Attenuation of NF-κB and activation of Nrf2 signaling by 1,2,4-triazine derivatives, protects neuron-like PC12 cells against apoptosis

Article abstract of DOI:10.1007/s10495-010-0496-6

Oxidative stress has been implicated in the etiology of neurodegenerative diseases and aging. Indeed, accumulation of reactive oxygen species, such as hydrogen peroxide, generated by inflammatory cells, leads to oxidative stress, which may contribute to the neuronal degeneration observed in a wide variety of neurodegenerative disorders of the central nervous system, such as Alzheimer's disease. The present study indicates that H₂O₂-induced cell death can be inhibited in the presence of 1,2,4-triazine derivatives, as measured by MTT and caspase-3 activity. We further show that these compounds exert their protective effect by up-regulation of hemeoxygenase-1, glutamylcysteine synthetase, glutathione peroxidase and nuclear factor-erythroid 2 p45-related factor 2 (Nrf2), while they inhibit NF-κB and decrease lipid peroxidation. It shows that there is a potential cross talk between NF-κB and Nrf2, an important cytoprotective transcription factor in the presence of these compounds. Moreover, in order for drugs to be effective in the treatment of neurodegenerative diseases, they must be capable of penetrating the blood-brain barrier, whereas more than 98% of all potential central nervous system drugs don't cross. Using a reliable model based on the artificial neural network indicated that these compounds satisfy this requirement.

Full text of DOI:10.1007/s10495-010-0496-6

Apoptosis (2010) 15:738–751
DOI 10.1007/s10495-010-0496-6
ORIGINAL PAPER
Attenuation of NF-jB and activation of Nrf2 signaling
by 1,2,4-triazine derivatives, protects neuron-like PC12
cells against apoptosis
•
Solaleh Khoramian Tusi Niloufar Ansari
•
•
Mohsen Amini Azim Dehghani Amirabad
•
•
Abbas Shafiee Fariba Khodagholi
Published online: 8 April 2010
Ó Springer Science+Business Media, LLC 2010
Abstract Oxidative stress has been implicated in the
etiology of neurodegenerative diseases and aging. Indeed,
accumulation of reactive oxygen species, such as hydrogen
peroxide, generated by inflammatory cells, leads to oxi-
dative stress, which may contribute to the neuronal
degeneration observed in a wide variety of neurodegener-
ative disorders of the central nervous system, such as
Alzheimer’s disease. The present study indicates that
H2O2-induced cell death can be inhibited in the presence of
1,2,4-triazine derivatives, as measured by MTT and cas-
pase-3 activity. We further show that these compounds
exert their protective effect by up-regulation of hemeoxy-
genase-1, glutamylcysteine synthetase, glutathione peroxi-
dase and nuclear factor-erythroid 2 p45-related factor 2
(Nrf2), while they inhibit NF-jB and decrease lipid per-
oxidation. It shows that there is a potential cross talk
between NF-jB and Nrf2, an important cytoprotective
transcription factor in the presence of these compounds.
Moreover, in order for drugs to be effective in the treat-
ment of neurodegenerative diseases, they must be capable
of penetrating the blood–brain barrier, whereas more than
98% of all potential central nervous system drugs don’t
cross. Using a reliable model based on the artificial neural
network indicated that these compounds satisfy this
requirement.
Keywords Alzheimer’s disease ꢀ Artificial neural
network ꢀ NF-jB ꢀ Nrf2 ꢀ PC12 cells ꢀ Triazine derivatives
Abbreviations
Ab
AD
Amyloid b
Alzheimer’s disease
ANN
AREs
BBB
CAT
CNS
Artificial neural network
Antioxidant responsive elements
Blood–brain barrier
Catalase
Central nervous system
Plasma protein binding ratio
Electrochemiluminescence
c-Glutamylcysteine synthetase
Glutathione peroxidase
Glutathione
CSBP
ECL
c-GCS
GPx-1
GSH
HBA
HBB
HO-1
H2O2
Keap1
MDA
MTT
Abraham’s hydrogen-bond acidity
Hydrogen-bond basicity
Hemeoxygenase-1
Hydrogen peroxide
S. K. Tusi ꢀ N. Ansari ꢀ A. D. Amirabad ꢀ F. Khodagholi (&)
Neuroscience Research Center, Shahid Beheshti University
of Medical Sciences, Tehran, Iran
Kelch-like ECH-associated protein 1
Malondialdehyde
e-mail: khodagholi@sbmu.ac.ir
3-[4,5-dimethylthiazol-2-yl]-2,5-dephenyl
tetrazolium bromide
N. Ansari ꢀ M. Amini
Department of Medicinal Chemistry, Faculty of Pharmacy,
and Drug Design & Development Research Center, Tehran
University of Medical Sciences, 14176 Tehran, Iran
NF-jB
NGF
Nuclear factor-jB
Nerve growth factor
NRB
Nrf2
Number of rotable bonds
Nuclear factor-erythroid 2 p45-related factor 2
A. Shafiee
Department of Medicinal Chemistry, Faculty of Pharmacy,
and Pharmaceutical Sciences Research Center, Tehran
University of Medical Sciences, 14176 Tehran, Iran
P-gp (H) High affinity p-gp substrate probability
PSA Polar surface area
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Apoptosis (2010) 15:738–751
739
Because the prerequisite to cure neurological disorders is the
ability of drug permeating across BBB.
RMS
ROS
SOD
Root-mean-sequare
Reactive oxygen species
Superoxide dismutase
Materials and methods
Materials
Introduction
Antibodies directed against caspase-3, NF-jB and b-actin
were obtained from Cell Signaling Technology. Glutam-
ylcysteine synthetase (c-GCS), Glutathione peroxidase-1
(GPx-1) and Hemeoxygenase-1 (HO-1) antibodies were
from ABCAM. The polyclonal Nrf2 (C-20) was purchased
from Santa Cruz Biotechnology. All the other reagents,
unless otherwise stated, were from Sigma-Aldrich (St.
Louis, MO).
Oxidative stress-induced cell damage has long been impli-
cated both in the physiological process of aging and in a
variety of neurodegenerative diseases such as Alzheimer’s
disease (AD) [1, 2]. Oxidative damage, mediated by reac-
tive oxygen species (ROS) which can be generated fol-
lowing cell lysis, oxidative burst or the presence of an
excess of free transition metals can attack proteins,
deoxynucleic acid and lipid membranes, thereby disrupting
cellular function and integrity. Hydrogen peroxide (H2O2),
one of the main ROS, is produced during the redox process
and is recently considered as a messenger in intracellular
signaling cascades [3]. In addition, it is well known that
H2O2 could cause lipid peroxidation and DNA damage, thus
induce apoptosis in many different cell types [4, 5].
Therefore, therapeutic strategies aimed at preventing or
delaying ROS-induced apoptosis might be a reasonable
choice for the treatment of these diseases. Among various
therapeutic strategies, inhibition of nuclear factor-jB
(NF-jB) activity is one of the most suitable. NF-jB, which
is a redox-sensitive transcription factor, regulates expres-
sion of genes that are involved in cellular differentiation,
proliferation, apoptosis, oxidative response, inflammation,
and immuno response [6]. Its activation was evident by
many distinct stimuli including bacterial lipopolysaccha-
ride, oxidative stress and amyloid beta (Ab) [7]. These
results suggest that NF-jB activation may act as a pro-
apoptotic factor.
Cell culture and PC12 differentiation
Rat pheochromocytoma (PC12) cells obtained from Pasteur
Institute (Tehran, Iran) were grown in Dulbecco’s modified
Eagle’s medium (DMEM) (Sigma, Aldrich), supplemented
with 10% fetal bovine serum and 1% antibiotic mixture
comprising penicillin–streptomycin, in a humidified atmo-
sphere at 37°C with 5% CO2. Growth medium was changed
three times a week. The cells were differentiated by treating
with nerve growth factor (NGF) (50 ng/ml) every other day
for 6 days.
Synthesis
The synthetic reactions used for the synthesis of 5,6-diaryl-
1,2,4-triazine derivatives are outlined in Fig. 1. The first
step of our strategy was the formation of compound 2. Its
preparation is well described in the literature [12]. Briefly,
a mixture of compound 1 and sodium cyanide was refluxed
in alcohol for 2 h. 1,2-bis(4-methoxyphenyl)ethandione (3)
was prepared according to the known method of prepara-
tion of benzyl [13]. The treatment of 1,2-bis(4-methoxy-
phenyl)ethandione with thiosemicarbazide, according to
the previously described procedure [9], resulted in 1,2,4-
triazine ring closure and provided 5,6-diaryl-1,2,4-triazine-
3-thiol (4). This key intermediate was separately reacted
with three alkyliodides (methyliodide, ethyliodide and
n-propyliodide), in the presence of triethylamine in meth-
anol, to give compounds 5a–c, respectively.
Triazine derivatives are one group of compounds pos-
sessing a wide array of biological activities. Many inves-
tigations revealed the anticancer potential of various types
of triazine derivatives [8]. Some of the others have been
introduced as anti inflammatory [9], radical scavenger [10],
and b-sheet breaker [11] agents. 3-(methylthio)-5,6-di-(4-
methoxyphenyl)-1,2,4-triazine is one of the thioalkyl 5,6-
diphenyl triazine derivatives that its antiinflammatory
action has been determined by Saxena et al. [9].
In the present investigation we determined the neuro-
protective effect of this compound and two of its derivatives
and showed that there is a potential cross talk between
NF-jB and nuclear factor-erythroid 2 p45-related factor 2
(Nrf2), an important cytoprotective transcription factor in
their presence. In addition, we developed a reliable model
based on the artificial neural network (ANN) to predict
blood-brain barrier (BBB) permeability of these compounds.
General procedures for preparation of 5a: One mmole of
compound 4 was dissolved in 50 ml of methanol and 1.2
mmole methyliodide and 1 ml of triethylamine were
added. The mixture was stirred for 2 h and the solvent was
removed under reduced pressure. 20 ml of water and 50 ml
of dichloromethane were added to the mixture and mixed.
The organic phase was separated and dried with sodium
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Apoptosis (2010) 15:738–751
Fig. 1 Synthesis pathway for
the preparation of 5a–c
sulphate. Dichloromethane was removed under reduced
pressure and the residue was crystallized in methanol and
water to yield 5a. The structure of the product was con-
firmed by HNMR.
5a–c for 3 h prior to our experiments, then the cells were
treated with H2O2 (150 lM) for 24 h.
Measurement of cell viability
3-Thiomethyl-5,6-(dimethoxyphenyl)-1,2,4-triazine (5a),
m. p.; 152-153°C, HNMR (CDCl3) d: 2.75(s, 3H, SCH3),
3.80(s, 3H, OCH3), 3.85(s, 3H, OCH3), 6.85(d, J =
9.0 Hz, 2H), 6.90(d, J = 9.0 Hz, 2H), 7.50(d, J = 9.0 Hz, 2H),
7.57(d, J = 9.0 Hz, 2H).
Cell viability was determined by the conventional
MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium
bromide) reduction assay. The dark blue formazan crystals
formed in intact cells were solubilized in DMSO and the
absorbance was measured at 550 nm. Results were
expressed as the percentages of reduced MTT, assuming
the absorbance of control cells as 100%.
Drug treatment
All compounds were dissolved in dimethyl sulfoxide
(DMSO) at 10-2 M and then diluted in culture medium.
Western blot analysis
Treatment conditions
For western blot analysis, the cells were lysed in buffer
containing complete protease inhibitor cocktail. The total
proteins were electrophoresed in 12% SDS-PAGE gels,
transferred to polyvinylidene fluoride membranes and
Differentiated PC12 cells, plated in 75 cm2 culture flasks,
were incubated with different concentrations (5–20 lM) of
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Catalase activity assay
probed with specific antibodies. Immunoreactive polypep-
tides were detected by chemiluminescence using enhanced
ElectroChemiLuminescence (ECL) reagents (Amersham
Bioscience, USA) and subsequent autoradiography. Quan-
tification of the results was performed by densitometric scan
of films. Data analysis was done by Image. J, measuring
integrated density of bands after background subtraction.
Protein concentrations were determined according to Brad-
ford’s method [14]. A standard plot was generated using
bovine serum albumin. Nuclear and cytoplasmic proteins
were isolated as described by Kutuk and Basaga [15].
Catalase (CAT) activity was measured by the method of
Aebi [19]. Briefly, 200 ll of cell lysate was added to a
cuvette containing 1.995 ml of 50 mM phosphate buffer
(pH 7.0). Reaction was started by addition of 1.0 ml of
freshly prepared 30 mM H2O2. The rate of decomposi-
tion of H2O2 was measured spectrophotometrically at
240 nm.
Measurement of intracellular ROS
The fluorescent probe 20,70-dichlorofluorescein diacetate
(DCF-DA) was used to monitor intracellular accumulation
of ROS. For this purpose, the DCFH-DA solution (10 lM)
was added to the suspension of the cells (1 9 106/ml), the
mixture was incubated at 37°C for 1 h. The cells were then
washed twice with PBS and finally, the fluorescence
intensity was measured by Varian-spectrofluorometer,
model Cary Eclipse with excitation and emission wave-
lengths of 485 nm and 530 nm, respectively.
Measurement of glutathione levels
The concentration of glutathione (GSH) was determined in
whole cell lysate using dithionitrobenzoic acid (DTNB)
method at 412 nm [16].
Measurement of lipid peroxidation
Malondialdehyde (MDA) levels were measured by the
double heating method [17]. The method is based on
spectrophotometric measurement of the purple color gen-
erated by the reaction of thiobarbituric acid (TBA) with
MDA. Briefly, 0.5 ml of cell lysate was mixed with 2.5 ml
of tricholoroacetic acid (TCA, 10% w/v) solution followed
by boiling in a water bath for 15 min. After cooling to room
temperature, the samples were centrifuged at 3000 rpm for
10 min and 2 ml of each sample supernatant was trans-
ferred to a test tube containing 1 ml of TBA solution
(0.67% w/v). Each tube was then placed in boiling water for
15 min. After cooling to room temperature, the absorbance
was measured at 532 nm with respect to the blank solution.
Dataset description, ANN model training
and sensitivity analysis
In this investigation, dataset is composed of log BB as
output of ANN and seven descriptors as inputs of ANN.
Log BB shows the BBB permeability which is defined as
the logarithm of the blood/brain concentration ratios at
steady-statement.
The experimental log BB values of 153 molecules were
collected from the published papers [20, 21]. The 13
descriptors initially include polar surface area (PSA),
plasma protein binding ratio (CSBP), polarizability,
molecular weight, molecular volume, number of rotable
bonds (NRB), high affinity p-gp substrate probability (P-gp
(H)), molar refraction, log P, Abraham’s hydrogen-bond
acidity (HBA), Abraham’s hydrogen-bond basicity (HBB),
number of hydrogen-bond donor and number of hydrogen-
bond acceptor. Then the ANN model (7-5-1) with one
hidden layer was built. The 13 descriptors and the corre-
sponding logBB value were as inputs and output of ANN
model, respectively. For the model training, the datasets
were randomly divided into training set (n = 117) and
testing set (n = 36). The transfer function of hidden layer
and out layer was selected as tanhAxon. After correlation
coefficient analysis among the 13 descriptors, 7 descriptors
including PSA, molecular volume, log P, CSBP, NRB,
P-gp (H) and molecular weight were selected finally to
build dataset. After training our network based on these 7
descriptors, 3 compounds including mesoridazin, thiorida-
zine and gentistic acid were removed for the remarkable
difference between observed logBB and predicted logBB.
Superoxide dismutase activity assay
Superoxide dismutase (SOD) activity was measured based
on the extent inhibition of amino blue tetrazolium forma-
zan formation in the mixture of nicotinamide adenine
dinucleotide, phenazine methosulphate and nitroblue tet-
razolium (NADH-PMS-NBT), according to the method of
Kakkar et al. [18]. Assay mixture contained 0.1 ml of cell
lysate, 1.2 ml of sodium pyrophosphate buffer (pH 8.3,
0.052 M), 0.1 ml of phenazine methosulphate (186 lM),
0.3 ml of NBT (300 lM) and 0.2 ml of NADH (750 lM).
Reaction was started by addition of NADH. After incuba-
tion at 30°C for 90 s, the reaction was stopped by addition
of 0.1 ml of glacial acetic acid. Reaction mixture was
stirred vigorously with 4.0 ml of n-butanol. Color intensity
of the chromogen in butanol was measured spectrophoto-
metrically at 560 nm. One unit of enzyme activity was
defined as that amount of enzyme which caused 50%
inhibition of NBT reduction/mg protein.
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Then, the rest 150 compounds were randomized and 36
compounds were selected as testing set and 114 compounds
were used with sixfold cross-validation. The learning was
continued with the use of validation set until the root-
mean-sequare (RMS) error reached the smallest value with
regard to validation set of data. The structure of molecule is
drawn by using chemsketch. PSA, NRB and P-gp(H) are
calculated by using ADME Boxes v 4.0 (Trial version on
www.pharma-algorithms.com). The CSPB was obtained
using ChemSilico Property Prediction Software (free ver-
sion on www.chemsilico.com). Molecular geometry was
optimized based on AM1 methods by using Hyperchem
7.52 Evaluation Version. Then, the rest descriptors were
calculated by using hyperchem and chemsketch.
After incubation with 50 ng/ml NGF for 6 days, the PC12
cells displayed a neuronal phenotype that resembles that of
the sympathetic neurons. Morphological observation indi-
cated that a majority of NGF-differentiated PC12 cells
ceased division and extended neuritic processes. Exposure
to H2O2 for 24 h caused heterogeneity in their shape, loss
of connections between neural cells and mostly detachment
from the cultured plate surface. As shown in Fig. 2d, pre-
treatment of the cells with 5a–c, alleviated the cell damage.
According to the morphological change, 5a showed higher
protective effect compared to the others (5b and 5c).
Triazine derivatives inhibit caspase-3 activation
in neuron-like PC12 cells
Sensitivity analysis is a method that shifts descriptors
slightly to evaluate corresponding changes in output.
Sensitivity analysis gives the significance of each descrip-
tor. It also evaluates the rationality of the model developed.
Caspases play a major role in affecting cell death in cells
undergoing apoptosis [23] and previous studies demon-
strated efficacy of caspase inhibitors in preventing H2O2-
induced apoptosis in PC12 cells [24, 25]. To determine
whether these triazine derivatives have antiapoptic effect,
we measured the level of cleaved caspase-3 by western blot
analyses in the presence of the best protective concentra-
tion of these compounds. As shown in Fig. 3, H2O2
induced the appearance of cleaved active caspase-3, argu-
ing for involvement of caspase-3 in H2O2-induced cell
death in PC12 neurons. In those cells pretreated with 5a–c,
band of cleaved (active) caspase-3 was weaker compared
to H2O2-treated cells by about 3.91, 2.98 and 2.32 fold,
respectively, demonstrating the ability of these compounds
to suppress oxidative stress-induced activation of caspase-3
in differentiated PC12 cells.
Data analysis
All data are represented as the mean SD Comparison
between groups was made by one-way analysis of variance
(ANOVA) followed by a specific post-hoc test to analyze
the difference. The statistical significances were achieved
when P \ 0.05.
Results
Triazine derivatives suppress oxidative stress-induced
cell death in PC12 neurons
The present investigation was aimed to determine the
neuroprotective role of some triazine derivatives against
oxidative stress-induced cell death. Therefore we chose
differentiated PC12 cells as an in vitro model and H2O2 as
the oxidative agent [22]. To assess if triazine derivatives
can protect PC12 cells, cells were plated in 96-well plate,
treated with different doses of thriazine derivatives, and
exposed to H2O2 3 h later, because we found that this time
of pre-treatment was resulted in the most neuroprotective
effect (Fig. 2a). After 24 h, the cells were assayed for cell
viability. As the results show in Fig. 2c, pretreatment with
5a–c dose-dependently protected neurons against H2O2-
induced cell death. Treatment of the cells with 5a, 5b and
5c at their best protective concentration (10 lM), increased
cell viability to 80%, 70% and 65%, respectively. At this
concentration (10 lM), 5a–c alone did not have any
cytotoxic effect in PC12 cells, after 24 h (Fig. 2b).
Triazine derivatives treatment result in inhibition
of NF-jB in PC12 cells
To further explore the mechanisms underlying the protec-
tive effect of 5a–c against apoptosis, we analyzed NF-jB
activity, because one of the events that occurs following
oxidative damage is the activation of inflammatory path-
ways. The inflammatory response is mainly regulated by
NF-jB [26]. NF-jB is normally located in the cytosol,
bound and inhibited by I-jB. Following oxidative stress,
NF-jB is released from this inhibition, translocates to the
nucleus and activates transcription of downstream inflam-
matory mediators. We found that in those cells exposed to
H2O2 the level of NF-jB was increased 5.91 fold in
nucleus as determined by western blot (Fig. 4). Interest-
ingly, this increase was prevented by pretreating cells
directly with 5a–c by about 2.00, 1.63 and 1.60 fold,
respectively. This inhibition is associated with the pre-
vention of cell death induced by H2O2.
The neuroprotective effect of the compounds was fur-
ther confirmed by morphological observation (Fig. 2d).
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C
A 100
5a
5b
5c
90
80
70
60
50
40
30
20
10
0
-6
-3
0
1
Preincubation time (h)
D
5 μM
B
μM
μM
μM
10
15
20
100
80
60
40
20
0
*
*
*
*
*
**
Fig. 2 Effect of 5a–c on cell viability. a Triazine derivatives (5 lM)
were added to PC12 cells at different times followed by H2O2
treatment After 24 h, cell viability was determined by MTT assay.
b Differentiated-PC12 cells were treated with 5a–c at indicated
concentrations for 24 h and the cell viability was determined by MTT
test. c Differentiated-PC12 cells were pretreated with 5a–c at
indicated concentrations for 3 h and then exposed to H2O2 for 24 h.
Cell viability was determined by MTT test. Viability was calculated
as the percentage of living cells in treated cultures compared to those
in control cultures. Each value represents the mean SD (n = 3).
#
* significantly different from control cells. significantly different
from H2O2-treated cells. d Morphological evaluation of cells exposed
to 5a–c (10 lM) for 3 h followed by exposure to 150 lM H2O2 for
24 h. Photographs of the cells were taken by an invert microscope at
2009 magnifications. The cytotoxicity of H2O2 is evident as
heterogeneity in their shape, loss of connections between neural cells
and cell death (white arrows) relative to control cells
Triazine derivatives treatment result in induction
of HO-1 in PC12 neurons
pretreatment could increase HO-1 protein 1.48, 1.37 and
1.34 fold compared to the H2O2-treated cells, respectively.
Perhaps the first line of defense against oxidative injury
comes from the molecular switches that recognize redox
changes within the intracellular environment. In eukaryotes
HO-1 is a ubiquitous and redox-sensitive enzyme that
catabolizes heme into carbon monoxide (CO), iron and
biliverdin [27–29]. HO-1 is usually expressed at low levels
under basal conditions, but is highly inducible in response
to various agents causing oxidative stress. Because CO and
bilirubin may act as adaptors to provide cytoprotection
against various environmental stresses, induction of HO-1
by chemopreventive agents has been postulated to be part
of the chemoprotective mechanisms [30, 31]. To determine
whether triazine derivatives induce HO-1, PC12 neurons
were pretreated with 5a–c for 3 h and then exposed to
H2O2 for 24 h. HO-1 protein level in the cell lysates was
detected by western blot and as seen in Fig. 5, 5a–c
Triazine derivatives treatment result in induction
of Nrf2 in PC12 neurons
It is generally accepted that Nrf2 plays a key role in the
adaptive response to oxidative and electrophilic stress,
maintaining the cellular self-defense. To establish whether
the Nrf2 pathway was involved in HO-1 induction by 5a–c
in PC12 neurons, we examined protein levels and nuclear
localization in response to 5a–c. The level of Nrf2 protein
was kept relatively low under basal conditions due to
proteasomal degradation of Nrf2. Activation of the kelch-
like ECH-associated protein 1 (Keap1)-Nrf2 pathway leads
to stabilization of Nrf2 protein by detaching it from Keap1
repression [32]. As the results in Fig. 6a show, a low level
of Nrf2 was detected in control PC12 cells, while pre-
treatment with 5a–c for 3 h before exposure to H2O2 with
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Apoptosis (2010) 15:738–751
Fig. 3 Decrease of caspase-3
expression in PC12 cells
pretreated with 5a–c.
A
Procaspase-3
35 kDa
a Procaspase-3 response to 5a–c
(10 lM) on PC12 cells
pretreated for 3 h and then
exposed to H2O2 for 24 h.
Twenty lg proteins were
separated on SDS-PAGE,
western blotted, probed with
anti-caspase antibody and
reprobed with anti-b-actin
antibody. (One representative
western blot was shown;
n = 3). b The densities of
corresponding bands were
measured and the ratio was
calculated. The median of three
independent experiments is
shown. * significantly different
from control cells.
19 kDa
17 kDa
Caspase-3
β-actin
45 kDa
B
1.8
1.6
1.4
1.2
1
Procaspase-3
Caspase-3
**
##
##
#
##
significantly different from
H2O2-treated cells
0.8
0.6
0.4
0.2
0
##
##
##
**
the dose that induced HO-1 caused a significant increase in
Nrf2 nuclear protein levels. As shown in Fig. 6b, densito-
metric analyses revealed about 13.61, 11.49 and 10.62 fold
increase in Nrf2 level in the presence of 5a–c compared to
H2O2-treated cells, respectively. Time-dependent induction
of Nrf2 by western blot showed the same amounts of Nrf2
exposure to H2O2 in those cells pretreated by 5a–c. How-
ever in the absence of 5a–c a significant increase in the
amount of Nrf2 was observed after 30 min in a way that
reached to the basal level after 2 h (Fig. 6c, d). It suggests
that one of the main mechanisms involved in the cyto-
protective effect of 5a–c is through its ability to stabilize
Nrf2 in the nucleus and upregulation of the Nrf2-target
genes.
with our hypothesis that triazine derivatives can activate the
antioxidant system, indicating that GSH metabolism can be
regulated by these compounds. This observation is likely
due to mediated expression of c-GCS, because the rate
limiting step in GSH biosynthesis is mediated by c-GCS,
which is upregulated by Nrf2 [33–35]. Here the role of
c-GCS was confirmed by determination of its amount. As is
illustrated in Fig. 7, 5a–c stimulated an increase in the
amount of c-GCS. From the other side, glutathione perox-
idase (GPx-1) is another factor that is regulated by Nrf2 [26]
and is involved in the elimination of intra- and extracellular
H2O2 by converting GSH to the oxidized form (GSSG). Our
study showed that GPx-1 expression decrease significantly
at 150 lM H2O2, while preincubation of the cells with 5a–c
(10 lM) attenuated the decrease of GPx-1 expression
(Fig. 8).
Triazine derivatives increase the glutathione level
in differentiated-PC12 cells through c-GCS
Triazine derivatives reduce lipid peroxidation
and rescued loss of antioxidant enzyme activities
in H2O2-treated PC12 cells
As is indicated in Table 1, PC12 cells treated with H2O2
caused the decrease in GSH content by 2.63 fold, however,
pretreatment of PC12 cells by 5a–c resulted in more than
2.51, 2.20 and 1.91 fold increase in GSH level compared to
the H2O2-treated cells, respectively. This result is consistent
Treatment of PC12 cells with 150 lM H2O2 caused the
increase in the intracellular MDA level by 1.78 fold, while
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Apoptosis (2010) 15:738–751
745
Fig. 4 Western blot analysis to
measure the effects of 5a–c on
the nuclear levels of NF-jB in
PC12 cells. a NF-jB response
to 5a–c (10 lM) on PC12 cells
pretreated for 3 h and then
exposed to H2O2 for 24 h.
Twenty lg proteins were
separated on SDS-PAGE,
western blotted, probed with
anti-NF-jB antibody and
reprobed with anti-b-actin
antibody. (One representative
western blot was shown;
A
NF-κB
65 kDa
45 kDa
β-actin
B
2.8
2.6
2.4
2.2
2
1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0
***
n = 3). b The densities of
NF-jB bands were measured
and the ratio was calculated.
The median of three
#
#
#
independent experiments is
shown. * significantly different
from control cells.
#
significantly different from
H2O2-treated cells
Fig. 5 Western blot analysis to
measure the effects of 5a–c on
HO-1 in PC12 cells. a HO-1
response to 5a–c (10 lM) on
PC12 cells pretreated for 3 h
and then exposed to H2O2 for
24 h. Twenty lg proteins were
separated on SDS-PAGE,
A
HO-1
32 kDa
45 kDa
β-actin
western blotted, probed with
anti-HO-1 antibody and
reprobed with anti-b-actin
antibody. (One representative
western blot was shown;
#
B
#
0.7
#
0.6
0.5
0.4
0.3
0.2
0.1
0
n = 3). b The densities of
corresponding bands were
measured and the ratio was
calculated. The median of three
independent experiments is
#
shown. significantly different
from H2O2-treated cells
preincubation of cells with 5a–c markedly attenuated the
change of MDA level. In these condition increase in MDA
level was restored by about 1.47, 1.41 and 1.34 fold,
respectively (Table 1). In addition, PC12 cells treated with
150 lM H2O2 caused the decrease in the activities of SOD
and CAT by about 3.70 and 2.94 fold, respectively.
However, pretreatment with 5a–c significantly attenuated
the decrease of SOD activity by about 4.71, 4.38 and 4.19
123

746
Apoptosis (2010) 15:738–751
Triazine derivatives inhibit H2O2-induced ROS
A
57 kDa
45 kDa
Nrf2
production
ROS levels produced by H2O2 were determined based on
DCF fluorescence. The fluorescence intensity of DCF in
cells treated with H2O2 (150 lM) was increased by 5.07
fold compared to untreated control cells. Pretreatment of
the cells with 5a–c (10 lM) prevented the accumulation of
ROS relative to H2O2-treated cells (Fig. 9).
β-actin
##
B
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
#
#
Triazine derivatives can cross BBB based on ANN
method
*
The performance of this model is given in Table 2. For the
testing set data, the relationship between observed and
predicted values is shown in Fig. 10a, which indicates our
model is valid and can be used to predict logBB of new
compounds. As the results in Table 3 show, the logBB
values of 5a–c were calculated as 0.0549, 0.028 and -0.021,
respectively, which indicate these neuroprotective com-
pounds have good permeability values compared to low
permeable compounds such as cimetidine and icotidine
with logBB values of -0.973 and -1.39, respectively and
impermeable compounds that their log BB are less than
-8.0 [21]. In these conditions the logBB values of good
permeable compounds such as desmethyldiazepam, pergo-
lide and toluene were 0.231, 0.402 and 0.226, respectively.
The sensitivity analysis report was generated as Fig. 10b
From the results, it can be seen that the most important
factor for BBB permeability is PSA. In addition, the
influencing trend of variable factors on logBB can also be
shown.
C
Nrf2
57 kDa
45 kDa
β-actin
0.8
D
**
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
*
*
Discussion
Fig. 6 Western blot analysis to measure the effects of 5a–c on the
nuclear levels of Nrf2 in PC12 cells. a Nrf2 response to 5a–c (10 lM)
on PC12 cells pretreated for 3 h and then exposed to H2O2 for 24 h.
Twenty lg proteins were separated on SDS-PAGE, western blotted,
probed with anti-Nrf2 antibody and reprobed with anti-b-actin
antibody. (One representative western blot was shown; n = 3).
b The densities of Nrf2 bands were measured and the ratio was
calculated. c Nrf2 response to H2O2 after different times. (One
representative western blot was shown; n = 3). d The densities of
corresponding bands were measured and the ratio was calculated. The
median of three independent experiments is shown. * significantly
Many studies have shown that oxidative stress is a major
cause of cellular injuries in a variety of human diseases
including neurodegenerative disorders. ROS such as
hydrogen peroxide, superoxide anion and hydroxyl radical
readily damage biological molecules, which can ultimately
lead to apoptotic or necrotic cell death [4, 5]. Thus,
removal of excess ROS or suppression of their generation
may be effective in preventing oxidative cell death.
Recently, researchers have made considerable efforts to
search for compounds with neuroprotective potential. In
addition, the prerequisite to cure neurological disorders is
that the drug distribution in central nervous system (CNS)
can reach effectively therapeutic concentrations. BBB as
shield not only maintains the homeostasis of the CNS, but
also refuses many potentially important diagnostic and
therapeutic agents from entering into the brain. Thus, the
#
different from control cells. significantly different from H2O2-
treated cells
fold and increased CAT activity by about 2.87, 2.48 and
2.28 fold, respectively (Table 1). In the absence of oxida-
tive stress, these compounds did not significantly alter the
level of MDA as well as the activities of SOD and CAT in
differentiated PC12 cells.
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Apoptosis (2010) 15:738–751
747
Table 1 Effects of 1,2,4-triazine derivatives on lipid peroxidation, glutathione level and antioxidant enzyme activities in H2O2-treated PC12
cells
Treatment
MDA (nmol/mg protein)
SOD (U/mg protein)
CAT (lmol/mg protein)
GSH (lmol/mg protein)
Control
H2O2
0.61 0.02
1.09 0.07*
0.59 0.04
0.74 0.07#
0.62 0.05
0.77 0.01#
0.60 0.04
0.81 0.03#
41.7 1.18
11.26 0.37*
37.67 3.18
53.11 1.51#
34.03 2.83
49.43 3.58#
32.25 0.92
47.24 2.30#
2.50 0.13
0.85 0.06*
2.34 0.15
2.44 0.18#
2.59 0.09
2.11 0.13#
2.47 0.16
1.94 0.15#
6.50 0.13
2.47 0.12*
6.91 0.22
6.22 0.07#
6.71 0.21
5.44 0.22#
6.66 0.09
4.73 0.08#
5a
5a ? H2O2
5b
5b ? H2O2
5c
5c ? H2O2
Cells were incubated with 150 mM H2O2 for 24 h for assay of MDA, GSH content, SOD and CAT activities. 1,2,4-triazine derivatives were
added to the culture 3 h prior to H2O2 addition. The data were presented as means SD (n = 6)
#
* P \ 0.05 versus control; P \ 0.05 versus H2O2 group
Fig. 7 Determination of 5a–c
effects on c-GCS in PC12 cells.
A
γ-GCS
a Western blot analysis to
measure the effects of 5a–c
(10 lM) on c-GCS in PC12
cells pretreated for 3 h and then
exposed to H2O2 for 24 h. b The
densities of corresponding
bands were measured and the
ratio was calculated. The
73 kDa
45 kDa
β-actin
B
0.6
0.5
0.4
0.3
0.2
0.1
0
median of three independent
experiments is shown.
* significantly different from
##
#
##
control cells. significantly
different from H2O2-treated
cells
##
*
ability of drug permeating across BBB becomes critical in
the development of new medicines, especially in the design
of new drugs which are active in brain tissue. Therefore in
this study we synthesized these compounds and wished to
examine whether these 1,2,4-triazine derivatives have
neuroprotective effect in NGF-differentiated PC12 cells
and can penetrate the BBB, as well.
metabolism and the deleterious effects of hydroxyl and
peroxyl radicals on membrane lipids and proteins. Our data
confirmed that treatment of cells with H2O2 resulted in cell
death, which was greatly decreased in the presence of 5a–c.
This result indicated that 5a–c did significantly protect
PC12 neurons from H2O2-induced cytotoxicity.
To further examine the involvement of the signaling
pathway, we focused first on NF-jB, as there was a report
of anti inflammatory effect of 5a [9]. Indeed, the link
between oxidative stress and NF-jB was established
mainly from the inhibition of NF-jB activation by cellular
antioxidants [37–40]. It is also true that reduction of NF-jB
activation protects Ab-induced PC12 cell and hippocampal
The PC12 cell line is a useful and widespread model for
studying neuronal differentiation into sympathetic neurons
and other neurobiochemical and neurobiological events
[36]. H2O2 has been extensively used as an inducer of
oxidative stress in vitro model [3]. The exposure of cul-
tured cell to H2O2 results in an imbalance in energy
123

748
Apoptosis (2010) 15:738–751
Fig. 8 Western blot analysis to
measure the effects of 5a–c on
GPx-1 in PC12 cells. a GPx-1
response to 5a–c (10 lM) on
PC12 cells pretreated for 3 h
and then exposed to H2O2 for
24 h. Twenty lg proteins were
separated on SDS-PAGE,
western blotted, probed with
anti-GPx-1 antibody and
A
GPX1
73 kDa
45 kDa
β-actin
B
1
reprobed with anti-b-actin
antibody. (One representative
western blot was shown;
##
##
0.8
0.6
0.4
0.2
0
##
n = 3). b The densities of
corresponding bands were
measured and the ratio was
calculated. The median of three
independent experiments is
shown. * significantly different
from control cells.
*
#
significantly different from
H2O2-treated cells
Control
makes 5a–c applicable agents. It seems that in this sig-
naling pathway ROS act as second messengers. Because,
ROS have been associated with neurodegenerative disease
[3] and recent studies have demonstrated that ROS, over a
narrow concentration range, might function as second
messengers in cell-signaling pathways [3]. Several data
have shown that ROS activates NF-jB [43]. In addition,
caspase-3 a family of cysteine protease, can be activated by
ROS. So the suppressive effect of our compounds on
translocation of NF-jB and also the expression of active
forms of caspase-3 shows that these triazine deriva-
tives suppress NF-jB activating via suppression of ROS
production.
300
250
200
150
100
50
H2O2
5a+H2O2
5b+H2O2
5c+H2O2
0
500
510
520
530
540
550
Wavelength (nm)
Fig. 9 Inhibitory effect of 5a–c on the accumulation of intracellular
ROS. The PC12 cells were incubated 3 h with or without 5a–c
(10 lM) and then exposed to H2O2 for 24 h. Intracellular levels of
reactive oxygen species were measured with DCFH-DA. The median
of three independent experiments is shown
Although many signals and metabolic events are
important in the regulation of cell death, the intracellular
redox level, in particular, has been shown to play a critical
role. Redox regulation is an interesting and important issue
which tightly associates with oxidative stress. In intercel-
lular redox modulation system, HO-1 is one of the anti-
oxidant proteins in response to oxidative stress. HO-1, also
known as heat shock protein 32 (Hsp-32), belongs to a
family of cytoprotective and detoxification genes that
possess antioxidant responsive elements (AREs) in their
regulatory regions. The Nrf family of transcription factors
can bind the ARE [32]. Previous studies suggest that Nrf2
plays a key role in antioxidant-induced HO-1 expression.
In this study, 1,2,4-triazine derivatives induced Nrf2
nuclear translocation. In addition 5a–c increased c-GCS
level (the rate limiting step in GSH biosynthesis), one of
the other Nrf2-target genes, suggesting that Nrf2 is indeed
important for triazine derivatives-induced cytoprotective
effect in neuron-like PC12 cells.
Table 2 Regression statistics of model
Parameters
Sixfold cross-validation
Testing set
Correlation coefficient (r)
Mean square error
Number
0.85
0.24
114
0.73
0.23
36
neuron cell death [41]. Kutuk and Basaga have also
reported that aspirin prevents H2O2-induced apoptosis in
Hela cell through NF-jB inactivation [42].
Our results also clearly indicate that inhibition of NF-jB
is concomitant with decrease of caspase-3 level, which
123

Apoptosis (2010) 15:738–751
749
Fig. 10 a Observed versus
predicted values of logBB.
b Sensitivity of each descriptor
A
B
0.4
0.35
0.3
0.25
0.2
0.15
0.1
0.05
0
PSA
logP
M.W
CSPB
Volum
p-gp(H)
NRB
Descriptor
Table 3 Predicted logBB of
1,2,4-triazine derivatives
elimination of H2O2 is therefore, critical for reducing
oxidative stress, and the action of CAT provides a repair
mechanism for oxidized membrane components.
Compound
logBB
0.0549
5a
5b
5c
0.028
BBB is another problem in AD drug development, as it is
a stubbornly real obstacle for potential drugs against many
disorders of CNS, in a way that more than 98% of all
potential CNS drugs don’t cross the BBB [47]. To design or
select compounds with desirable brain penetration proper-
ties, there is a great demand in today’s drug discovery
setting for rapid and reliable approaches to predict brain
penetration for a vast number of compounds. One approach
is to use ANN method that is rapid and cost effective.
Besides, this model has shown better results in comparison
to the other softwares [21]. Using this approach showed that
our attempt to select neuroprotective compounds having the
ability of penetrating into CNS was successful. For this
purpose we used medical chemistry to synthesize com-
pounds that are small and lipid-friendly enough to pass
through one side of the endothelial cells and out the other.
Collectively, we provide the documentation of the
neuroprotective effect of some diaryl triazine derivatives.
It seems that in this cytoprotection, HO-1 and c-GCS
-0.021
Besides, in contrast to most other Nrf2-activators like
flavonoids, sulforaphane or curcumin [44, 45] which are
already toxic at low concentration, these compounds
showed no toxicity in our assays in vitro and were able to
stabilize Nrf2 at low concentrations.
In addition, the oxidation of membrane lipids, one of the
primary events in oxidative cellular damage can be asses-
sed by measurement of MDA, a breakdown product of lipid
peroxidase. On the other hand, cells are often equipped
with several antioxidant mechanisms that serve as a
detoxifying system to prevent damage caused by ROS.
Among the most important defenses against oxygen radi-
cals are CAT and SOD enzymes. SOD catalyzes the
breakdown of superoxide into H2O2 and water and is
therefore of central regulator of ROS levels [46]. The
123

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Apoptosis (2010) 15:738–751
activation of nuclear factor kappaB. Free Radic Biol Med
28(9):1317–1327
upregulation through Nrf2 pathway play the key roles. In
addition in this system, inhibition of NF-jB is the other
main factor that protects neurons against oxidative stress
induced caspase-3 activity. These results underlied the
possibility that Nrf2-mediated neuroprotective effect
maybe achieved by suppression of NF-jB. Therefore in
oxidative stress conditions, different cell defense mecha-
nisms act synergistically to protect cell against death. It has
been shown that lipopolysaccharide and/or H2O2-induced
NF-jB activation could be attenuated by diverse Nrf2
activators, such as curcumin [48], alginate [49] and chito-
san [50].However, definite evidence showing that Nrf2 can
inhibit NF-jB signaling is still required. On the contrary, it
was reported recently that NF-jB could directly repress
Nrf2 signaling at the transcription level. NF-jB competes
against Nrf2 for transcription co-activator CREB binding
protein [51]. In addition, NF-jB recruits histone deace-
tylase 3 to cause local hypoacetylation to hamper Nrf2
signaling [51]. However we can not rule out the possibility
of involvement of other mechanisms. In fact, the complete
molecular milieu that links all these events needs to be
elucidated. Continued attempts to identify novel target
molecules responsible for this protective effect and also to
clarify their cross talk with upstream and downstream
signaling molecules will pave the way for exploiting
prevention and/or therapeutic strategies for the manage-
ment of oxidative stress-mediated disorders. A further
study of the detailed mechanisms is now in progress in our
laboratory.
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