Polysaccharide peptides from Coriolus versicolor exert differential immunomodulatory effects on blood lymphocytes and breast cancer cell line MCF-7 in vitro

Abstract

The protein-bound polysaccharides (PBP), isolated from Coriolus versicolor (CV) fungus, are considered as natural compounds with potential therapeutic applications. The immunopotentiating and antitumor activity of polysaccharopeptides has been previously examined, however similar findings could not be achieved. The source of PBP, variations in extraction process as well as environmental factors seems to affect the biological properties of these active CV components. Since further analysis are needed to draw more definite conclusion, the present study aimed to investigate the immunomodulatory properties of the PBP extract, isolated from commercially available capsules of Coriolus versicolor. Our results revealed that the effect mediated by PBP extract depends on the target cells. We reported that the polysaccharopeptides induced a significant decrease in breast cancer MCF-7 cells growth, which was TNF-α-dependent phenomenon. Interestingly, the level of two others cytokines, IL-1β and IL-6 was not affected. On the other hand, in this study we noticed that protein-bound polysaccharides extracted from CV significantly augmented the proliferative response of blood lymphocytes in a time-dependent manner, which was associated with IL-6 and IL-1β mRNA upregulation. Moreover we found that the cells response to PBP stimuli might be inversely related to its concentration.

Full text

Immunology
Letters
174
(2016)
37–44
Contents lists available at ScienceDirect
Immunology
Letters
journal homepage: www.elsevier.com/locate/immlet
Polysaccharide
peptides
from
Coriolus
versicolor
exert
differential
immunomodulatory
effects
on
blood
lymphocytes
and
breast
cancer
cell
line
MCF-7
in
vitro
Małgorzata
Kowalczewskaa,∗,
Jakub
Piotrowskia,b,
Tomasz
J˛
edrzejewskia,
Wiesław
Kozaka
aDepartment
of
Immunology,
Faculty
of
Biology
and
Environment
Protection,
Nicolaus
Copernicus
University,
Lwowska
1
Street,
87-100
Torun,
Poland
bCentre
for
Modern
Interdisciplinary
Technologies,
Nicolaus
Copernicus
University,
Wilenska
4
Street,
87-100
Torun,
Poland
a
r
t
i
c
l
e
i
n
f
o
Article
history:
Received
11
February
2016
Received
in
revised
form
5
April
2016
Accepted
13
April
2016
Available
online
14
April
2016
Keywords:
Protein-bound
polysaccharides
Coriolus
versicolor
MTT
test
qRT-PCR
Cytokine
Lymphocytes
MCF-7
cells
a
b
s
t
r
a
c
t
The
protein-bound
polysaccharides
(PBP),
isolated
from
Coriolus
versicolor
(CV)
fungus,
are
considered
as
natural
compounds
with
potential
therapeutic
applications.
The
immunopotentiating
and
antitumor
activity
of
polysaccharopeptides
has
been
previously
examined,
however
similar
findings
could
not
be
achieved.
The
source
of
PBP,
variations
in
extraction
process
as
well
as
environmental
factors
seems
to
affect
the
biological
properties
of
these
active
CV
components.
Since
further
analysis
are
needed
to
draw
more
definite
conclusion,
the
present
study
aimed
to
investigate
the
immunomodulatory
properties
of
the
PBP
extract,
isolated
from
commercially
available
capsules
of
C.
versicolor.
Our
results
revealed
that
the
effect
mediated
by
PBP
extract
depends
on
the
target
cells.
We
reported
that
the
polysaccharopep-
tides
induced
a
significant
decrease
in
breast
cancer
MCF-7
cells
growth,
which
was
TNF-␣-dependent
phenomenon.
Interestingly,
the
level
of
two
others
cytokines,
IL-1␤
and
IL-6
was
not
affected.
On
the
other
hand,
in
this
study
we
noticed
that
protein-bound
polysaccharides
extracted
from
CV
significantly
augmented
the
proliferative
response
of
blood
lymphocytes
in
a
time-dependent
manner,
which
was
associated
with
IL-6
and
IL-1␤
mRNA
upregulation.
Moreover
we
found
that
the
cells
response
to
PBP
stimuli
might
be
inversely
related
to
its
concentration.
©
2016
Published
by
Elsevier
B.V.
on
behalf
of
European
Federation
of
Immunological
Societies.
1.
Introduction
Protein
bound
polysaccharides
(PBP),
among
them
PSP
(polysac-
charide
peptide)
and
PSK
(Krestin)
derived
from
Coriolus
versicolor
(CV)
fungus
are
classified
as
biological
response
modifiers
(BRMs),
with
potential
therapeutic
applications
[1,2].
Both,
PSP
and
PSK,
are
proteoglycans
of
approximately
100
kDa
with
a
bioactive
␤-d-
glucan
region
bound
to
a
protein
core
[3].
These
components
of
CV,
are
found
to
possess
a
wide
range
of
beneficial
medical
proper-
ties,
including
anti-cancer,
anti-bacterial
and
anti-viral,
as
well
as
immune-potentiating
[4–7].
Moreover,
PBP
possess
the
ability
to
counter
immunosuppressive
effects
of
chemotherapy
and
radio-
∗Corresponding
author.
E-mail
addresses:
m
kowalczewska@umk.pl
(M.
Kowalczewska),
piotrowski
jak@umk.pl
(J.
Piotrowski),
tomaszj@umk.pl
(T.
J˛
edrzejewski),
wkozak@umk.pl
(W.
Kozak).
therapy
and
reduce
cancer
treatment-related
symptoms
such
as
fatique,
loss
of
appetite
and
pain
[6,7].
Despite
the
fact
that
PBP
are
widely
used
as
an
immunomodi-
fiers,
in
both
healthy
individuals
and
patients
with
different
cancer
diseases
[8],
the
molecular
mechanisms
of
action
of
these
BRMs
is
still
poorly
understood.
Moreover,
although
few
research
groups
have
formerly
examined
the
immunomodulatory
activities
of
CV
extracts,
they
failed
to
reach
a
similar
conclusion
[9].
Some
stud-
ies
revealed
that
protein-bound
polysaccharides
derived
from
C.
versicolor
has
the
ability
to
increase
the
proportion
of
mono-
cytes
(CD14+/CD16−)
subsets
of
peripheral
blood
mononuclear
cells
(PBMCs)
with
no
effect
on
B-
and
T-cells
[3].
Others
have
shown
a
significant
stimulatory
effect
of
PSP
on
T
cell
prolifer-
ation
[4,10].
Differences
in
the
results
can
also
be
observed
in
terms
of
cytokines
profile
induced
by
CV
extracts.
Some
investiga-
tors
showed
that
PSP
exhibit
suppressive
effects
on
Th1
cytokines
production
(IL-2,
IFN-␥,
TNF-␣),
but
stimulates
the
production
of
IL-10
(Th2
cytokine),
[11],
whereas
others
implied
that
CV
extracts
differentially
enhanced
the
production
of
Th1-related
cytokines
http://dx.doi.org/10.1016/j.imlet.2016.04.010
0165-2478/©
2016
Published
by
Elsevier
B.V.
on
behalf
of
European
Federation
of
Immunological
Societies.

38
M.
Kowalczewska
et
al.
/
Immunology
Letters
174
(2016)
37–44
(including
IL-2,
IL-12,
IL-18,
IFN-␥),
without
any
effect
on
the
Th2-related
peptides
(IL-4
and
IL-6),
[10].
Discrepancies
are
also
observed
regarding
various
human
cancer
cells
in
both,
in
vitro
and
in
vivo
studies
[12].
Recent
evidence
indicated
that
broad
range
of
environmental
factors
and
medium
composition
[2],
various
PBP
source
(different
strains
of
C.
versicolor)
and
variations
in
extraction
process,
affect
the
activity
of
polysaccharide
peptides
and
therefore
may
stand
as
an
explanation
for
these
discrepancies
[9].
To
draw
more
defi-
nite
conclusions
further
studies
on
various
CV
extracts
have
to
be
performed.
Therefore,
in
this
study
we
examined
the
immunomod-
ulatory
properties
of
polysaccharide
peptides
extracted
from
the
C.
versicolor
capsules
(MycoMedica,
Czech
Republic),
which
are
commercially
available
and
have
not
yet
been
investigated.
Here,
we
tested
in
vitro
the
immunomodulating
and
anticancer
activi-
ties
of
PBP
directed
at
peripheral
blood
lymphocytes
and
breast
cancer
cells
(MCF-7).
Measured
parameters
included:
proliferative
response
and
the
expression
of
the
selected
cytokines:
interleukin
(IL)-1␤,
IL-6
and
tumor
necrosis
factor-␣
(TNF-␣).
The
results
of
our
investigation
have
shown
that
examined
PBP
induced
a
signif-
icant
decrease
in
growth
of
breast
cancer
cells,
which
was
TNF-␣
dependent.
In
contrast,
the
extract
exerted
a
strong
stimulatory
impact
on
blood
lymphocytes
proliferation
and
proinflammatory
cytokines
expression
in
inverted
dose
response
manner.
2.
Material
and
methods
2.1.
Preparation
of
PBP
solution
Polysaccharide
peptides
were
isolated
from
C.
versicolor
(MycoMedica,
Czech
Republic).
According
to
the
manufacture,
PBP
are
the
major
soluble
components
of
the
stock
CV
extract
con-
stituting
approximately
25%
by
weight.
Plain
RPMI
medium
1640
(Sigma
Aldrich;
Germany)
was
used
to
dissolve
the
CV
extract
as
stock
solution
of
8
mg/ml
and
4
mg/ml
respectively,
for
48
h
at
room
temperature
with
continuous
agitation.
Insoluble
material
was
removed
by
centrifugation
at
2000g
for
10
min.
The
soluble
supernatant,
containing
2
mg/ml
or
1
mg/ml
of
PBP
respectively,
was
sterilized
using
0.22
␮m
filter,
and
further
diluted
with
plain
culture
medium
to
the
defined
concentrations
as
indicated.
2.2.
MCF-7
cell
line
The
MCF-7
human
breast
cancer
cell
line
was
obtained
from
European
Collection
of
Cell
Cultures
(Lot.
13K023;
UK).
Cells
were
cultured
and
expanded
at
37 ◦C
in
a
humidified
atmosphere
of
5%
CO2in
culture
medium
RPMI
1640
(with
l-glutamine;
Sigma
Aldrich)
supplemented
with
10%
heat
inactivated
fetal
bovine
serum
(FBS,
PAA;
Austria),
100
IU/ml
penicillin
and
100
␮g/ml
streptomycin
(PAA
Laboratories
GmbH;
Germany),
and
non-
essential
amino
acids
1×
(Sigma
Aldrich).
Cells
were
removed
from
cell
culture
flasks
or
plates
by
0.25%
trypsin-EDTA
(Sigma
Aldrich)
digestion.
2.3.
Peripheral
blood
lymphocytes
isolation
Blood
was
sterile
collected
from
anesthetized
8
weeks
old
female
Wistar
rats
by
cardiac
puncture
into
the
solution
of
K3EDTA
(Sigma
Aldrich).
The
procedure
was
approved
by
the
local
Bioethi-
cal
Committee
for
Animal
Care
in
Bydgoszcz,
Poland
(permission
no.
35/2014).
Peripheral
blood
mononuclear
cells
isolation
was
performed
according
to
density
gradient
centrifugation
method.
Briefly,
the
collected
blood
was
diluted
1:1
(v/v)
with
PBS
(pH
7.4)
at
room
temperature
(RT).
Diluted
cell
suspension
was
carefully
layered
onto
the
separation
medium
(Ficoll-Paque
Plus,
Amersham
Biosciences)
in
a
15
ml
centrifuge
tube.
After
centrifugation
at
400g
for
35
min
at
RT,
the
PBMCs
fraction
was
collected
at
the
inter-
face
between
the
two
layers.
The
cells
were
than
washed
twice
with
RPMI
medium
(RPMI-1640
with
l-glutamine;
Sigma
Aldrich),
before
being
suspended
in
RPMI
medium
supplemented
with
10%
heat
inactivated
FBS
(PAA),
100
IU/ml
penicillin
and
100
␮g/ml
streptomycin
(PAA
Laboratories
GmbH).
After
2
h
of
preincubation,
peripheral
blood
lymphocytes
were
separated
from
the
adhered
monocytes
and
cultured
for
subsequent
experiments.
2.4.
Cell
culture
and
PBP
treatment
Before
culturing
in
cell
culture
flat-bottom
plates,
lymphocytes
and
MCF-7
cell
count
and
viability
were
determined
by
trypan
blue
exclusion
test,
using
the
LUNATM automated
cell
counter
(Logos
Biosystems,
USA).
For
cytokine
assessment
cells
were
adjusted
to
a
final
concentration
of
0.5
×
106/ml
and
incubated
in
24-well
culture
in
RPMI
supplemented
with
10%
FBS
and
antibiotics.
Before
stimu-
lation,
lymphocytes
and
MCF-7
cells
were
preincubated
at
37 ◦C
for
24
h
in
an
incubator
providing
a
humidified
atmosphere
containing
5%
CO2.
In
order
to
perform
proliferation
test,
9
×
104or
1
×
104cells
(lymphocytes
and
MCF-7
cells,
respectively)
were
seeded
in
96-
well
culture
plate
in
a
total
volume
of
100
␮l
supplemented
plain
RPMI
medium
and
preincubated
for
24
h.
After
preincubation,
cells
were
stimulated
with
sterile
PBP
solution
at
the
final
concentration
of
100
␮g/ml
and
300
␮g/ml
respectively,
without
adding
phytohemagglutinin
(PHA).
As
a
neg-
ative
control,
the
cells
were
added
with
plain
culture
medium
alone
(a
solvent
for
PBP).
2.5.
MTT
viability
assay
In
order
to
determine
the
growth-promoting
effects
of
PBP
on
lymphocytes
and
MCF-7
viability,
MTT
assay
was
used
to
detect
the
reduction
of
3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-
2H-tetrazolium
bromide
(MTT;
Sigma
Aldrich)
by
mitochondrial
dehydrogenase
to
blue
formazan
product,
which
reflects
the
nor-
mal
functioning
of
mitochondria
and
thus
the
metabolic
rate
of
cells
[10].
Following
incubation
of
indicated
cells
with
PBP
solution
(100
␮g/ml
and
300
␮g/ml
respectively)
for
18-,
24-,
48-
and
72
h,
10
␮l
of
filtered
(0,22
␮m)
5
mg/ml
MTT
dissolved
in
plain
RPMI
was
added
to
each
well
and
the
plate
was
incubated
in
dark,
at
37 ◦C,
humidified
atmosphere
containing
5%
CO2for
4
h.
After
the
incubation
period,
the
culture
supernatant
was
removed
using
a
multichannel
pipette.
One
hundred
microliters
of
dimethylsulfox-
ide
(DMSO,
Sigma
Aldrich)
were
then
added.
The
plate
was
mixed
horizontally
for
15
min
and
the
optical
density
was
measured
at
570
nm
(with
reference
wavelength
of
630
nm)
using
Synergy
HT
Multi-Mode
Microplate
Reader
(BioTek;
USA).
The
results
were
expressed
as
percentage
of
control
cells;
570/630
nm
ratio
in
con-
trol
wells
serves
as
100%.
2.6.
Cytokine
mRNA
expression
quantification
by
qRT-PCR
In
our
approach,
the
mRNA
level
of
cytokines:
IL-1␤,
IL-6
and
TNF-␣
was
determined
by
qRT-PCR.
After
indicated
times
of
incubation
with
PBP
solution,
total
RNA
was
extracted
from
the
cultured
cells
according
to
the
Chomczynsky
method
[13]
using
TRI
Reagent®Solution
(Life
Technologies,
USA).
The
RNA
sample
concentration
was
measured
using
Take3
Micro-Volume
Plate
of
Synergy
HT
Multi-Mode
Microplate
Reader
(BioTek;
USA).
First-
strand
cDNA
synthesis
was
carried
out
on
1
␮g
of
total
RNA
using
iScriptTM Reverse
Transcription
Supermix
for
RT-qPCR
(Bio-Rad,
USA),
according
to
the
manufacturer’s
instructions.
Quantitative
real-time
PCR
was
performed
in
a
20
␮l
mixture
containing
2
␮l
of
the
cDNA
and
iTaqTM Universal
SYBR®Green
Supermix
(Bio-

M.
Kowalczewska
et
al.
/
Immunology
Letters
174
(2016)
37–44
39
Table
1
PCR
primer
sequences.
Human
Forward
Reverse
␤-actin
TGGGCATGGGTCAGAAGGATTC
TAGCACAGCCTGGATAGCAACG
IL-1␤GACTTCACCATGCAATTTGTGTCTTCCTAA
ACAGGAGATCCTCTTAGCACTACCCTAAG
IL-6
GCTGCAGGACATGACAACTCATCTCATTC
ATGCCCATTAACAACAACAATCTGAGGTG
TNF-␣
GTGAAGTGCTGGCAACCACTAAGAATTC
CTCGCCACTGAATAGTAGGGCGATTAC
Rat
Forward
Reverse
18S
sRNA
CATGGCCGTTCTTAGTTGGT
GAACGCCACTTGTCCCTCTA
IL-1␤
ACCTGCTAGTGTGTGATGTTCC
TCTGAGAGACCTGACTTGGCA
IL-6
AGACTTCACAGAGGATACCACC
AACTCCAGAAGACCAGAGCA
TNF-␣AGACCCTCACACTCAGATCATC
TAGAGAACGGATGAACACGCC
Rad)
on
CFX
Connect
Real-Time
PCR
Detection
System
(Bio-Rad).
The
thermal
cycling
conditions
were
as
follows:
3
min
of
denatu-
ration
at
95 ◦C
followed
by
40
cycles
of
denaturation
at
95 ◦C
for
10
s,
annealing
at
52◦C
for
30
s
and
extension
at
72 ◦C
for
45
s.
The
primer
sequences
for
each
target
are
reported
in
Table
1.
Dilutions
of
standards
and
test
samples
were
run
in
duplicate.
Each
reaction
was
repeated
at
least
three
times.
To
analyze
the
specificity
of
the
products,
a
melt
curve
procedure
was
added
on
a
Bio-Rad
CFX96.
Standard
curves
were
prepared
for
target
(IL-1␤,
IL-6
and
TNF-␣)
and
reference
(␤-actin
or
18S
sRNA)
genes.
Calibrator-normalized
relative
quantification
was
carried
out
using
CFX
Manager
Software
3.1
(Bio-Rad).
2.7.
Effect
of
neutralizing
antibodies
to
TNF-˛
on
the
PBP
induced
anti-proliferative
response
of
breast
cancer
cells
MCF-7
cells
in
the
total
amount
of
1
×
104cells
per
well
were
seeded
in
96-well
culture
plate
in
a
final
volume
of
200
␮l
sup-
plemented
plain
RPMI
medium.
After
24
h
of
preincubation
the
anti-human
TNF-␣
monoclonal
mouse
antibody
(R&D
Systems,
cat.
nr
MAB210)
was
added
to
MCF-7
cells
at
concentrations
1
␮g/ml
and
5
␮g/ml,
respectively.
Control
cells
were
added
with
mouse
IgG1
isotype
control
(R&D
Systems,
cat.
nr
MAB002).
After
1
h
the
cells
were
stimulated
with
sterile
PBP
solution
at
the
final
concen-
tration
of
50-,
100-
and
200
␮g/ml.
As
a
negative
control,
the
cells
were
added
with
plain
culture
medium
alone
(a
solvent
for
PBP).
Following
cells
incubation
with
PBP
solutions
and
neutralizing
anti-
bodies
to
TNF-␣
for
48-
and
72
h,
the
cells
viability
was
measured
by
MTT
test
according
the
procedure
described
in
Section
2.5.
2.8.
Statistical
analysis
All
values
are
reported
as
means
±
standard
error
mean
(SEM).
Statistical
significance
was
determined
by
Student’s
t
test,
followed
by
ANNOVA
with
a
value
of
P
<
0.05
considered
to
be
statistically
significant.
3.
Results
3.1.
Anti-proliferative
activity
of
PBP
on
breast
cancer
cells
The
protein-bound
polysaccharides
at
the
concentration
of
100
␮g/ml
inhibited
the
proliferation
of
MCF-7
cells
in
time-
dependent
manner.
Significant
growth
suppression
of
cells
was
observed
after
48
h
incubation
with
PBP,
lasting
up
to
72
h
(75%
of
metabolically
active
cells
compared
to
100%
control
cells).
Inter-
estingly,
no
significant
changes
in
cells
viability
were
observed
when
cells
were
stimulated
with
higher
concentration
of
PBP
(300
␮g/ml),
(Fig.
1).
3.2.
Mitogenic
effect
of
PBP
on
peripheral
blood
lymphocytes
The
results
of
peripheral
blood
lymphocytes
proliferation
test
clearly
illustrated
that
PBP
possess
mitogenic
activity.
The
polysac-
charide
peptides
at
the
concentration
of
100
␮g/ml,
as
well
as
300
␮g/ml
were
found
to
stimulate
lymphocytes
in
time-
dependent
manner,
however
not
dose-dependent
fashion.
The
100
␮g/ml
dose
of
PBP
was
found
to
prominently
promote
the
cells
growth,
with
maximal
increase
between
24-
and
48
h
of
incubation,
lasting
up
to
72
h
when
achieving
645
±
24%
increase
compared
to
non-stimulated
cells
(Fig.
2).
The
enhancement
in
lymphocytes
pro-
liferation
after
cells
stimulation
with
PBP
in
dose
300
␮g/ml
was
also
detectable,
however
not
as
significant
as
observed
with
lower
concentration
of
PBP
solution.
The
maximal
increase
compared
to
control
cells
was
also
observed
after
72
h
of
incubation
(61
±
15%),
(Fig.
2).
3.3.
Differential
effect
of
MCF-7
cells
treatment
with
PBP
on
IL-ˇ,
IL-6
and
TNF-˛
mRNA
expression
Since
some
studies
have
reported
that
mushroom
extracts
show
anti-cancer
and
anti-inflammatory
effects
[12,14,15],
we
evaluated
the
relative
mRNA
expression
of
IL-1␤,
IL-6
and
TNF-␣
in
MCF-7
cancer
cells
after
treatment
with
PBP
derived
from
C.
versicolor.
Results
were
compared
with
untreated
MCF-7
cells
used
as
a
con-
trol.
The
experiments
data
revealed
that
the
levels
of
IL-1␤
and
IL-6
were
downregulated
after
18
h
of
incubation
with
both
solutions
of
PBP
(100
␮g/ml
and
300
␮g/ml)
compared
to
control
cells
(Fig.
3A).
However,
after
24
h
of
MCF-7
cells
incubation
with
PBP
at
the
con-
centration
of
100
␮g/ml,
the
relative
expression
of
IL-6
was
found
to
upregulate
and
reached
6.93
±
0.99
fold
of
control
(Fig.
3B).
Interestingly,
the
present
results
demonstrated
that
the
rela-
tive
expression
of
TNF-␣
was
significantly
altered,
however
only
when
cells
were
incubated
with
PBP
at
the
concentration
of
100
␮g/ml.
After
18
h
of
incubation,
the
TNF-␣
expression
increased
13.46
±
0.89
fold
compared
to
non-stimulated
cells,
whereas
24
h
incubation
resulted
in
significant
augmentation,
41.48
±
3.99
fold
of
control.
The
higher
concentration
of
PBP
(300
␮g/ml)
caused
a
slight
downregulation
of
TNF-␣
in
MCF-7
cells
(Fig.
3A
and
B).
3.4.
mRNA
expression
upregulation
of
IL-1ˇ
and
IL-6
after
lymphocytes
treatment
with
PBP
Regarding
the
cytokines
production,
elevated
mRNA
levels
of
IL-1␤
and
IL-6
were
found
in
blood
lymphocytes
after
exposure
to
the
PBP
solutions.
The
interval
changes
of
the
levels
of
exam-
ined
cytokines
are
shown
on
Fig.
4A
and
B.
Our
data
suggested
that
the
cells
stimulation
with
PBP
induce
a
significant
increase
of
IL-6.
However,
we
reported
an
inverse
association
between
PBP
con-
centration
and
mRNA
gene
expression
elevation.
Eighteen
hours
lymphocytes
exposure
to
100
␮g/ml
of
PBP
induces
an
increase
of
IL-6
by
18.74
±
2.57
fold
compared
to
control
(non-stimulated

40
M.
Kowalczewska
et
al.
/
Immunology
Letters
174
(2016)
37–44
Fig.
1.
Anti-proliferative
effects
of
PBP
extracted
from
Coriolus
versicolor
on
MCF-7
cell
line.
Cells
were
incubated
with
two
concentrations
(100
␮g/ml
and
300
␮g/ml)
of
PBP
in
culture
medium
for
18-,
24-,
48-
and
72
h
and
the
cells
viability
was
assessed
by
MTT
assay.
Results
are
expressed
as
the
mean%
MTT
absorbance
(ratio
of
absorbance
in
the
wells
with
PBP-treated
cells
to
that
with
non-stimulated,
control
cells
×
100%)
±
SEM
of
three
independent
experiments
with
six
wells
each.
Asterisks
indicate
significant
differences
compared
to
the
control
cells
(***P
<
0.001).
Fig.
2.
Mitogenic
effect
of
PBP
extracted
from
CV
on
blood
lymphocytes.
Rat
blood
lymphocytes
were
isolated
and
incubated
with
PBP
(100
␮g/ml
and
300
␮g/ml,
respectively)
in
culture
medium
for
18-,
24-,
48-
and
72
h.
Proliferative
response
was
evaluated
by
MTT
test.
Data
were
expressed
as
the
mean%
MTT
absorbance
(ratio
of
absorbance
in
wells
with
PBP-treated
cells
to
that
with
untreated
counterparts,
control
cells
×
100%)
±
SEM
of
three
independent
experiments
with
six
wells
each.
Asterisks
indicate
significant
differences
compared
to
the
control
cells
(*P
<
0.05,
***P
<
0.001).
cells),
whereas
stimulation
with
the
300
␮g/ml
of
PBP
resulted
in
12.82
±
2.29
fold
increase
(Fig.
4A).
In
the
case
of
24-h
incubation,
an
increase
by
8.93
±
0.39-
and
only
1.83
±
0.47-fold
correspondingly
was
observed
in
comparison
to
naive
cells
(Fig.
4B).
The
analysis
of
IL-1␤
revealed
that
lymphocytes
treatment
with
PBP
resulted
in
cytokine
mRNA
expression
upregulation.
Similar
to
IL-6
assessment,
we
observed
an
inverse
association
between
PBP
concentration
and
IL-1␤
mRNA
relative
expression.
Eighteen-
hour
incubation
with
the
100
␮g/ml
of
PBP
was
found
to
induce
12.11
±
2.04
fold
increase
in
IL-1␤
concentration
compared
to
control,
which
was
approximately
four
times
stronger
than
in
cell
cultures
stimulated
with
PBP
at
concentration
of
300
␮g/ml
(2.84
±
0.07),
(Fig.
4A).
Likewise
in
other
measurements,
24-h
expo-
sure
to
PBP
resulted
in
downregulation
of
IL-1␤
(3.19
±
0.49
and
1.66
±
0.21
for
PBP
at
the
doses
of
100
␮g/ml
and
300
␮g/ml,
accordingly),
(Fig.
4B).
The
presented
results
demonstrate
that
there
was
no
signifi-
cant
alteration
in
the
relative
TNF-␣
expression
in
the
lymphocytes,
except
with
a
slight
upregulation
after
18
h
of
incubation
with
100
␮g/ml
of
the
PBP
(2.71
±
0.32),
(Fig.
4A).
However,
after
24
h
incubation
period,
the
TNF-␣
mRNA
level
significantly
decreased
(0.41
±
0.09
fold),
(Fig.
4B).
3.5.
Neutralizing
antibodies
to
TNF-˛
inhibit
anti-proliferative
effect
of
PBP
on
breast
cancer
cells
We
have
shown
that
PBP
significantly
upregulate
the
expression
of
TNF-␣
in
breast
cancer
cells
(Fig.
3).
The
signaling
pathway
medi-
ated
by
TNF-␣
has
been
implicated
in
the
cancer
cells
response
to
fungal
products,
so
we
questioned
whether
this
upregulation
might
be
related
with
the
inhibition
of
MCF-7
proliferation
induced
by
PBP.
Prior
to
cells
stimulation
with
PBP
solutions,
we
incubated
MCF-7
with
neutralizing
antibodies
to
TNF-␣
in
a
final
amount
of
1
␮g/ml
and
5
␮g/ml
according
to
the
procedure
described
by
Navenot
et
al.
[16].
Since,
the
significant
growth
suppression
of
cells
was
observed
after
48-
and
72
h
of
incubation
with
PBP
(Fig.
1),
the
assay
with
TNF-␣
neutralizing
antibodies
was
performed
in
the
same
time
intervals.
In
previous
experiments,
we
have
also
found
that
PBP
at
the
concentration
of
300
␮g/ml
is
ineffective,
hence
in
this
investigation
we
used
solutions
of
50-,
100-
and
200
␮g/ml,
respectively.
Compared
with
the
control
group
(added
with
control
IgG),
the
TNF-␣
neutralizing
antibodies
significantly
inhibited
the
anti-proliferative
effects
mediated
by
PBP
solution
in
a
dose-dependent
fashion
(Fig.
5).
The
effect
mediated
by
anti-
TNF-␣
antibodies
was
the
strongest
in
cell
samples
stimulated
with
polysaccharidopeptides
at
the
concentration
of
200
␮g/ml.
This
PBP

M.
Kowalczewska
et
al.
/
Immunology
Letters
174
(2016)
37–44
41
Fig.
3.
The
differential
effects
of
PBP
on
the
mRNA
expression
level
of
IL-1␤,
IL-6
and
TNF-␣
in
the
MCF-7
cells.
MCF-7
cells
were
incubated
with
PBP
(100
␮g/ml
and
300
␮g/ml)
in
culture
medium.
RNA
was
isolated
after
18
h
(A)
and
24
h
(B)
of
incubation
time
and
the
mRNA
level
of
cytokines
was
specifically
determined
by
qRT-
PCR.
The
level
of
␤-actin
mRNA
of
each
sample
was
used
as
an
internal
reference
to
normalize
the
data.
The
mRNA
fold
changes
(mRNA
relative
expression)
were
expressed
relative
to
the
corresponding
mRNA
mean
value
found
in
the
control
cells.
Results
are
given
as
mean
±
SEM.
Asterisks
indicate
significant
differences
between
sample
and
the
control
(*P
<
0.05,
**P
<
0.01,
***P
<
0.001).
Hash
marks
indicate
significant
differences
between
paired
group
(#P
<
0.5,
##P
<
0.01,
###P
<
0.001).
concentration,
together
with
100
␮g/ml
caused
significant
inhibi-
tion
of
MCF-7
cells
proliferation
(Fig.
5).
4.
Discussion
Loss
of
immune
competence
is
consider
as
an
important
risk
factor
of
cancer
development
and
progression
[17].
Polysaccharide
peptides
(PBP),
bioactive
ingredients
extracted
from
C.
versicolor,
are
found
to
play
an
important
role
in
supporting
the
immune
sys-
tem.
Despite
the
fact
that
PBP,
acting
as
immune-enhancers,
are
widely
used
as
a
therapeutic
adjuvant
for
cancer
immunotherapy
in
China
and
Japan
[8],
an
exact
mechanisms
leading
to
favorable
clinical
results
have
not
yet
been
identified.
Extensive
research
studies
including
clinical
trials,
have
been
conducted
over
the
past
25
years,
however
a
similar
conclusion
from
the
results
has
failed
to
be
drawn.
Since
additional
studies
on
various
CV
extracts
are
nec-
essary,
in
this
study
we
have
analyzed
the
immunomodulatory
and
antitumor
activities
of
PBP
extracted
from
commercially
available
C.
versicolor
capsules
(MycoMedica),
which
has
not
been
previously
studied.
In
the
present
investigation
we
assessed
the
proliferative
response
of
MCF-7
cancer
cells
and
blood
lymphocytes
after
stim-
ulation
with
two
different
concentrations
of
extracted
PBP.
The
metabolic
activity
of
cells
was
estimated
with
MTT
test.
This
col-
orimetric
assay
is
deemed
to
be
a
versatile
method,
thus
allowing
the
evaluation
of
cells
viability
upon
various
treatments.
The
pro-
duction
of
resultant
formazan
appears
to
be
proportional
to
the
level
of
energy
metabolism
in
the
living
cells.
Hence,
it
is
possi-
ble
to
assess
the
metabolically
activated
cells
even
in
the
absence
of
cells
proliferation
[18].
The
results
of
MTT
assay
showed
that
PBP
extracted
from
CV
at
the
concentration
of
100
␮g/ml
signif-
icantly
inhibited
the
proliferation
of
human
breast
cancer
MCF-7
cells
(Fig.
1).
The
obtained
data
are
in
parallel
with
others,
sug-
gesting
that
CV
extracts
possess
selective
antiproliferative
activity
against
certain
tumor
cells
[12,14,15,19,20].
Previous
studies
concerning
breast
tumor
cells
demonstrated
that
anticancer
mechanism
of
CV
extracts
include
retardation
of
cancer
proliferation
by
delaying
cell
cycle
and
induction
of
apopto-
sis
processes
[12,15].
Ho
et
al.
[15]
have
suggested
that
C.
versicolor
extract
could
trigger
the
apoptosis
of
ER-positive
MCF-7
cells
partly
via
upregulation
of
the
p53
protein
expression,
which
is
followed
by
a
significant
downregulation
of
Bcl-2
protein
leading
to
final
cell
death.
Our
findings
showed
that
MCF-7
cells
treatment
with
PBP
extract,
induced
decrease
of
MCF-7
cells
viability
(Fig.
1),
which
was
preceded
by
significant
upregulation
in
the
expression
of
TNF-
␣
(Fig.
3).
The
tumor
necrosis
factor-␣
is
able
to
induce
apoptotic
cell
death
via
activation
of
p38
MAPK
[21].
This
kinase,
in
apoptotic
processes,
is
reported
to
phosphorylate
Bcl-2
and
thus
inhibiting
its
anti-apoptotic
properties,
leading
finally
to
the
activation
of
caspase-9
and
in
consequence
caspase
3,7
[22].
Therefore,
we
con-
sider
that
the
polysaccharopeptides
mediated
apoptosis-inducing
effect
against
MCF-7
cells
might
be
due
to
direct
activity
of
CV
compounds
as
well
as
indirect
action
through
TNF
receptor.
This
assumption
seems
to
be
appropriate
since
blocking
of
TNF-␣
activ-
ity
with
monoclonal
antibody
strongly
inhibited
the
PBP-induced
cells
apoptosis
(Fig.
5).
The
obtained
results
suggest
that
activa-

42
M.
Kowalczewska
et
al.
/
Immunology
Letters
174
(2016)
37–44
Fig.
4.
The
effects
of
PBP
on
mRNA
cytokines
expression:
IL-1␤,
IL-6
and
TNF-␣
in
the
lymphocytes.
Blood
lymphocytes
were
isolated
and
incubated
with
PBP
extracted
form
CV
(100
␮g/ml
and
300
␮g/ml,
respectively)
in
culture
medium
without
PHA.
RNA
was
isolated
after
18
h
(A)
and
24
h
(B)
of
incubation
time,
and
the
mRNA
level
of
cytokines
was
specifically
determined
by
real-time
PCR.
The
level
of
18S
sRNA
mRNA
of
each
sample
was
used
as
an
internal
reference
to
normalize
the
data.
The
mRNA
fold
changes
(mRNA
relative
expression)
were
expressed
relative
to
the
corresponding
mRNA
mean
value
found
in
the
control
cells.
Results
are
expressed
as
mean
±
SEM.
Asterisks
indicate
significant
differences
between
sample
and
the
control
(*P
<
0.05,
**P
<
0.01,
***P
<
0.001).
Hash
marks
indicate
significant
differences
between
paired
group
(#P
<
0.5,
##P
<
0.01,
###P
<
0.001).
tion
of
TNF-␣
expression
in
breast
cancer
cells
might
be
one
of
the
mechanism
of
cytotoxic
activity
induced
by
fungus
protein-bound
polysaccharides.
In
this
study,
we
have
also
evaluated
the
effect
of
MCF-7
cells
stimulation
with
PBP
extract
on
the
relative
IL-1␤
and
IL-6
mRNA
level,
the
cytokines,
which
are
also
engaged
in
tumor
cells
growth
and
proliferation
processes.
The
outcomes
of
our
assay
showed
that
exposure
to
polysaccharopeptides
resulted
in
reduction
of
IL-
6
mRNA
level
in
MCF-7
cells,
compared
to
control
cells
(Fig.
3A).
The
only
upregulation
of
IL-6
mRNA
expression
was
noticed
in
cells
treated
with
PBP
at
the
concentration
of
100
␮g/ml
for
24
h
(Fig.
3B),
however
was
significantly
lower
that
this
observed
with
TNF-␣.
Likewise
IL-6,
interleukin-1␤,
a
pleiotropic
cytokine,
has
been
implicated
as
a
cancer
progression
promoting
factor
of
many
tumor
types
[23].
In
this
study,
we
have
shown
that
MCF-7
cells
stimulation
with
CV
polysacharopeptides
did
not
induce
any
IL-1␤
mRNA
elevation
(Fig.
3).
Among
various
activities,
protein-bound
polysaccharides,
the
bioactive
components
of
CV,
were
found
to
be
the
most
promi-
nent
in
immunomodulatory
efficiency
[10].
Here,
we
have
shown
that
polysaccharopeptides
derived
from
CV
capsules
(MycoMed-
ica),
which
previously
have
not
been
examined
in
in
vitro
studies,
exerted
strong
mitogenic
effects
on
blood
lymphocytes.
The
cur-
rent
data
displayed
that
PBP,
at
the
concentration
of
100
␮g/ml,
significantly
augmented
the
proliferative
response
of
lymphocytes.
We
noticed
time-dependent
increase
(Fig.
2),
greater
than
that
induced
by
LPS
at
the
concentration
of
100
ng/ml
(data
not
shown).
After
18
h
of
cells
incubation
with
100
␮g/ml
of
PBP,
the
amount
of
metabolically
active
cells
was
2
times
higher
than
in
the
control
samples,
whereas
24
h
incubation
resulted
in
a
fourfold
increase.
This
expansion
was
maintained
over
time
of
incubation
and
after
72
h
the
number
of
cells
reached
7.5
fold
of
non-stimulated
cells.
These
results
demonstrated
that
the
same
dose
of
CV
extract,
which
significantly
inhibited
the
proliferation
of
MCF-7
cells
(Fig.
1),
has
a
strong
stimulatory
influence
on
blood
lymphocytes
(Fig.
2),
sug-
gesting
that
the
extract
differentially
modulated
the
proliferation
of
normal
immunocompetent
cells
and
cancer
ones.
Since
cytokines
play
an
important
role
in
modulating
the
pro-
liferation
and
activation
of
lymphocytes,
the
regulatory
effects
of
tested
PBP
extract
on
the
IL-1␤,
IL-6
and
TNF-␣
expression
in
the
activated
lymphocytes
were
also
examined
using
real-time
PCR.
Our
results,
being
in
line
with
other
reports
[2,10,14]
demonstrated
that
protein-bound
polysaccharides
can
stimulate
IL-1␤
and
IL-6
expression
in
lymphocytes.
We
have
also
noticed
that
the
produc-
tion
of
these
cytokines
was
inversely
related
to
PBP
exposure
time.
Lymphocytes
stimulated
with
100
␮g/ml
of
PBP
for
18
h
expressed
almost
19-times
more
IL-6
than
non-treated
cells.
The
mRNA
level
diminished
after
24
h
incubation,
however
was
still
9-fold
higher
compared
with
control.
Interleukin-6
is
a
molecule,
which
induces
various
biological
responses
depending
on
the
target
cells
[24].
On
the
one
hand,
it
acts
as
B-cell
differentiation
factor,
on
the
other,
exerts
stimulatory
effects
on
proliferation
of
CD8+
T
cells
[25,26].
Similar,
IL-1␤
is
produced
by
a
large
number
of
immunocompetent
cells
[27].
Here
we
have
shown
that
18
h
lymphocytes
incubation
with
100
␮g/ml
of
PBP
resulted
in
12-times
upregulation
of
IL-1␤
mRNA
level
compared
to
non-stimulated
cells.
After
24
h
exposure

M.
Kowalczewska
et
al.
/
Immunology
Letters
174
(2016)
37–44
43
Fig.
5.
Inhibition
of
anti-proliferative
effect
mediated
by
CV
protein-bound
polysaccharides
by
TNF-␣
neutralizing
antibodies
in
MCF-7
cells.
The
breast
cancer
cells
were
incubated
with
neutralizing
antibodies
to
TNF-␣
(1-
or
5
␮g/ml,
respectively)
and
stimulated
with
PBP
at
the
concentration
of
50-,
100-
and
200
␮g/ml.
Control
cells
were
added
with
IgG
control
antibodies
and
subsequent
stimulated
with
PBP
at
indicated
concentrations.
Cells
proliferation
was
assessed
using
MTT
test
after
48
h
(A)
and
72
h
(B)
of
treatment.
Data
are
expressed
as
the
mean%
MTT
absorbance
(ratio
of
absorbance
in
wells
PBP-
and
antibody-treated
cells
to
that
with
untreated
counterparts,
control
cells
×
100%)
±
SEM
of
three
independent
experiments
with
six
well
each.
Asterisks
indicate
significant
differences
compared
to
the
control,
IgG
antibody
and
PBP
treated
cells
(*P
<
0.05,
**P
<
0.01,
***P
<
0.001).
The
white
bars
represents
the
amount
of
metabolically
active
cells
after
PBP
stimulation
and
incubation
with
IgG
control
antibodies
at
the
concentration
of
1
␮g/ml
(the
differences
between
the
IgG
concentration
of
1
␮g/ml
and
5
␮g/ml
were
not
observed).
to
PBP,
the
expression
level
of
this
cytokine
declined,
nonethe-
less
was
still
increased
in
comparison
to
control
cells.
These
data
indicate
that
the
tested
polysaccharopeptides
might
activate
lym-
phocytes
and
trigger
the
inflammatory
cytokines
expression,
hence
may
counteract
the
loss
of
immune
competence
in
patients
with
depressed
immunity.
It
is
surprising
that
although
the
IL-1␤
and
IL-6
mRNA
expres-
sion
was
significantly
augmented
in
PBP
treated
lymphocytes,
the
level
of
TNF-␣
was
practically
unchanged
(Fig.
4A
and
B).
The
PBP
extract,
which
has
been
tested,
significantly
augmented
the
expression
of
TNF-␣
in
cancer
cells,
without
affecting
its
level
in
lymphocytes
derived
from
peripheral
blood.
Therefore,
it
seems
possible
that
PBP
extract
might
promote
TNF-␣
secretion
and
its
cytotoxic
activity
within
tumor
tissue,
while
eliminating
the
toxic-
ity
which
is
associated
with
systemic
TNF-␣
increase
[21].
However
this
latter
assumption
has
to
be
further
investigated.
Our
results,
concerning
both
cell
types,
MCF-7
cells
and
lympho-
cytes,
have
revealed
that
higher
concentration
of
PBP
(300
␮g/ml),
unlike
the
lower
one
(100
␮g/ml),
hardly
affects
proliferative
response
neither
cytokine
expression.
This
observation
is
con-
firmed
by
a
growing
body
of
evidence
that
the
presence
of
small
stimulus,
such
as
low
concentration
of
polysaccharopeptides,
is
in
fact
able
to
induce
a
wide
range
of
defense
mechanisms.
The
basis
for
this
phenomenon
may
be
the
concept
of
hormesis
[28],
a
dose
response
relationship
which
has
been
documented
in
variety
bio-
logical
and
pharmacological
investigations.
In
this
state
the
low
doses
of
agents
or
stressors
generate
favorable
biological
responses,
but
little
effect
or
inhibitory
response
is
formed
by
high
doses
[29,30].
The
occurrence
of
this
phenomenon
was
previously
noticed
in
investigation
concerning
DNA
protection
abilities
of
C.
versi-
color
(Yunzhi)
extract
[29].
The
inverted
U-shape
response
was
also
presented
in
other
studies
concerning
cytokine
release
after
cells
treatment
with
CV
extract
[10,14],
however
has
not
been
discussed.
Our
assay
with
TNF-␣
neutralizing
antibodies,
where
other
con-
centrations
of
PBP
solutions
(50-
and
200
␮g/ml)
were
used,
also
confirmed
the
inverted
dose
response
after
PBP
treatment.
In
conclusion,
the
present
data
illustrate
that
polysaccharopep-
tides,
extracted
from
commercially
available
C.
versicolor
capsules,
induce
differential
effects
depending
on
target
cells.
Acting
as
BRMs,
PBP
have
strong
immune-enhancing
abilities
as
well
as
tumor-selective
inhibitory
properties,
which
seem
to
be
TNF-␣
dependent.
However,
further
molecular
studies
are
still
needed

44
M.
Kowalczewska
et
al.
/
Immunology
Letters
174
(2016)
37–44
to
explore
the
molecular
mechanism
by
which
tested
PBP
exert
beneficial
effects
in
treatment
and
prevention
of
human
cancers.
Conflict
of
interests
The
authors
declared
no
financial
or
commercial
conflict
of
interest.
Acknowledgement
This
study
was
supported
by
the
Nicolaus
Copernicus
Univer-
sity
Intramural
Grant
1928-B
to
Malgorzata
Kowalczewska
(Torun,
Poland).
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