Roswell Park Memorial Institute cell culture medium 1640; TNF-α: tumor
necrosis factor alpha; TNF-R: tumor necrosis factor receptor.
I. INTRODUCTION
The preventive and therapeutic effects of medicinal
mushrooms Agaricus bisporus (J. Lge) Imbach
(Agaricaceae, Agaricomycetideae) and Phellinus
linteus (Berk. et M.A. Curt.) Teng (Hymeno-
chaetaceae, Aphyllophoromycetideae) and their
components have been shown to be associated
with immunomodulatory effects.
1,2
This involves
proinfl ammatory activity or adjuvant effects, as
well as apoptosis, mostly with the involvement of
chemokines as mediators, produced by the cells of
the immune system. Mushroom extracts contain sev-
eral biologically or medicinally active components.
The infl uence of mushroom extracts on oxygen
metabolism, both in vitro and in vivo, may be a
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168 International Journal of Medicinal Mushrooms
A. V. SHNYREVA, W. SONG, & L. J. L. D. VAN GRIENSVEN
common denominator. Redox reactions strongly
infl uence the majority of physiological processes.
Several glucans of higher Basidiomycetes were
found to have a direct antioxidant effect in vitro.
3,4
Agaricus bisporus, which contains high amounts
of antioxidants, has been reported to additionally
harbor a pro-oxidative phenolic compound, that
is, the 4-(hydroxymethyl)-phenyl radical. Pheno-
lic compounds generally have anti- and/or pro-
oxidative properties, depending on the oxidative
status of their OH groups. Some are therefore able
to activate the intracellular formation of reactive
oxygen species (ROS). The most fundamental role
of ROS might be signal transduction and regulation
of diverse processes, including phagocyte activa-
tion, cell proliferation and migration, and apoptosis
(or programmed cell death). Molecular damage
caused by ROS in normal cells challenges the cells
to repair the damage. Abnormal or affected cells
are activated towards the cell death program, that
is, to either apoptosis or autophagous cell death.
Tumor cell lines appear particularly sensitive to
ROS-induced apoptosis. In fact, antitumor agents
exhibit their activity via ROS-dependent activation
of apoptotic cell death.
5
We demonstrated that partially purifi ed poly-
saccharide extracts of A. bisporus and Ph. linteus
are highly active in ROS generation.
6
When hot
water–extracted polysaccharides of A. bisporus
were subjected to DEAE cellulose adsorption
chromatography, a nonadsorbed, colorless, high
molecular weight (HMW) 1,4,α-glucan was obtained
that was unable to generate ROS, suggesting the
inability of some glucans to generate ROS.
7
The
DEAE-adsorbed fractions that were eluted by salt
gradient and consisted of a hazel-colored polysac-
charide-polyphenol complex were very active in
ROS generation. This suggests that the A. bisporus
glucan needs to be associated with polyphenolic
compounds to become an active ROS generator.
To study whether mushroom polyphenols have
a direct effect on cell growth and metabolism, we
applied Q-PCR assays to monitor alterations of gene
expression in the human leukemia cell line K562.
The objective of the research was to analyze pos-
sible effects of A. bisporus and Ph. linteus ethanol
extracts on the transcriptional regulation of genes
involved in cytokine release and apoptosis.
II. MATERIALS AND METHODS
A. Chemicals
RPM1640 with L-glutamine and sodium bicarbon-
ate was from Sigma Chemical Corp. (St. Louis,
MO, USA), serum supreme and fetal bovine serum
(South American origin) was from BioWhittaker
(Walkersville, MD, USA), the Trizol agent was
from Life Technologies (Rockville, MD, USA),
and the DNase I Amp Grade and Superscript II
reverse transcriptase were both from Invitrogen
(Carlsbad, CA, USA).
B. Cancer Cells
The human erythroid leukemia cell line K562 was
cultured in RPMI-1640 medium supplemented
with 10% fetal calf serum (fcs) and 100 U/mL
–1
penicillin + 100 μg/mL
–1
streptomycin and main-
tained in a humidifi ed incubator with 5% CO
2
at
37°C. Cell viability was assessed by the trypan
blue exclusion test.
Acridine orange/ethidium bromide staining
(AO/EB) was used to visualize the induction of
apoptotic bodies and was performed on live cells
in cultivation medium by the addition of an equal
volume of a mixture of 10 μg/mL
–1
acridine orange
and 1 μg/mL
–1
ethidium bromide in phosphate
buffered saline (PBS). The cells were then imme-
diately observed in a Zeiss Axioscope fl uorescence
microscope (Carl Zeiss AG, Oberkochen, Germany)
using a 40 × Neofl uar objective.
C. Mushroom Extracts and Treatment
Fresh fruiting body tissue of cultivated Agaricus
bisporus (Sylvan, strain A15) was obtained from
Innerlife B.V. (Venlo, The Netherlands). Dry
powdered wild-type Phellinus linteus was kindly
provided by Amazing Grace Ltd. (Thailand). Water
extracts were prepared as described previously.
6
Ethanol extracts were prepared as follows: 10 g
of lyophilized or dry powdered fruiting body were
homogenized to a fi ne powder in a mortar and
then suspended in 100 mL of 96% ethanol. The
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Volume 12, Issue 2, 2010 169
EXTRACTS OF A. BISPORUS AND PH. LINTEUS INDUCE PROAPOPTOTIC EFFECTS IN A HUMAN LEUKEMIA CELL LINE
mixture was stirred at 70°C for 2 hours and then
fi ltered and centrifuged to remove solid materi-
als. The supernatant was concentrated to a small
volume in a Buchi rotary evaporator at 45°C. The
concentrate was diluted to a clear 1% (w/v) solu-
tion in absolute ethanol.
1. Cell Stimulation and Treatment
K562 cells, at a fi nal concentration of 0.5 × 10
6
cells/
mL
–1
, were stimulated with 100 ng/mL
–1
IFN-γ for
20 hours and then incubated (treated) with mush-
room ethanol extracts for another 4 hours in 6- and
12-well culture plates. Various doses of mushroom
ethanol extracts (1, 5, and 10 μg/mL
–1
) and 200 μg/
mL
–1
of hot water extract were tested. Untreated
and unstimulated control cells were examined at
corresponding times.
D. RNA Isolation and cDNA Synthesis
Total RNA was extracted from K562 cells with
Trizol according to the manufacturer’s protocol. All
RNA extractions were treated with 1 μL of DNase I
Amp Grade for 15 min at room temperature and then
heated for 10 min at 65°C to remove any traces of
genomic DNA contamination. RNA concentration
and purity (260/280/230 ratio) were measured by
the NanoDrop ( NanoDrop Tecnologies, Thermo
Fisher Scientifi c Inc., Waltham, MA, USA), achiev-
ing a mean concentration of 191 ng/μL
–1
, a 260/280
ratio of 2.03, and a 260/230 ratio of 1.89.
One microgram of total RNA was converted
into cDNA using the Superscript II reverse tran-
scriptase. For each reaction, 4 μL 5 fi rst-strand
buffer (50 mM Tris–HCl, pH 8.3, 375 mM KCl,
15 mM MgCl
2
), 2 mL of 0.1 M dTT, 5 U RNAsin,
500 μM dNTP mix, 200 pmol Oligo-dT, 25 U
Superscript II reverse transcriptase, and MQ-
water were added to the RNA to a fi nal volume
of 20 μL. This reaction was then incubated at
42°C for 1 h. The reaction was carried out for
5 min at 25°C, followed by 30 s at 42°C, and
was terminated at 85°C for 5 min. The fi nished
cDNA products were stored in aliquots at –80°C
until needed.
E. Primer Design and Real-Time
Q-PCR Assays
Quantitative real-time polymerase chain reaction
(Q-PCR) was used for monitoring alterations of gene
expression in K562 cells stimulated with mushroom
ethanol extracts. Q-PCR enables a sensitive and
accurate quantifi cation of mRNA transcription lev-
els. Selection of a housekeeping (reference) gene
as an internal control is of crucial importance for
data interpretation. The β-actin gene, Actb, was
selected as a housekeeping gene from those most
commonly used in the literature.
8
The expression
of six genes involved in cell immune responses
and apoptosis regulation (Bcl-2, Casp-9, NF-κB,
TNF-α, IFN-γ, and IL-10) was evaluated. Specifi c
cDNA sequences were retrieved from the GenBank
sequence database of the National Center for Bio-
technology Information (www.ncbi.nih.gov), and
primers were designed by using BEACON software.
Criteria for primer selection were as described in
detail by Giguere and Prescott.
9
Briefl y, primers
were designed to span the exon-exon boundary to
eliminate the possible infl uence of the contamination
of genomic DNA. Primers were constructed to have
a length of 18–25 bp, 40–60% GC content, and to
produce amplicons 150–220 bp in length.
The Q-PCR was performed using SYBR Green
fl uorescence and a MJQ BioRad iCycler (USA).
After an initial step at 95°C for 10 min, amplifi -
cation was performed with 40 cycles denaturing
at 95°C for 30 s, annealing at 60°C for 40 s, and
extension at 72°C for 40 s. Amplifi cation was fol-
lowed by a melting curve analysis to confi rm PCR
product specifi city. A melting curve analysis was
performed after the fi nal amplifi cation cycle via a
temperature gradient from 60°C to 95°C. A non-
template control (NTC) was run with each assay,
and all determinations were performed at least in
duplicate to achieve reproducibility.
During the protocol optimization, all Q-PCR
amplifi ed products were loaded on 1.6% agarose
gel using the appropriate DNA ladder to confi rm
appropriate fragment sizes and lack of primer dimers.
No signals were detected in nontemplate controls.
Calibration curves were generated using rela-
tive concentration versus the threshold cycle (C
t
).
Raw data were analyzed using the 2
–∆∆Ct
method,
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170 International Journal of Medicinal Mushrooms
A. V. SHNYREVA, W. SONG, & L. J. L. D. VAN GRIENSVEN
also known as the comparative threshold cycle (C
t
)
or ∆∆Ct method.
10
The relative gene expression
was quantifi ed based on the results obtained for
β-actin as the endogenous control (the housekeep-
ing gene).
III. RESULTS
A. Design and Quality Assessment of
Q-PCR Protocol and Q-PCR Effi ciency
We evaluated the expression of Bcl-2, Casp-9,
NF-κB, TNF-α, IFN-γ, and IL-10 genes in K562 leu-
kemia cells under the following conditions: (1) cells
stimulated with mushroom ethanol extracts; (2) cells
stimulated with ethanol extracts and preactivated
(preincubated) with interferon-γ (IFN-γ); (3) cells
stimulated with hot water extracts. First, we analyzed
gene expression at different time points of stimula-
tion/treatment in the time-course experiment (data
not shown) in order to choose the optimal incubation
time. As a result, cells stimulated at 4-h time points
were subjected to comparative analysis of mRNA
transcriptional level in Q-PCR experiments.
Then, we tested various doses of A. bisporus
ethanol extract on gene expression in order to choose
the optimal concentration of mushroom extracts for
the induction of apoptosis and therefore applicable
for the analysis (Fig. 1). In the dose-response study,
A.
K562 unstimulated with IFN-Ȗ
0
0,5
1
1,5
2
2,5
3
3,5
control 1 ug/mL 5 ug/mL 10 ug/mL 200 ug/mL
Ethanol extract Water extract
Relative gene expression
(fold change)
Bcl-2
Casp-9
NF-kB
TNF-Į
B.
K562 stimulated with IFN-Ȗ
0
0,5
1
1,5
2
2,5
3
3,5
4
control 1 ug/mL 5 ug/mL 10 ug/mL 200 ug/mL
Ethanol extract Water extract
Relative gene expression
(fold change)
Bcl-2
Casp-9
NF-kB
TNF-Į
FIGURE 1. Effect of various doses of Agaricus bisporus extracts on gene expression in leukemia cell line K562. (A)
Cells were treated with mushroom extracts (without IFN- stimulation). (B) Cells were stimulated with IFN- for 20 h
and then treated with mushroom extracts. Control—untreated K562 cells without stimulation with IFN-. The optimal
concentration of ethanol extract is 10 g/mL
–1
.
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Volume 12, Issue 2, 2010 171
EXTRACTS OF A. BISPORUS AND PH. LINTEUS INDUCE PROAPOPTOTIC EFFECTS IN A HUMAN LEUKEMIA CELL LINE
K562 cells were initially treated using 1, 5, and 10
μg/mL
–1
of
A. bisporus alcohol extract. Our results
suggested that activation of gene transcription was
maximal at 10 μg/mL
–1
of A. bisporus ethanol
extract treatment.
We also investigated the IFN-γ synergetic effect
on the modulation of gene transcription in tumor
cells treated with mushroom ethanol extracts. K562
cells were activated (preincubated) with IFN-γ at
the concentration 100 ng/mL
–1
for 20 h and then
stimulated with mushroom extracts for another
4 h, followed by Q-PCR analysis of mRNA tran-
scripts of genes involved in cytokine release and
apoptosis (Bcl-2, Casp-9, NF-κB, TNF-α, IFN-γ,
and IL-10). At time-point zero (prior to IFN-γ
activation) and after the 20-h preincubation with
IFN-γ and subsequent 4-h stimulation by mushroom
ethanol extracts, the total RNA was isolated, reverse
transcribed, and gene expression was quantitated
by real-time Q-PCR. There were no apparent dif-
ferences in mRNA transcription of the reference
gene, β-actin, between IFN-γ-stimulated cells,
mushroom extract-stimulated, and nonstimulated
cells, indicating constitutive expression and the
reliability of the reference housekeeping gene used
in the experiment.
Therefore, the concentrations of 10 μg/mL
–1
of ethanol extract and 200 μg/mL
–1
of hot water
extract and 20-h stimulation with 100 ng/mL
–1
IFN-γ followed by 4-h incubation with mushroom
extracts were chosen as the optimal experimental
conditions for the subsequent investigation.
Agarose gel electrophoresis of PCR prod-
ucts during initial optimization experiments was
performed to reveal single bands corresponding
to amplifi ed genes for all primer sets tested (not
shown). Moreover, melting curve analysis was per-
formed after each Q-PCR run. This demonstrated a
single homogenous melting peak, confi rming highly
specifi c amplifi cation.
B. Gene Expression Analysis in K562
Cells Treated with Ethanol Extracts
No signifi cant differences in the level of nuclear
factor-kappaB (NF-κB) and tumor necrosis factor-
alpha (TNF-α) transcripts stimulated with A.
bisporus alcohol extract were noticed between
K562 cells left untreated or treated for 20 hours
with IFN-γ (Fig. 1). Transcription of these genes
remained at control values or was downregulated.
The Bcl-2 gene transcription underwent positive
regulation under the increased concentration of
A. bisporus alcohol extract (at concentration 10
μg/mL
–1
): more than 3.5-fold increase in IFN-γ-
stimulated K562 cells and 2.8-fold increase in cells
left nonstimulated. IFN-γ is shown to have immu-
noregulatory, antiviral, and anticancer properties.
It alters transcription in up to 30 genes, producing
a variety of physiological and cellular responses,
including inhibition of cellular proliferation and
effects on apoptosis.
11
As another biomarker of apoptosis induction,
caspase-9 activity was also slightly increased (up to
2.2.-fold) in K562 cells stimulated with A. bisporus
alcohol extract without IFN-γ pretreatment, indicat-
ing a proapoptotic effect of the mushroom extract
in K562 cells (Fig. 1A). Caspase-9 is known to
be activated during programmed cell death, or
apoptosis, and to be linked to the mitochondrial
death pathway.
12
Agaricus bisporus hot water extract (at concen-
tration 200 μg/mL
–1
) did not perform much activity
either in cells stimulated with IFN-γ or in unstimu-
lated cells: only the transcription of the Bcl-2 gene
was slightly induced (1.6-fold increase), whereas the
other tested genes were downregulated, including
the proapoptotic gene Casp-9 (Fig. 1). This result
suggested that the hot water extract of A. bisporus
is more likely to have antiapoptotic activity and
protects K562 cells against apoptosis.
In the subsequent set of experiments, K562 cells
were stimulated with ethanol extracts of A. bisporus
and Ph. linteus (at concentration 10 μg/mL
–1
). The
expression of six genes involved in cell immune
responses and apoptosis regulation (Bcl-2, Casp-9,
NF-κB, TNF-α, IFN-γ, and IL-10) was evaluated.
The gene transcriptional profi les differed in K562
cells treated with Ph. linteus or A. bisporus ethanol
extracts (Fig. 2). The Ph. linteus alcohol extract
demonstrated stronger activity: an approximately
4.4-fold increase of Bcl-2 gene transcription and
a 2.1-fold growth of Casp-9 in treated K562 cells.
Bcl-2 family members are known to play a major
role in apoptosis regulation, and they are responsive
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172 International Journal of Medicinal Mushrooms
A. V. SHNYREVA, W. SONG, & L. J. L. D. VAN GRIENSVEN
to many cytotoxic stimuli, including limited cyto-
kine levels, drugs, etc.
12,13
When the K562 cells were fi rst stimulated with
IFN-γ, followed by Ph. linteus alcohol extract treat-
ment, transcription of the infl ammatory cytokine
TNF-α was elevated up to 4.5-fold, suggesting
a quite strong proinfl ammatory effect of the Ph.
linteus extract on K562 cells (Fig. 2). The high
expression of TNF-α transcripts stimulated by
IFN-γ treatment may also indicate a proapoptotic
effect of TNF-α on K562 cells. One of the possible
mechanisms is that IFN-γ could induce sensitivity
of the cells to the proapoptotic effects of TNF-α
by promoting the surface expression of a TNF-α
receptor on tumor cells.
11
The transcription of proapoptotic genes (Bcl-
2, Casp-9, and NF-κB) in K562 cells treated with
A. bisporus ethanol extract was not much affected
by IFN-γ stimulation (Fig. 2). The transcription of
TNF-α and Bcl-2 genes was even higher in cells
without IFN-γ preincubation: 3.2- and 2.3-fold in-
crease, respectively, whereas the mRNA transcripts
of Casp-9 and NF-κB genes were increased less than
1.5-fold (Fig. 2). Upregulation of Bcl-2 and Casp-9
gene expression indicates a proapoptotic effect of
A. bisporus alcohol extract. Interestingly, transcrip-
tion of NF-κB was higher in cells stimulated with
Ph. linteus alcohol extract without preincubation
with IFN-γ (1.7-fold increase), suggesting a posi-
tive transcriptional regulation in the K562 leukemia
cells in response to treatment by the mushroom
alcohol extract only.
The interleukin-10 (IL-10) gene was slightly
upregulated by A. bisporus ethanol extract in K562
cells. The IL-10 mRNA transcription level was
increased 1.7-fold in cells activated with IFN-γ
for 20 h, whereas the transcription of the IFN-γ
gene was always downregulated in the experiment
(Fig. 2). In cells treated with the Ph. linteus alcohol
extract, the transcription of IL-10 and IFN-γ genes
was also downregulated.
IL-10 is an anti-inflammatory cytokine that
has pleiotropic effect in immune regulation. Inter-
estingly, the lack of IL-10 transcription in K562
cells was always correlated with down regulated
IFN-γ transcription. This is not surprising because
K562
0
0,5
1
1,5
2
2,5
3
3,5
4
4,5
5
A.bisporus Ph.linteus A.bisporus Ph.linteus control
Relative gene expression (fold increase
)
Bcl-2
Casp-9
NF-kB
TNF-Į
IFN-y
IL-10
INF-Ȗ Stimulated Unstimulated
FIGURE 2. Induction of gene expression in leukemia cells K562 stimulated in vitro with ethanol extracts of Agaricus
bisporus and Phellinus linteus. mRNA transcripts were quantifi ed without stimulation and after 20 h of IFN- stimula-
tion followed by 4-h treatment with 10 g/mL
–1
ethanol extracts. For each sample, one cDNA was prepared (Q-PCR
assay for each gene was performed in duplicate).
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Volume 12, Issue 2, 2010 173
EXTRACTS OF A. BISPORUS AND PH. LINTEUS INDUCE PROAPOPTOTIC EFFECTS IN A HUMAN LEUKEMIA CELL LINE
IL-10 is able to inhibit proinflammatory cyto -
kines (including IFN-γ),
10
and, vice versa, IFN-γ
itself can specifically downregulate the cyto kine
IL-10 transcription.
14
IV. DISCUSSION
The K562 cell line, derived from a chronic myeloid
leukemia (CML) patient, is known to be particularly
resistant to apoptotic death.
15
However, analysis of
gene expression in Q-PCR experiments has demon-
strated that K562 cells that were preactivated with
IFN-γ for 20 h can be triggered to apoptosis (or at
least become more responsive to apoptosis) by a
4-h mushroom alcohol extract treatment.
Apoptosis, or programmed cell death, is a form
of cell deletion/elimination aimed at the control of
cell differentiation and proliferation that can be trig-
gered by either intracellular or extracellular signals,
followed by activation of a group of enzymes called
caspases.
16
Caspases work like a cascade pathway
by targeting and cleaving key cellular structures
and thus promoting cell decomposition.
17
Apoptosis can be considered as a promising
mechanism to fi ght tumor cells. That is why it
was of interest to evaluate the variety of genes
involved in the programmed cell death. For
example, TNF-α, when it binds to the TNF-R1
receptor, can act as an extracellular trigger for
apoptosis.
18
TNF is shown to be a major media-
tor of apoptosis as well as of infl ammation and
immunity. TNF-α is a multifunctional cytokine
that stimulates the acute-phase reaction in the in-
fl ammatory process
19
and can cause apoptotic cell
death and activation of the NF-κB transcriptional
factor.
20
The central role of TNF in infl ammation
has been demonstrated by the ability of agents
that block the activity of TNF to treat a range of
infl ammatory conditions, including rheumatoid
arthritis, infl ammatory bowel disease, and multiple
sclerosis.
21
TNF-α can deliver both proapoptotic
and antiapoptotic signals to the cells. For example,
interaction of TNF-α with its membrane receptor
TNF-R1 can activate the NF-κB pathway that, in
turn, leads to death receptor-mediated apoptosis.
18
However, some other factors, such as the amount
of reactive oxygen species (ROS), can shift the
inter cellular balance in favor of the proinfl amma-
tory or proapoptotic pathway.
The considerably high transcription of TNF-α
and Bcl-2 genes stimulated by alcohol extracts of
A. bisporus and Ph. linteus in our experiments
indicates their proapoptotic effect on K562 leu-
kemia cells (Fig. 2). The highest level of Bcl-2
transcripts (4.4-fold increase) was observed in
cells treated with the Ph. linteus extract, which
indicates its stronger effect on apoptosis induction
as compared to the A. bisporus ethanol extract.
Morphologically, no obvious signs of increased
apoptotic activity were observed after 4 h of incuba-
tion, when live stimulated K562 cells were stained
with ethidium bromide and immediately visualized
microscopically. After overnight incubation with
ethanol extracts, the situation was quite different:
most IFN-γ-stimulated and ethanol extract–treated
K562 cells had died, mostly through apoptosis,
leaving apoptotic cells and granular nuclear rem-
nants in the culture. The apoptotic cells and the
nuclear remnants incorporated ethidium bromide
and showed bright orange fl uorescence under the
fl uorescence microscope.
The increased level of TNF-α transcripts ob-
served in cells preincubated with IFN-γ also proves
the proapoptotic effect of IFN-γ stimulation. This
trend was clearly observed in cells treated with the
Ph. linteus alcohol extract: in cells preincubated
with IFN-γ, the transcription of Bcl-2 was decreased,
whereas the transcription of TNF-α was elevated
up to 4.5-fold as compared to cells without IFN-γ
stimulation. Therefore, it is suggested that IFN-γ
stimulation induces the sensitivity of K562 leukemia
cells to the proapoptotic action of TNF-α. Many
reports indicate that IFN-γ can arrest the cell cycle
and provide a proapoptotic signal.
11,22
In our experiment, NF-κB transcription was
slightly upregulated in K562 cells treated with
A. bisporus and Ph. linteus alcohol extracts (from
1.2- to 1.7-fold increase), the gene transcription be-
ing not much affected by IFN-γ activation (Fig. 2).
NF-κB is a universal transcriptional factor that
regulates the expression of a large number of genes
involved in immune-infl ammatory responses, cell
cycle progression, and inhibition of apoptosis.
23
The NF-κB transcriptional factor can be activated
in response to various stimuli, including cytokines,
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174 International Journal of Medicinal Mushrooms
A. V. SHNYREVA, W. SONG, & L. J. L. D. VAN GRIENSVEN
viral infection, oncoproteins, membrane receptor’s
overexpression, and other stressful conditions (e.g.,
ROS). All these processes require rapid reprogram-
ming of gene expression.
As shown in a number of research studies,
NF-κB is constitutively expressed in some tumor
cells.
24
In our experiment, the level of NF-κB
transcripts was slightly decreased in K562 cells
preactivated with IFN-γ, as compared to the cells
stimulated with mushroom ethanol extracts only:
from 1.6- to 1.4-fold for Ph. linteus and from 1.4-
to 1.2-fold for the A. bisporus extract (Fig. 2).
This might suggest the potent antitumor activity of
mushroom ethanol extracts applied in combination
with IFN-γ stimulation because the repression of
NF-κB gene transcription is believed to stop the
proliferation of tumor cells. Therefore, it might
be suggested that the mushroom ethanol extracts
are able to inhibit/repress NF-κB transcription
and to block activation of the NF-κB signaling
pathway. For example, many natural products,
including antioxidants that were suggested to have
anticancer and anti-infl ammatory activity, have
also been shown to inhibit the transcriptional fac-
tor NF-κB.
25
Several recent studies on medicinal
mushrooms (Ganoderma lucidum, Pleurotus spp.,
Schizophyllum commune, Marasmius oreades, Tra-
metes versicolor, etc.) also indicate that fungal low-
molecular-weight metabolites are able to modulate
the activity of NF-κB.
26
In conclusion, we demonstrated a suppressive
effect/activity of Ph. linteus and A. bisporus alcohol
extracts on erythroid leukemia cell line K562 (pre-
activated with IFN-γ). Mushroom alcohol extracts
are suggested to exert their anticancer effect via
the induction of apoptosis. The Ph. linteus alcohol
extract demonstrated stronger proapoptotic activity
on K562 leukemia cells than the alcohol extract of
A. bisporus. Therefore, even tumor cells generally
resistant to programmed cell death, such as K562,
can be triggered to apoptosis by treatment with
Ph. linteus alcohol extract. The K562 cells were
more responsive to transcriptional induction of
proapoptotic genes (particularly, Bcl-2 and TNF-α)
when stimulated with IFN-γ. Treatment of K562
cells with A. bisporus extract selectively promoted
transcription of the cytokine gene IL-10.
ACKNOWLEDGMENTS
This work was assisted by an Erasmus Mundus
grant, External Cooperation Window, to Dr. A. V.
Shnyreva (Russia) who would like to express her
thanks for the hospitality she received at Plant
Research International WUR (The Netherlands).
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