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Pinealon Increases Cell Viability by Suppression of Free
Radical Levels and Activating Proliferative Processes
V. Khavinson,
1
Y. Ribakova,
2
K. Kulebiakin,
3
E. Vladychenskaya,
3
L. Kozina,
1
A. Arutjunyan,
1
and A. Boldyrev
2,3
Abstract
The synthetic tripeptide pinealon (Glu-Asp-Arg) demonstrates dose-dependent restriction of reactive oxygen
species (ROS) accumulation in cerebellar granule cells, neutrophils, and pheochromocytoma (PC12) cells, in-
duced by oxidative stress stimulated by receptor-dependent or -independent processes. At the same time,
pinealon decreases necrotic cell death measured by the propidium iodide test. The protective effect of pinealon is
accompanied with a delayed time course of ERK 1/2 activation and modification of the cell cycle. Because
restriction of ROS accumulation and cell mortality is saturated at lower concentrations, whereas cell cycle
modulation continues at higher concentrations of pinealon, one can conclude that besides its known antioxidant
activity, pinealon is able to interact directly with the cell genome.
Introduction
Under the influence of environmental factors, emo-
tional stress, and/or progression of age pathologies, the
regulation of body functions is disturbed. The role of regu-
latory neuropeptides in the formation of the adaptive re-
sponse of an organism to stress and the disturbance of
homeostasis is now discussed broadly.
1
The endogenous
components of living cells, peptide bioregulators, demon-
strate diverse biological effects; they are effective at low
concentrations and show no side effects.
2–4
However, their
therapeutic use is limited by their permeability through the
blood–brain barrier, relatively rapid metabolization, and ef-
fect on the immune system. Short synthetic analogs of neu-
ropeptides that preserve their specific activity do not have
these restrictions. Among these short peptides, which are
potential modulators of regulatory functions, is the tripep-
tide pinealon (Glu-Asp-Arg), which has been synthesized
after analysis of amino acid composition of bovine brain
extracts.
5
The Glu-Asp-Arg sequence is the most common
motif in a complex peptide from the cerebral cortex called
cortexin that demonstrates neuroprotective properties.
6
This
compound was synthesized recently and was found to be
capable of stimulating neuronal regeneration
5
and protecting
brain neurons from hypoxia.
7
The aim of present study was
to characterize the effect of pinealon on cell metabolism
under oxidative stress conditions in vitro.
Materials and Methods
Three different kinds of cell preparations were used in
these experiments: (1) Granule cells isolated from cerebel-
lums of 10- to 12-day-old rats, (2) neutrophils isolated from
peripheral blood of intact adult rats, and (3) a commercially
available culture of pheochromocytoma (PC12) cells. Cere-
bellar granule cells represent a standard model for the study
of oxidative stress,
8
including their induction of the intra-
cellular accumulation of reactive oxygen species (ROS).
9,10
In
contrast, when neutrophils are activated, they generate ROS
in the surrounding medium.
11
We have used these two
models to estimate the ability of pinealon to diminish the
levels of free radicals both inside and outside the cells. To
estimate the effect of pinealon on cell cycle division, PC12
cells were used because they are characterized precisely in
the literature and the role of ROS in regulation of the PC12
cell cycle is well known.
12
Experiments using Wistar rats were carried out according
to international rules of working with laboratory animals
(http://www.nap.edu/books/0309083893/html/R1.html).
Dissociated cerebellar granule cells
Cerebellar granule cells were derived from 7- to 10-day-
old pups. A suspension of surviving neurons was obtained
after treatment of cerebellar slices by dispase, followed by a
wash with standard Tyrode solution (148 mM NaCl, 5 mM
1
Saint-Petersburg Institute of Bioregulation and Gerontology, and
2
Research Center of Neurology Russian Academy of Medical Sciences,
St. Petersberg, Russian Federation.
3
Department of Biochemistry, School of Biology, M.V. Lomonosov Moscow State University, Moscow, Russian Federation.
REJUVENATION RESEARCH
Volume 14, Number 5, 2011
ªMary Ann Liebert, Inc.
DOI: 10.1089/rej.2011.1172
535
KCl, mM CaCl
2
, 1 mM MgCl
2
, 10 mM glucose, 10 mM
HEPES, pH 7.4) and filtration through a 53-mm nylon filter.
10
Oxidative stress was induced in the presence or absence of
pinealon by a 30-min exposure of the cells to 100 nM ouabain
or 500 mM homocysteine (HC). Both compounds are known
to be able to induce oxidative stress in cerebellum granule
cells by interacting with specific receptors on the neuronal
membrane. Ouabain affects sodium/potassium–adenosine
triphosphatase (Na/K-ATPase) of the outer cell membrane.
13,14
HC is a congener of N-methyl-D-aspartate (NMDA), and it
acts via the glutamate receptor of the NMDA class.
15
In both
cases, an increase in intracellular ROS levels and activation
of the key enzyme of the mitogen-activated protein kinase
(MAPK) cascade ERK1/2 kinase (extracellular regulated
kinase, isoforms 1 and 2) is observed.
14,15
Each ligand was
used in a concentration sufficient to induce oxidative stress
via intracellular accumulation of ROS.
14,15
The cell suspen-
sion was incubated with ouabain or HC (in the presence or
absence of pinealon), and then flow cytometry analysis was
performed.
Primary culture of cerebellar granule cells
Cerebellums from 7- to 10-day-old rats were washed with
cold Hanks’ solution (PanEko, Russia) and incubated 20 min
with 0.05% trypsin solution (PanEko, Russia). The neurons
were then washed and cultivated for 11 days in Neuroba-
sal
TM
-A cultivation medium, with 2% Supplement B-27 used
as a serum substitute (Invitrogen, USA), GlutaMax (Invitro-
gen, USA), 50 U/mL penicillin, 50 U/mL streptomycin, and
20 mM KCl (at 5% CO
2
and 378C). Before the following ex-
periment, cells were removed from the substrate with tryp-
sin-EDTA (PanEko, Russia) and exposed to 500 mM HC for
5–30 min in the presence or absence of pinealon. Then cell
suspension was used in western blotting experiments.
Experiments with neutrophils
Before the experiment, adult rats weighing 200–250 grams
were treated with chloral hydrate (500 mg/kg intravenous-
ly). A blood sample was collected from the jugular vein in a
heparin-containing syringe (Spofa, Czech Republic, 50 U/mL
blood). Intact neutrophils were obtained by centrifuging the
blood samples in MonoPoly medium (ICN Biomedicals,
USA) and suspending them in Hanks’ solution. Neutrophils
were activated by adding of 2 mg/mL zymosan to the in-
cubation medium.
To obtain a suspension of neutrophils activated in vivo
(peritoneally induced neutrophils), animals were treated
with 500 mL of zymosan suspension (4.5 mg/mL of Hanks’
solution, intraperitoneally) to induce a local inflammatory
area. Activated neutrophils were isolated from peritoneal
fluid, and a cell suspension with a purity of 95–98% was
obtained. The cells were sedimented by centrifugation, re-
suspended in 1 mL of Hanks’ solution, and stored at 378C for
up to 3 hr.
16
The functional activity of neutrophils was evaluated by
production of ROS, measured as a chemiluminescence re-
sponse in the presence of 1 mM luminol (Sigma-Aldrich,
Germany) on a SmartLum 5773 chemiluminometer (St.-
Petersburg, Russia). Neutrophils were activated using HC
(Sigma-Aldrich, Germany). In preliminary experiments, we
tested the effect of HC on the chemiluminescence of intact
cells in the range of 5–30 min and found no effect in this time
interval. Thus, for characterization of HC action, we used
30 min of incubation.
PC-12 cell culture
Cells were cultured in 75-mm
3
flasks in RPMI-1640 me-
dium (PanEko, Russia) supplemented with 10% fetal calf
serum (FCS; PanEko, Russia), 0.2 mg/mL L-glutamine (Pa-
nEko, Russia), and 20 mg/mL gentamicin at 5% CO
2
and
378C. Cells were removed from the substrate with trypsin-
EDTA (PanEko, Russia).
Measuring the levels of free radicals
in cerebellar neurons
To determine the intracellular ROS levels, cerebellar gran-
ule cells were loaded with the fluorescent dye 2,7-dihlor-
odihydrofluoresceine-diacetate (DCF-DA; Molecular Probes,
USA) at a final concentration 100 mM.
8
Cells were removed
from the substrate, washed free of trypsin, and resuspended
in Hanks’ solution. They were then placed in 1.5-mL Eppen-
dorf tubes, allowed to rest for 30min, and incubated for
40 min with DCF-DA. After dye loading, the cells were ex-
posed for 20 min to 1 mM hydrogen peroxide (H
2
O
2
;Sigma,
Germany) in the presence or absence of pinealon (Garmonika,
Russia) at concentrations noted in the legends to the figures.
For 1 min before the measurement, the samples were sup-
plemented with 10 mM propidium iodide (PI; Sigma, Ger-
many). Measurements were performed on BD FACSCalibur
flow cytometer (Becton, Dickinson, USA) after gating the cell
population corresponding in size to neuronal cells.
8–10
Western blotting
The activity of ERK 1/2, which is tightly connected with
cell viability,
17
was measured as a ratio between the phos-
phorylated form of ERK 1/2 and an endogenous control
(total actin level). Quantification was performed using
western blotting. Cells were washed with cold Hanks’ so-
lution and were lysed using RIPA buffer (Sigma-Aldrich,
USA). The lysates were then subjected to sodium dodecyl
sulfate polyacrylamide gel electrophoresis (SDS-PAGE) with
10% separating and 6% concentrating gels. A prestained
protein molecular weight marker (Fermentas, Germany) was
run in parallel. After SDS-PAGE, the proteins were electro-
blotted onto polyvinylydine difluoride (PVDF) membranes
(Thermo Scientific, USA). Detection of proteins was per-
formed as described by the manufacturers of the antibodies
anti-ERK 1/2 (Thr202/Tyr204) (1:1,000, Cell Signaling
Technology, USA), anti-phospho-ERK 1/2 (Thr202/Tyr204)
(1:1,000, Cell Signaling Tecnology, USA), and anti-pan-actin
(1:2,000 Cell Signaling Tecnology, USA). Secondary anti-
bodies were conjugated with horseradish peroxidase (HRP;
1:1,000, Cell Signaling Tecnology, USA). Membranes were
visualized via enhanced chemiluminescence (ECL) with Su-
perSignal West Femto solution (Thermo Scientific, USA).
Data were analyzed using TotalLab Quant software (TotalLab
Limited, UK).
Study of the PC12 cell cycle
To measure the parameters of the cell cycle, cells were
stained with PI.
18
Cells were removed from the substrate,
536 KHAVINSON ET AL.
washed free of trypsin, and then resuspended in phosphate-
buffered saline (PBS; PanEko, Russia); fixation was per-
formed with 70% ethanol (at 208C overnight). The next day,
cells were washed free of ethanol with PBS, and staining was
performed in citrate buffer containing 100 mg/mL RNase A
from bovine pancreas (Sigma, Germany) (40 mg PI per 1
million cells, 40 min in the dark at 378C).
Results
As shown earlier, incubation of neurons with a specific
inhibitor of Na/K-ATPase ouabain results in accumulation
of free radicals and activation of ERK 1/2 kinase,
14
which
induces switching on of the so-called early response genes.
18
Incubation of DCF-DA pre-loaded rat cerebellum granule
cells with 100 nM ouabain causes an increase in their fluo-
rescence, which is a sign of increasing intracellular levels of
free radicals (Fig. 1).
Addition of pinealon to the incubation medium at dif-
ferent concentrations leads to suppression of the level of
free radicals in a dose-dependent manner (Fig. 1), which
corresponds to the earlier described antioxidant action of
pinealon.
19
A 100 nM concentration of pinealon is enough to
prevent ouabain-induced ROS accumulation completely.
Thus, pinealon in a dose-dependent manner prevents an
increase in the ROS accumulation induced by ouabain.
When cerebellum cells were incubated with 500 mMHC
under the same conditions as above, the stationary ROS
level was increased by 92 5%, and this activation was
abolished in the simultaneous presence of HC and pinealon
(500 nM).
It is known that ROS can act as a secondary messenger in
the cells, triggering cascades of cellular signaling, in partic-
ular, that of MAPK pathway ERK 1/2.
18,20
Therefore, we
evaluated the effect of pinealon on the neuronal level of
ERK 1/2 which is activated by HC (Fig. 2). In control sam-
ples containing only HC, activation of ERK 1/2 is observed
within 2.5 min, whereas in the presence of HC and pinealon,
an increase in the level of active forms of ERK 1/2 occurs
20 min later. Thus, pinealon has a suppressing effect on ac-
tivation of ERK 1/2 in rat cerebellar granule cells exposed to
HC.
The ability of pinealon to suppress intracellular levels of
free radicals in neurons indicates its possible effects on the
other ROS-generating systems, especially those that generate
them for extracellular use. To this purpose, we investigated
the ability of pinealon to affect the respiratory burst of
neutrophils activated by two different modes—in vitro by
adding zymosan to the reaction medium with freshly
isolated neutrophils, and in vivo by measuring the chemilu-
minescence response of neutrophils isolated from rats after
24-hr of intraperitoneal administration of zymosan, as de-
scribed above. Intraperitoneal administration of zymosan to
intact animals promotes inflammation, which accumulates
intraperitoneally induced neutrophils. Thus, the cells pre-
pared after such a procedure are characteristic of high sta-
tionary levels of ROS production.
Figure 3 shows that under the conditions used both neu-
trophils activated by zymosan in vitro (Fig. 3A) and those
prepared from zymosan-treated animals (Fig. 3B) demon-
strate a similar ability to generate a chemiluminescent signal.
In both models of neutrophil activation, pinealon showed a
dose-dependent ability to inhibit accumulation of ROS, and
its effect was carried out in the same concentration range
(Fig. 3).
In both previous models of oxidative stress, we observed
no cell death. In further experiments on pheochromocytoma
PC12 cell cultures, we used a less physiological, but a
stronger, stress agent, H
2
O
2
, which induced not only ROS
accumulation but also cell death. Exposure of PC12 cells to
1mM H
2
O
2
for 20 min resulted in a five-fold increase in ROS
levels (Fig. 4). Preincubation of the cells with pinealon for
60 min decreased the accumulation of ROS induced by H
2
O
2
(Fig. 4, gray bars). At the same time, pinealon had no sig-
nificant impact on the level of ROS in intact cells (Fig. 4,
white bars).
FIG. 1. Pinealon restricts reactive oxygen species (ROS) accumulation in cerebellar granule cells induced by 30 min of
incubation of cells with 100 nM ouabain. (#) Significant difference in relation to control ( p<0.05; (*) significant difference in
relation to samples with ouabain ( p<0.05). CDF, 2,7-Dihlorodihydrofluoresceine.
PINEALON INCREASES CELL VIABILITY 537
Under these conditions, incubation of cells with 1 mM
H
2
O
2
resulted in a loss of about half of the entire cell pop-
ulation, while increasing concentrations of pinealon pro-
gressively increased the proportion of cells remaining alive,
despite the presence of H
2
O
2
in the medium (Fig. 5). Thus,
pinealon neutralizes effects of toxic compounds that stimu-
late the development of oxidative stress and protects cells
from necrotic death.
It is known that the intracellular ROS level affects distri-
bution of cells between different phases of cell cycle
18
; i.e.,
FIG. 2. Effect of pinealon (10 nM) on activation of ERK 1/2 in cerebellar granule cells in the presence of 500 mM homo-
cysteine (HC). (A) Results of analysis. (White bars) Activation of ERK 1/2 after incubation of cells in the presence of HC; (gray
bars) the same in the presence of HC and pinealon. (*) Significant difference between groups with p<0.05. (B) Western
blots; rows 1 and 2, samples with HC (correspond to white bars); rows 3 and 4, samples with HC and pinealon (correspond to
grey bars).
FIG. 3. Effect of pinealon on generation of free radicals by rat neutrophils activated in vitro (A, arrows correspond to adding
zymosan), and in vivo (B, arrows correspond to start of measurement).
538 KHAVINSON ET AL.
ROS are capable of regulating mechanisms that promote the
cell cycle from one stage to another.
20
Taking into account
the ability of pinealon to reduce intracellular levels of free
radicals, we studied its possible effect on the advancement of
cells through the cell cycle. Using flow cytometry, we have
demonstrated that a 24-hr incubation of PC12 cells in pine-
alon-containing medium leads to a distinct redistribution of
cells through the phases of the cell cycle (Fig. 6A and B). This
effect has a clear dose dependence: An increase in the con-
centration of pinealon from 50 to 500 nM leads to a decrease
the number of cells in the G
1
phase and an increase in the
number of cells in the G
2
and S phases, indicating the
modulating effect of pinealon on the proliferative activity of
cells (Fig. 6C).
Discussion
It was shown earlier that pinealon has a pronounced an-
tihypoxic effect on neurons that is explained by restriction of
excitoxicity of NMDA and by inhibition of ROS accumula-
tion.
7
Pinealon also stimulates the activity of the anti-
oxidative enzymes superoxide dismutase and glutathione
peroxidase in the rat brain under hypobaric hypoxia.
19
These
data point out the ability of pinealon to diminish oxidative
stress.
In this paper, we studied the effects of a synthetic tri-
peptide pinealon on properties of living cells. Using rat cer-
ebellar granule cells, we have demonstrated the ability of
pinealon to reduce stationary ROS levels caused by the ac-
tion of both receptor-dependent (ouabain, HC) and non-
receptor (H
2
O
2
) activators of oxidative stress. On surviving
cultures of neutrophils activated by zymosan, we also
showed the ability of pinealon to reduce ROS production
during the inflammatory response.
ROS take part in a number of physiological processes,
such as inflammation, apoptosis, neoplastic transformation,
and aging. In recent years, the amount of evidence regarding
the functioning of ROS as second messengers has in-
creased.
21,22
It is known that the redox potential of the cells
changes as they move through several phases of cell cycle.
For example, in the G
1
phase, ROS control the activity of
cyclin-dependent kinases (CDKs) and phosphorylation of the
retinoblastoma protein (pRb), thereby adjusting the entrance
into the S phase of the cell cycle. Induction of oxidative stress
often leads to cell cycle arrest in G
2
. Thus, one can assume
that adjusting the ROS level within proliferating cells by
specific substances can affect the cell function.
Comparison of the effect of pinealon on ROS accumulation
and cell death in cultured PC12 cells demonstrated that the
increase in survival roughly corresponded to suppression of
ROS levels. At the same time, it is seen from comparison of
Figs. 4 and 5 that when pinealon concentration reaches from
FIG. 4. Effect of pinealon on intracellular reactive oxygen species (ROS) levels in PC12 cells, as measured in the presence or
absence of 1 mM hydrogen peroxide (H
2
O
2
). (White bars) Sample containing only pinealon at various concentrations as
indicated; (grey bars) samples containing the same concentrations of pinealon and 1 mM H
2
O
2
, the incubation time of 60 min.
(*) Significant difference from the sample containing H
2
O
2
. alone ( p<0.05). DCF, 2,7-Dihlorodihydrofluoresceine.
FIG. 5. Influence of pinealon on cellular death of PC12 cells
after 30 min of exposure to 1 mM hydrogen peroxide (H
2
O
2
).
(*) Significant decrease from sample with H
2
O
2
(p<0.05).
PINEALON INCREASES CELL VIABILITY 539
100 nM to 500 nM, the ROS level stops decreasing whereas
the number of dead cells continues to decelerate. This ob-
servation suggests some additional points regarding the
pinealon effect.
We have also showed that simultaneously with suppres-
sion of ROS accumulation pinealon delays the time course of
ERK 1/2 activation. Such an effect is in a good correlation
with its action on cell cycle division, suggesting its possible
influence on complex intracellular processes. It is noteworthy
that when pinealon concentration increases from 100 nM to
500 nM (which corresponds to saturating area for ROS ac-
cumulation) both necrotic death prevention and modulation
of cell cycle continue. This observation is in accordance with
data concerning the protective action of pinealon on neuro-
nal survival at oxidative stress levels induced by hypoxia
19
and suggests some additional mechanisms of its antioxidant
action.
It suggests that, in addition to the antioxidant effect re-
sulting in ROS neutralization, pinealon may act directly with
the cell genome and/or gene expression factors. Our paper is
the first demonstration that pinealon is able to modulate ERK
1/2 activity and thus activate proliferative processes in PC12
cell culture, thus showing pinealon to be a useful tool for
modulating cell metabolism.
Acknowledgments
The work was partially supported by RFBR Grants, ## 09-
04-00507, 10-04-01461 and 10-04-00906.
Author Disclosure Statement
The authors have no conflict of interests to disclose.
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Address correspondence to:
Alexander Boldyrev
Department of Biochemistry
School of Biology
M.V. Lomonosov Moscow State University
Lenin’s Hills, Building 1/2
119992 Moscow
Russia
E-mail: aaboldyrev@mail.ru
Received: February 19, 2011
Accepted: April 29, 2011
PINEALON INCREASES CELL VIABILITY 541
