Evaluation of two novel antioxidants with differential effects on curcumin-induced apoptosis in C2 skeletal myoblasts; involvement of JNKs

Article abstract of DOI:10.1016/j.bmc.2014.12.046

Excessive levels of reactive oxygen species (ROS) result in numerous pathologies including muscle disorders. In essence, skeletal muscle performance of daily activities can be severely affected by the redox imbalances occurring after muscular injuries, su

Full text of DOI:10.1016/j.bmc.2014.12.046

Bioorganic & Medicinal Chemistry 23 (2015) 390–400
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Evaluation of two novel antioxidants with differential effects on
curcumin-induced apoptosis in C2 skeletal myoblasts; involvement
of JNKs
Maria Peleli a, , Ioanna-Katerina Aggeli a, , Alexios N. Matralis b, Angeliki P. Kourounakis b, Isidoros Beis a,
Catherine Gaitanaki a,
⇑
a Department of Animal and Human Physiology, School of Biology, University of Athens, University Campus, Athens 157 84, Greece
b Department of Medicinal Chemistry, School of Pharmacy, University of Athens, Athens 157 71, Greece
a r t i c l e i n f o
a b s t r a c t
Article history:
Excessive levels of reactive oxygen species (ROS) result in numerous pathologies including muscle disor-
ders. In essence, skeletal muscle performance of daily activities can be severely affected by the redox
imbalances occurring after muscular injuries, surgery, atrophy due to immobilization, dystrophy or
eccentric muscle contraction. Therefore, research on the potential beneficial impact of antioxidants is
of outmost importance. In this context, aiming at further exploring the mechanisms of action of our
newly synthesized antioxidant compounds (AK1 and AK2) in a skeletal muscle experimental setting,
we initially investigated their scavenging effect on 2,2-diphenyl-1-picrylhydrazyl (DPPH) and subse-
quently assessed their effect on the viability of C2 skeletal myoblasts in the presence of two pro-oxidants:
H2O2 and curcumin (MTT assay). Interestingly, while both compounds reversed the detrimental effect of
H2O2, only AK2 was cytoprotective in curcumin-treated C2 cells. We next confirmed the immediate acti-
vation of extracellular signal-regulated kinases (ERKs) and the more delayed activation profile of c-Jun
NH2-terminal kinases (JNKs) in C2 skeletal myoblasts exposed to curcumin, by Western blotting. In cor-
relation with the aforementioned results, only AK2 blocked the curcumin-induced activation of JNKs
pathway. Furthermore, JNKs were revealed to mediate curcumin-induced apoptosis in C2 cells and only
AK2 to effectively suppress it (by detecting its effect on poly(ADP-ribose) polymerase fragmentation).
Overall, we have shown that two similar in structure novel antioxidants confer differential effects on
C2 skeletal myoblasts viability under oxidative stress conditions. This result may be attributed to these
antioxidants respective diverse mode of interaction with the signaling effectors involved in the observed
responses. Future studies should further evaluate the mechanism of action of these compounds in order
to support their potential application in therapeutic protocols against ROS-related muscle disorders.
Ó 2015 Published by Elsevier Ltd.
Received 30 October 2014
Accepted 18 December 2014
Available online 29 December 2014
Keywords:
C2 skeletal myoblasts
Antioxidant
ROS
ERKs
JNKs
Curcumin
Apoptosis
1. Introduction
and Gilbert and published in 1954.1 Pro-oxidants are classified into
radicals and non-radicals comprising of: nitric oxide radical (NOÅ),
ꢀÅ
Reactive oxygen species (ROS) were suggested for the first time
as significant players in biological systems, bearing salutary or
damaging effects in the cell, by the pioneering work of Gersham
superoxide ion radical (O2 ), hydroxyl radical (OHÅ), as well as
hypochlorous acid (HClO), hydrogen peroxide (H2O2) and organic
peroxides, respectively. Mitochondria serve as the major organelles
responsible for ROS production, with enzymes such as NADPH
oxidase, xanthine oxidase and nitric oxide synthase, also
functioning as endogenous sources of ROS.2–4 Antioxidant enzymes
(superoxide dismutase, catalase, thioredoxins, peroxidases) and
low-molecular-weight antioxidants (glutathione, melatonin)
defend redox equilibrium against any interference that could
jeopardize cell function and survival.
Abbreviations: BSA, bovine serum albumin; DMSO, dimethyl sulphoxide; DPPH,
2,2-diphenyl-1-picrylhydrazyl; DTT, dithiothreitol; ECL, enhanced chemilumines-
cence; ERK, extracellular signal-regulated kinase; JNK, c-Jun NH2 terminal kinase;
MAPK, mitogen-activated protein kinase; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide; PAGE, polyacrylamide gel electrophoresis; PARP,
poly(ADP-ribose) polymerase; PMSF, phenyl methyl sulphonyl fluoride; ROS,
reactive oxygen species; TBS, Tris-buffered saline.
⇑
Corresponding author. Tel.: +30 210 72 74 136; fax: +30 210 72 74 635.
E-mail address: cgaitan@biol.uoa.gr (C. Gaitanaki).
Nevertheless, when endogenous antioxidative mechanisms are
impaired, ROS accumulate and exert detrimental effects on lipids,
The authors contributed equally to the present study.
http://dx.doi.org/10.1016/j.bmc.2014.12.046
0968-0896/Ó 2015 Published by Elsevier Ltd.

M. Peleli et al. / Bioorg. Med. Chem. 23 (2015) 390–400
391
proteins and DNA,5 potentially leading to cell death via necrosis or
apoptosis.6 Apoptosis is a morphologically and biochemically
defined form of cell death, characterized by chromatin condensa-
tion, membrane blebbing, nuclear breakdown and by cleavage of
poly(ADP-ribose) polymerase (PARP). PARP is responsible for the
poly(ADP-ribosyl)ation of various nuclear proteins and is inacti-
vated after proteolysis.7,8 The apoptotic mechanism can be initi-
ated by two pathways: (a) the extrinsic pathway, mediated by
activation of cell surface death receptors and (b) the intrinsic
pathway, principally related with mitochondrial membrane
permeabilization and release of pro-apoptotic factors.9,10
polyphenol derived from the root of Curcuma longa, that possesses
antioxidant properties, has been shown to attenuate oxidative
stress-induced muscle damage after downhill running,28 in addition
to promoting skeletal muscle recovery following immobilization.24
In a previous study, aiming to develop multifunctional agents
against the multifactorial nature of atherosclerosis, we designed
and synthesized novel 1,4-benzo(xa/thia)zine derivatives AK1
and AK2 (Fig. 1), by combining structural features of known anti-
oxidant and antihyperlipidemic lead compounds in one structure.
The pharmacological evaluation of these derivatives proved their
significant antioxidant activity both in vitro and in vivo.29 The
key role of oxidative stress in the onset and progression of many
myopathies, along with the salutary potential of antioxidants
against muscle pathologies, prompted us to investigate the biolog-
ical effect of our novel antioxidant compounds on a skeletal muscle
experimental setting. Given that differentiated skeletal muscle
cells have lost ability to proliferate, any regeneration can princi-
pally be achieved by myoblasts.30 Therefore, after evaluating the
radical scavenging (reducing) capacity of AK1 and AK2 (DPPH
method), we looked into the effect of these compounds on the
viability of C2 skeletal myoblasts exposed to oxidative stress and
on the signaling pathways mediating this response.
Oxidative stress exerts its effects by activating a plethora of sig-
naling pathways, including mitogen-activated protein kinases
(MAPKs). In essence, MAPKs interact and cross-talk with various
effectors, transducing the stressful stimulus to the nuclear and other
cellular compartments, ultimately favoring cell survival or
death.11,12 c-Jun NH2-terminal kinases (JNKs) and extracellular
signal-regulated kinases (ERKs) comprise two well-studied MAPK
family members. JNKs were identified as protein kinases that phos-
phorylate the NH2-terminal of the transcription factor c-Jun13 and
regulate diverse biological processes through modulating the
activity of numerous substrates including: transcription factors or
other proteins.14,15 JNKs have also been demonstrated to determine
cell fate by either inducing mitochondria-dependent apoptosis or
promoting survival of cells exposed to stress, that is, in response to
UV-irradiation, hyperosmolarity or oxidative stress.15,16 On the
other hand, ERKs are primarily activated by growth factors and
mitogens and are mainly involved in anabolic processes, including
cell cycle regulation as well as cell proliferation and differentia-
tion.14 Additionally, ERKs have been found to respond to various
forms of stress, that is, hypoxia and osmotic or redox imbalances,
principally playing a cytoprotective and anti-apoptotic role.17–19
If not effectively counterbalanced, excessive levels of ROS con-
tribute to the pathogenesis of various diseases including neurode-
generative, cardiovascular and muscular disorders.20,21 Skeletal
muscle in particular, often encounters oxidative stress conditions
at the cellular level. Redox imbalances may occur during intense
muscle exercise leading to injury,22 during the ischemia/
reperfusion phase in vascular and muscular traumas or reconstruc-
tive surgeries promoting inflammation,23 as well as during muscle
immobilization, resulting in atrophy due to increased protein
breakdown.24 With numerous reports noting that increased levels
of ROS are implicated in muscle wasting-related pathological dis-
orders such as sarcopenia or Duchenne dystrophy5,25 and severely
impair skeletal muscle performance, the use of antioxidants as a
potential therapeutic strategy, may prove beneficial. In fact, studies
have already demonstrated antioxidants to protect against
ROS-induced muscle injuries.26,27 For example, curcumin, a natural
2. Materials and methods
2.1. Chemicals
Curcumin was purchased from Cayman Chemical (Ann Arbor,
MI, USA). Catalase, Leupeptin, trans-epoxysuccinyl-L-leucylamido-
(4-guanidino) butane (E-64) were obtained from Sigma–Aldrich
(St Louis, Missouri, USA). Dithiothreitol (DTT), phenylmethylsul-
fonyl fluoride (PMSF), aprotinin, dimethyl sulfoxide (DMSO) and
Bradford protein assay reagent were purchased from Applichem
(Darmstadt, Germany). Melatonin was obtained from Merck KGaA
(Darmstadt, Germany). AS601245 was purchased from Calbio-
chem-Novabiochem (La Jolla, CA, USA). Hydrogen peroxide was
purchased from Chem-lab, NV (Zedelgem, Belgium). Nitrocellulose
(0.45 lM) was obtained from Macherey-Nagel GmbH (Duren,
Germany). Prestained molecular mass markers were from New
England Biolabs (Beverly, MA, USA). Rabbit polyclonal antibodies
specific for the dually phosphorylated forms of ERKs and JNKs,
phosphorylated c-Jun, total c-Jun and poly(ADP-ribose) polymer-
ase were all purchased from Cell Signaling Technology (Beverly,
MA, USA). Anti-actin antibody (A2103) was from Sigma. Secondary
antibody (horseradish peroxidase-conjugated anti-rabbit anti-
body) was from Dako A/S (Glostrup, Denmark). The enhanced
chemiluminescence (ECL) kit was from Amersham International
(Uppsala, Sweden). Super RX film was purchased from Fuji photo
OH
O
O
O
COOH
OH
N
B
H2N
N
H
HO
HBr
Trolox
A
OH
OH
O
S
N
N
AK1
1
AK2
2
Figure 1. Design and structures of AK1 and AK2: incorporation of structural features of lead compounds (Trolox, antioxidant A and anti-hyperlipidemic B) in the final
antioxidant derivatives AK1 and AK2.

392
M. Peleli et al. / Bioorg. Med. Chem. 23 (2015) 390–400
film GmbH (Dusseldorf, Germany). Cell culture supplies were from
PAA Laboratories (Pasching, Austria). Dulbecco’s modified Eagle’s
medium (DMEM) was from Sigma. All other general chemicals
were purchased from Applichem (Darmstadt, Germany).
with water and saturated NaHCO3 solution, dried and the solvent
is distilled off. The residue is purified by flash column chromatogra-
phy on silica gel (dichloromethane–petroleum ether 1:1) to give
AK2 (60%) as a yellow semisolid: 1H NMR (400 MHz, CDCl3)
d(ppm): 3.03 (s, 3H, N-CH3), 3.26 (d, J = 11.81 Hz, 1H, 3-H benzothi-
azine), 3.60 (d, J = 11.84 Hz, 1H, 3-H benzothiazine), 4.01 (br s, 1H,
OH), 6.85 (t, J = 7.36 Hz, 1H, 7-H benzothiazine), 6.89 (d,
J = 8.56 Hz, 1H, 5-H benzothiazine), 7.09–7.14 (m, 2H, 6,8-H benzo-
thiazine), 7.27–7.30 (m, 1H, 40-H biphenyl), 7.41 (t, J = 7.60 Hz, 2H,
30,50-H biphenyl), 7.51–7.55 (2ꢁ d, 4H, 2,6,20,60-H biphenyl), 7.71
(d, J = 8.11 Hz, 2H, 3,5-H biphenyl). 13C NMR (200 MHz, CDCl3)
d(ppm): 40.78 (N-CH3), 64.16 (3-C benzothiazine), 82.07 (2-C ben-
zothiazine), 113.36 (5-C benzothiazine), 119.89 (7-C benzothiazine),
125.40 (6-C benzothiazine), 126.39 (3,5-C biphenyl), 126.64 (40-C
biphenyl), 127.19 (2,6,20,60-C biphenyl), 127.50 (8-C benzothiazine),
128.81 (30,50-C biphenyl), 130.32 (8a-C benzothiazine), 139.59 (4-C
biphenyl), 140.57 (1-C biphenyl), 141.21 (10-C biphenyl), 143.23
(4a-C benzothiazine). Elemental analysis (C, H): C21H19NOS. 0.5
H2O Calculated (%) C: 73.65, H: 5.89. Found (%) C: 73.95, H: 6.00.
2.2. Design of AK-AM1 and AK-AM2
AK1 was designed via the incorporation of structural features of
(a) Trolox (a hydrophilic derivative of Vitamin E that maintains its
strong antioxidant activity) and A (that inhibits the apoptosis of
endothelial cells induced by oxidized LDL) and (b) the antihyper-
lipidemic compound B (an inhibitor of Squalene Synthase, enzyme
that participates in the cholesterol biosynthesis pathway).31 AK2
resulted from the isosteric replacement of oxygen of the benzox-
azine ring of AK1 with sulfur (Fig. 1).29
2.3. Synthesis
2.3.1. Synthesis of AK1
Compound AK1 [2-(4-biphenyl)-4-methyl-3,4-dihydro-2H-
benzo[1,4]oxazin-2-ol] was synthesized and characterized as pre-
viously described.29
2.4. Interaction with the stable free radical DPPH
Compounds AK-AM1 and AK-AM2, dissolved in absolute etha-
2.3.2. Synthesis of AK229,32
nol, at concentrations of 25–400
lM, were added to an equal vol-
2.3.2.1.
(6).
3-(4-Phenyl-phenacyl)benzothiazolium
A mixture of benzothiazole 5 (7.5 mmol), 4-bromoacetylbi-
bromide
ume of an ethanolic solution of 2,2-diphenyl-1-picrylhydrazyl
(DPPH-final concentration 400 lM) at room temperature. Absorbance
phenyl 4 (5 mmol) and absolute EtOH (4 mL) is heated in an oil
bath at 100–110 °C for 2 h. On completion of the reaction, the
reaction mixture turned into a solid. Ethyl ether (50 mL) is then
added and the mixture is sonicated for 30 min. The solid is filtered,
washed with ethyl ether and dried. White solid, yield: 93%,
mp = 245–246 °C (248 °C).33 IR (Nujol): 1683 cmꢀ1 (C@O). 1H NMR
(400 MHz, DMSO-d6) d(ppm): 6.73 (s, 2H, CH2), 7.37–7.41 (m, 1H,
40-H biphenyl), 7.47 (t, J = 7.46 Hz, 2H, 5,6-H benzothiazole), 7.73–
7.77 (m, 2H, 30,50-H biphenyl), 7.79–7.85 (m, 2H, 20,60-H biphenyl),
7.91 (d, J = 8.56 Hz, 2H, 3,5-H biphenyl), 8.11 (d, J = 8.56 Hz, 2H,
2,6-H biphenyl), 8.32 (dd, J1 = 1.34 Hz, J2 = 7.46 Hz, 1H, 7-H benzo-
thiazole), 8.51 (dd, J1 = 1.47 Hz, J2 = 7.83 Hz, 1H, 4-H benzothiazole),
10.56 (s, 1H, 2-H benzothiazole).
(517 nm) was recorded at different time intervals for 90 min.34
2.5. Cell culture and treatment
In all experiments, C2 murine myoblasts were used, a kind gift
from Prof. Yaffe.35 Cells were grown in DMEM supplemented with
15% (v/v) heat-inactivated fetal bovine serum and antibiotics,
under a humidified atmosphere of 95% air/5% CO2 at 37 °C. Exper-
iments were carried out at a 70% confluence and after at least 3 h of
serum deprivation. Curcumin, dissolved in DMSO, was added to the
medium for the times and at the doses indicated. Exogenous H2O2
was used as an oxidant agent at 1 mM for the times indicated.
AS601245, AK1 and AK2 were dissolved in DMSO, catalase was dis-
solved in DMEM and melatonin in ethanol. All these compounds
were added to the medium 30 min prior to treatment with curcu-
min or H2O2. Cells were left untreated (control) or incubated with
DMSO, ethanol, or the aforementioned compounds alone, for the
appropriate time intervals.
2.3.2.2. 4-Formyl-2-(4-biphenylyl)-3,4-dihydro-2H-1,4-benzothiazin-
2-ol (7).
To a solution of 6 (2 mmol) in MeOH (150 mL), an
aqueous solution of NaOH 1% (2.2 mmol) is added portionwise
under argon. The reaction mixture is then stirred at room temper-
ature and under argon for 2 h. Cold water is added and the solid
separated is filtered off, washed with cold acetonitrile and dried.
White solid, yield: 94%, mp 159–160 °C: IR (Nujol): 3216 cmꢀ1
2.6. Protein extraction
(OH), 1652 cmꢀ1 (C@O). 1H NMR (400 MHz, DMSO-d6) d(ppm):
Cells were washed twice with ice-cold PBS, harvested by scrap-
ing the dishes, lysed in ice-cold buffer G [20 mM Tris–HCl pH 7.5,
20 mM b-glycerophosphate, 2 mM EDTA, 10 mM benzamidine,
⁄
3
3
3.09 (s, ꢁ 1H, OH), 3.34 (d, J = 13.31 Hz, 4 ꢁ 1H, 3-H benzothi-
4
azine),⁄ 3.45 (s,
ꢁ 1H, OH),⁄ 3.63 (d, J = 13.50 Hz,
ꢁ 1H, 3-H
1
1
4
4
benzothiazine),⁄ 3.83 (d, J = 13.69 Hz, 1 4 ꢁ 1H, 3-H benzothiazine),⁄
20 mM NaF, 0.2 mM Na3VO4, 200
lM leupeptin, 10 lM E-64,
benzothiazine),⁄ 7.08–7.17 (m, 4H,
5 mM DTT, 300 M PMSF and 0.5% (v/v) Triton X-100] and then
l
3
4.92 (d, J = 13.30 Hz,
4
5,6,7,8-H benzothiazine), 7.31 (t, J = 7.34 Hz, 1H, 40-H biphenyl),
7.40 (t, J = 7.43 Hz, 2H, 30,50-H biphenyl), 7.55 (d, J = 7.82 Hz, 2H,
incubated on ice for 30 min. Lysates were centrifuged (20,800g,
4 °C, 10 min) and the supernatant (total protein extract) was col-
lected. Protein concentration was determined using the Bradford
assay. After quantification, 0.33 vol. of sodium dodecyl sulphate
(SDS) sample buffer [SB4X: 0.33 mol/L Tris–HCl (pH 6.8), 10%
(w/v) SDS, 13% (v/v) glycerol, 20% (v/v) 2-mercaptoethanol, 0.2%
(w/v) bromophenol blue] was added to the samples which were
then boiled and stored at ꢀ80 °C until use.
20,60-H biphenyl), 7.59 (d, J = 8.41 Hz, 2H, 2,6-H biphenyl), 7.73
ꢁ 1H, CHO),⁄ 8.77
1
(d, J = 8.22 Hz, 2H, 3,5-H biphenyl), 8.22 (s,
4
3
(s,
ꢁ 1H, CHO).⁄ Elemental analysis (C, H): C21H17NO2S. Calcu-
4
lated (%) C: 72.60, H: 4.93. Found (%) C: 72.44, H: 4.39. ⁄Due to
the existence of two rotamers, two signals are observed in 1H
NMR spectrum of 7.29
2.3.2.3. 4-Methyl-2-(4-biphenylyl)-3,4-dihydro-2H-1,4-benzothia-
zin-2-ol (AK2). Benzothiazine derivative 7 (1 mmol) is dissolved
in anhydrous THF under argon, and BH3ꢂTHF (1 M, 7 mL) is added
dropwise and stirred at room temperature for 1 h. Water is then
added and the mixture extracted with dichloromethane, washed
2.7. Subcellular fractionation: preparation of nuclear extracts
Cells were washed twice with ice-cold PBS, harvested by scrap-
ing the dishes, lysed in ice-cold buffer A [10 mM HEPES pH 7.9,
10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1.5 mM MgCl2, 10 mM

M. Peleli et al. / Bioorg. Med. Chem. 23 (2015) 390–400
393
NaF, 1 mM Na3VO4, 20 mM b-glycerophosphate, 1 mM DTT,
0.5 mM PMSF, 2 g/ml leupeptin, 4 g/ml aprotinin] and incubated
3 with 4-bromoacetylbiphenyl 4 (Fig. 2). Compound AK2 was syn-
thesized according to a synthetic procedure recently applied for
similar other derivatives32 that proved more efficient, involving
fewer steps and higher yields than previously described.29 Thus,
the synthesis of AK2 (Fig. 2) includes first the preparation of the
N-substituted benzothiazolium salt 6 (via the reaction of benzothi-
azole 5 with 4-bromoacetylbiphenyl 4 in refluxing EtOH) followed
by the hydrolysis of 6 with an aqueous solution of NaOH affording
7. Reduction of the formyl group of 7 using BH3:THF produced the
final derivative AK2.
l
l
on ice for 15 min. Samples were centrifuged (10,000g, 5 min, 4 °C)
in a BR4i Jouan centrifuge, and the supernatants (cytosolic extract)
discarded. Pellets were re-suspended in buffer A containing 0.6%
(v/v) Nonidet P40, incubated on ice (10 min) and centrifuged
(10,000g, 5 min, 4 °C). Supernatants were discarded, pellets were
re-suspended in buffer B [20 mM HEPES pH 7.9, 400 mM NaCl,
1 mM EGTA, 0.1 mM EDTA, 1.5 mM MgCl2, 10 mM NaF, 1 mM
Na3VO4, 20 mM b-glycerophosphate, 20% (v/v) glycerol 0.2 mM
DTT, and 0.5 mM PMSF, with 2 lg/ml leupeptin and 4 lg/ml apro-
tinin] and incubated 1 h on ice. After centrifugation (20,800g,
10 min, 4 °C), protein concentrations were determined using the
Bradford assay and supernatants (nuclear extract) were boiled
with 0.33 vol. of SB4X.
3.2. Interaction of AK1 and AK2 with DPPH
As the first step in the present study, we examined the time
course of the interaction of AK1 and AK2 with the stable free
radical DPPH (Fig. 3). The DPPH radical-scavenging method is a
common spectrophotometric procedure for determination of
antioxidant capacity.34 Both compounds showed a very efficient
2.8. SDS–PAGE and immunoblot analysis
Protein samples (30
(w/v), 10% (w/v) or 15% (w/v) polyacrylamide gels and transferred
electrophoretically onto nitrocellulose membranes (0.45 M).
l
g) were separated by SDS–PAGE on 8%
interaction with
a
relatively high concentration of DPPH
M). Since this
(400 M) at relatively low concentrations (up to 50
l
l
l
ability is considered an indicator of reducing potential and radical
Membranes were then incubated in TBST [20 mM Tris–HCl pH
7.5, 137 mM NaCl, 0.05% (v/v) Tween 20] containing 5% (w/v)
non-fat milk powder for 60 min at room temperature. Subse-
quently, membranes were incubated overnight with the appropri-
ate antibody, according to the manufacturer’s instructions (1:1000
dilution). After washing in TBST (4 ꢁ 5 min) blots were incubated
with the respective horseradish peroxidase-conjugated secondary
antibody (1:5000 dilution) in TBST containing 1% (w/v) nonfat milk
powder (60 min at room temperature). After washing in TBST
(4 ꢁ 5 min), bands were detected using enhanced chemilumines-
cence (ECL) and quantified by scanning densitometry (Gel Analyzer
v. 1.0). Equal protein loading was verified by probing identical
samples with an anti-actin antibody or with an antibody against
total c-Jun levels for the nuclear extracts. Normalization was car-
ried out by dividing the average value of each protein studied, with
the respective levels of actin or with total c-Jun levels in the case of
phosphorylated c-Jun.
scavenging properties, these compounds seem quite active in this
respect. The IC50 values for AK1 (119
30 min are comparable to the activity of reference antioxidant res-
veratrol (IC50 = 120 M), under the same experimental conditions.
lM) and AK2 (271 lM) after
l
We also observed that AK1 exhibited its antioxidant activity more
rapidly compared to AK2, as seen by the steeper decline in the
absorbance of the mixture in the presence of compound AK1, com-
pared to that of AK2 (Fig. 3).
3.3. AK1 and AK2 inhibit H2O2-induced cell death of C2
myoblasts
In an effort to further characterize and decipher the mecha-
nisms of the antioxidant activity of both AK1 and AK2, we decided
to investigate their effect on an experimental model of skeletal
myoblasts exposed to oxidative stress conditions. To this end, C2
skeletal myoblasts were treated for 24 h with 1 mM H2O2, a con-
centration previously shown to affect C2 viability in a moderate
but statistically significant manner,36 in the presence of increasing
concentrations of AK1 or AK2. Findings of the MTT analysis
2.9. Cell viability assay
Cell viability was evaluated using a 3-(4,5-dimethylthiazol-
2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cells were
seeded in 96-well culture plates (5 * 103 cells/well), and treated
with various concentrations of curcumin for 24 h. Untreated cells
served as a control group. Four hours before the end of treatment,
revealed that incubation with 1
on H2O2-treated cells viability, while 15
enhanced cell survival, with viability levels reaching 97.1 2.3%
compared to 83.1 3.5% in the H2O2-treated cells (P <0.05). Higher
concentrations resulted in decreased viability levels (76.2 1.6% at
l
M AK1 had no substantial effect
lM of AK1 considerably
50
lg MTT was added to each well. Subsequently cells were lysed
30 lM and 71.6 2.0% at 50 lM, respectively) (Table 1). A higher
in 0.1 M HCl/isopropanol, and OD was measured in an ELISA micro-
concentration of AK2 was needed so as to reverse the deleterious
plate reader (DENLEY, West Sussex, UK) using a 540-nm filter.
effect of H2O2, with a marked, statistically significant increase in
viability being observed at 30
(97.6 2.0%) of the compound (P <0.05, Table 2). Both AK1
(15 M) and AK2 (30 M) had no effect on cell viability when used
lM (99.1 1.8%) and 50 lM
2.10. Statistical evaluations
l
l
Western blot images and MTT data shown are representative of
at least three independent experiments. Each data point represents
the mean sem. Statistical analyses were performed using the
oneway ANOVA multiple comparison test (Graph Pad Prism
Software, San Diego, CA) followed by appropriate post-hoc tests
(Bonferroni) for group comparisons. A value of P <0.05 was consid-
ered to be statistically significant.
on their own (last lanes in Tables 1 and 2, respectively).
3.4. Dose-dependent effect of curcumin on cell viability of H2O2-
treated C2 myoblasts
Having evaluated the cytoprotective/antioxidant potential of
AK1 and AK2 against the detrimental effect of H2O2 on C2 skeletal
myoblasts viability, we next decided to compare it with the respec-
tive effect of a natural antioxidant. In particular, we probed into
the dose-dependent effect of curcumin on the cell viability of
H2O2-treated C2 myoblasts. As depicted in Table 3, curcumin pro-
tected cells against H2O2-conferred death, since treatment with
3. Results
3.1. Chemistry
Compound AK1 was synthesized and characterized as previ-
15
lM as well as 30 lM of curcumin, significantly restored C2
ously described,29 that is, via the reaction of 2-methylaminophenol
myoblasts viability at 90.2 2.2% and 93.1 1.8%, respectively

394
M. Peleli et al. / Bioorg. Med. Chem. 23 (2015) 390–400
OH
NaHCO3
DMF, RT
O
N
OH
NH
O
+
Br
AK1
3
4
O
S
S
N
EtOH
100-110 o
aq. NaOH
+
Br
N
MeOH, Ar, rt
C
Br
5
4
O
6
OH
OH
S
N
S
N
BH3:THF
THF, Ar, rt
H
O
AK2
7
Figure 2. Synthetic route for AK1 and AK2 (described in Section 2).
B
A
AK1
AK2
C
AK1 - IC50 (30 min) = 119 µM
AK2 - IC50 (30 min) = 271 µM
Figure 3. (A, B) Time-dependent interaction of DPPH (400
lM) with various concentrations (25–400 lM) of AK1 and AK2, respectively. Absorbance (517 nm) was recorded at
different time intervals and values are means sem for at least two independent experiments. (C) % reducing capacity of AK1 and AK2 after 30 min of interaction with DPPH.
(P <0.05). We also confirmed the beneficial effect of another two
established antioxidants: catalase (94.2 2.8%) and melatonin
(92.5 3.3%).
found to dramatically enhance the effect of H2O2, with C2 viability
levels dropping to 53.6 1.4% (P <0.05). Given this synergistic
effect of H2O2 with curcumin and based on other studies reporting
ROS generation by this polyphenol,37–39 we next assessed the effect
Of note, curcumin alone at 50 lM reduced C2 myoblasts viabil-
ity to a level similar to that of H2O2 (83.8 0.4% and 83.1 3.5%,
of all antioxidants under investigation, using 50
lM curcumin as a
respectively). Furthermore, at this concentration, curcumin was
pro-oxidative agent (Table 4). To our surprise, while the damaging

M. Peleli et al. / Bioorg. Med. Chem. 23 (2015) 390–400
395
Table 1
Table 4
Assessment of (%) cell viability by MTT assay
Assessment of (%) cell viability by MTT assay
Conditions
% cell viability (means sem)
Conditions
Ctr
% cell viability (means sem)
Ctr
100
100
1 mM H2O2
83.1 3.5⁄
84.6 1.0
97.1 2.3\
76.2 1.6\
71.6 2.0\
98.1 1.3
50
l
M curcumin
83.8 0.4⁄
99.5 1.8\
98.5 1.3\
53.6 1.0\
99.1 2.3\
1
l
M AK1 + 1 mM H2O2
50 U/ml catalase + 50
l
M curcumin
15
30
50
15
lM AK1 + 1 mM H2O2
lM AK1 + 1 mM H2O2
lM AK1 + 1 mM H2O2
lM AK1
15
15
30
l
l
l
M melatonin + 50 lM curcumin
M AK1 + 50
M AK2 + 50
l
l
M curcumin
M curcumin
C2 skeletal myoblasts were left untreated (Ctr) or treated with curcumin (50
lM) in
C2 skeletal myoblasts were left untreated (Ctr) or treated with H2O2 (1 mM) in the
the presence or absence of catalase (50 U/ml), melatonin (15 M), 15 M AK1 and
l
l
presence or absence of increasing doses of AK1 ranging from 1
lM to 50
lM for
30 lM AK2 for 24 h. Values are means sem for at least three independent
24 h. Values are means sem for at least three independent experiments. ⁄P <0.05
compared with untreated cells (Ctr), \P <0.05 compared with H2O2-treated cells.
experiments. ⁄P <0.05 compared with untreated cells (Ctr), \P <0.05 compared with
curcumin-treated cells.
tions used in other studies in various experimental models.40,41
Hence, we examined the time-dependent effect of curcumin on
the phosphorylation pattern of ERKs and JNKs, two known MAPK
subfamilies, in C2 skeletal myoblasts. In particular, cells were
Table 2
Assessment of (%) cell viability by MTT assay
Conditions
% cell viability (means sem)
Ctr
100
1 mM H2O2
1 lM AK2 + 1 mM H2O2
83.1 3.5⁄
81.6 1.0
87.1 3.3
99.1 1.8\
97.6 2.0\
99.5 1.2⁄
treated with curcumin (50 lM) for increasing time intervals
(15 min up to 4 h). Under these conditions, phosphorylation levels
of ERKs attained maximal value as early as 15 min of treatment
(4.2 0.2-fold, P <0.001; Fig. 4A and B) and remained significantly
elevated after 1 h of exposure (3.1 0.1-fold, P <0.001; Fig. 4A and
B), declining thereafter to control values. As depicted in Figure 4C,
JNKs followed a more delayed and sustained activation pattern,
with maximal induction observed after 2 h (4.3 0.2-fold,
P <0.001 and Fig. 4D). Equal protein loading was verified by immu-
noblotting identical samples with a specific anti-actin antibody
(Fig. 4A bottom panel).
15
30
50
30
lM AK2 + 1 mM H2O2
lM AK2 + 1 mM H2O2
lM AK2 + 1 mM H2O2
lM AK2
C2 skeletal myoblasts were left untreated (Ctr) or treated with H2O2 (1 mM) in the
presence or absence of increasing doses of AK2 ranging from 1 lM to 50 lM for
24 h. Values are means sem for at least three independent experiments. ⁄P <0.05
compared with untreated cells (Ctr), \P <0.05 compared with H2O2-treated cells.
Table 3
Assessment of (%) cell viability by MTT assay
3.6. Diverse effects of various antioxidant agents on curcumin-
induced activation of ERKs and JNKs in C2 skeletal myoblasts
Conditions
% cell viability (means sem)
Ctr
1 mM H2O2
50 U/ml catalase + 1 mM H2O2
15
100
We subsequently looked into the effects of various antioxidants
on ERKs and JNKs activation by H2O2 or curcumin. All compounds
tested were found to markedly suppress H2O2-induced phosphor-
ylation of ERKs (by approximately 55 1.5%, Fig. 5A and B) and
JNKs (by approximately 81 1.7%, Fig. 5C and D). Curcumin-
induced phosphorylation of ERKs was partially inhibited by cata-
lase, melatonin and AK2 (by approximately 53 1.2%, Fig. 6A and
B); AK1 inhibited curcumin-induced phosphorylation of ERKs by
79 0.8% (Fig. 6A: 5th lane and B). Interestingly, curcumin-induced
activation of JNKs was significantly inhibited in the presence of
catalase, melatonin and AK2 (by approximately 50 1.3%,
Fig. 6C: 3rd, 4th and 6th lanes, respectively, and D), while AK1
had no effect (Fig. 6C: 5th lane and D). So as to shed light onto this
finding, we focused on JNKs signaling pathway, and next examined
the possible activation of the transcription factor c-Jun, an estab-
lished substrate of JNKs.13 To this end, cells were incubated with
curcumin for increasing time periods from 0.5 up to 4 h and Wes-
tern blot analysis was performed after subcellular fractionation, in
the nuclear fraction of the respective samples. As shown in
83.1 3.5⁄
94.2 2.8\
92.5 3.3\
85.5 1.0
90.2 2.2\
93.1 1.8\
53.6 1.4\
97.1 1.8
96.9 2.2
83.8 0.4⁄
l
M melatonin + 1 mM H2O2
1
lM curcumin + 1 mM H2O2
15
30
50
lM curcumin + 1 mM H2O2
lM curcumin + 1 mM H2O2
lM curcumin + 1 mM H2O2
50 U/ml catalase
15
50
l
l
M melatonin
M curcumin
C2 skeletal myoblasts were left untreated (Ctr) or treated with H2O2 (1 mM) in the
presence or absence of increasing doses of curcumin ranging from 1 M to 50 M as
M) for 24 h. Values are means sem
l
l
well as catalase (50 U/ml) and melatonin (15
l
for at least three independent experiments. ⁄P <0.05 compared with untreated cells
(Ctr), \P <0.05 compared with H2O2-treated cells.
effect of curcumin on C2 survival was abrogated in the presence of
catalase (99.5 1.8%), melatonin (98.5 1.3%) as well as AK2
(99.1 2.3%), it was further enhanced in the presence of AK1
(53.6 1.0%).
Figure 7A (top panel) 50 lM curcumin induced maximal phosphor-
ylation of c-Jun after 4 h of treatment (3.0 0.2 fold relative to
control, P <0.001 and Fig. 7B). Equal protein loading was verified
by immunoblotting identical samples with a specific antibody
raised against total c-Jun levels (Fig. 7A bottom panel). What is
more, mirroring the findings presented in Figure 6D, c-Jun phos-
phorylation after treatment with curcumin, was considerably sup-
pressed in the presence of catalase (by approximately 74 1.4%),
melatonin (by approximately 57 1.1%) and AK2 (by approxi-
mately 40 0.7%) (Fig. 7C: 3rd, 4th and 6th lanes respectively
and D) while AK1 had no effect (Fig. 7C: 5th lane and D).
3.5. Curcumin stimulates activation of ERKs and JNKs in C2
skeletal myoblasts
The ability of AK2 along with catalase and melatonin to inhibit
the pro-oxidative activity of both H2O2 and curcumin, in contrast
to the differential effect of AK1, prompted us to further investigate
the signaling mechanisms induced by curcumin, so as to elucidate
this intriguing observation. For all ensuing experiments described
in this study, curcumin was used at a concentration of 50
lM,
based on the above results as well as on the range of concentra-

396
M. Peleli et al. / Bioorg. Med. Chem. 23 (2015) 390–400
A
B
C
p-ERKs
p-JNKs
Ctr 0.25 0.5
β-actin
1
2
4
Time (hours)
Ctr 0.25 0.5
1
2
4
Time (hours)
D
Time (hours)
Time (hours)
Figure 4. Curcumin induces ERKs and JNKs phosphorylation in a time-dependent manner in C2 skeletal myoblasts. C2 cells were left untreated (Ctr) or treated with 50
lM
curcumin for increasing time periods ranging from 0.25 to 4 h. Whole cell extracts (30 g) were subjected to SDS–PAGE and immunoblotted with antibodies for
l
phosphorylated ERKs (A top panel) and JNKs (C). Equal protein loading was verified by immunoblotting identical samples with a specific anti-actin antibody (A bottom panel).
The respective protein bands are indicated on the right of the panels. Bands were quantified by laser scanning densitometry and plotted (B for p-ERKs and D for p-JNKs).
Western blots are representative of at least three independent experiments with overlapping results and data are means sem for at least three independent experiments.
Statistical analyses were performed using one way ANOVA with Bonferroni post-test ( P <0.001 vs control).
A
C
p-ERKs
β-actin
p-JNKs
Ctr
cat mel cur AK1 AK2
H2O2 (1mM)
Ctr
cat mel cur AK1 AK2
H2O2 (1mM)
B
D
Ctr
cat mel cur AK1 AK2
H2O2 (1mM)
Ctr
cat mel cur AK1 AK2
H2O2 (1mM)
Figure 5. Effect of various antioxidants on H2O2-induced ERKs and JNKs phosphorylation in C2 cells. C2 skeletal myoblasts were left untreated (Ctr) or were pre-incubated
with catalase (50 U/ml-cat), melatonin (15 M-mel), curcumin (15 M-cur), AK1 (15 M) or AK2 (30 M) for 30 min and then exposed to 1 mM H2O2 for 15 min, in the
absence or presence of the inhibitors. Extracts from cells lysed with buffer G (30 g) were subjected to SDS–PAGE and immunoblotted with antibodies for phosphorylated
l
l
l
l
l
ERKs (A top panel) and JNKs (C). Equal protein loading was verified by immunoblotting identical samples with a specific anti-actin antibody (A bottom panel). The respective
protein bands are indicated on the right of the panels. Bands were quantified by laser scanning densitometry and plotted (B for p-ERKs and D for p-JNKs). Western blots are
representative of at least three independent experiments with overlapping results and data are means sem for at least three independent experiments. Statistical analyses
were performed using one way ANOVA with Bonferroni post-test ( P <0.001 vs control and ⁄P <0.01 vs identically treated cells in the absence of the antioxidants).

M. Peleli et al. / Bioorg. Med. Chem. 23 (2015) 390–400
397
C
A
p-JNKs
β-actin
p-ERKs
β-actin
Ctr
cat mel AK1 AK2
curcumin (50μM)
Ctr
cat mel AK1 AK2
curcumin (50μM)
B
D
Ctr
cat mel AK1 AK2
curcumin (50μM)
Ctr
cat mel AK1 AK2
curcumin (50μM)
Figure 6. Effect of various antioxidants on curcumin-induced ERKs and JNKs phosphorylation in C2 cells. C2 skeletal myoblasts were left untreated (Ctr) or were pre-incubated
with catalase (50 U/ml-cat), melatonin (15 M-mel), AK1 (15 M) or AK2 (30 M) for 30 min and then exposed to 50 M curcumin for 15 min (ERKs) or 2 h (JNKs), in the
absence or presence of the inhibitors. Extracts from cells lysed with buffer G (30 g) were subjected to SDS–PAGE and immunoblotted with antibodies for phosphorylated ERKs
l
l
l
l
l
(A top panel) and JNKs (C). Equal protein loading was verified by immunoblotting identical samples with a specific anti-actin antibody (A, C: bottom panels). The respective
protein bands are indicated on the right of the panels. Bands were quantified by laser scanning densitometry and plotted (B for p-ERKs and D for p-JNKs). Western blots are
representative of at least three independent experiments with overlapping results and data are means sem for at least three independent experiments. Statistical analyses
were performed using one way ANOVA with Bonferroni post-test ( P <0.001 vs control and ⁄P <0.01 vs identically treated cells in the absence of the antioxidants).
A
C
p-c-Jun
t-c-Jun
p-c-Jun
t-c-Jun
Ctr 0.5
1
2
4
Ctr
cat mel AK1 AK2
curcumin (50μM)
Time (hours)
D
B
Ctr
cat mel AK1 AK2
curcumin (50μM)
Time (hours)
Figure 7. Time-dependent profile of c-Jun phosphorylation in response to curcumin in C2 skeletal myoblasts and effect of various antioxidants. (A) C2 cells were left
untreated (Ctr) or treated with 50 M curcumin for increasing time periods ranging from 0.5 to 4 h. (C) C2 cells were left untreated (Ctr) or were pre-incubated with catalase
(50 U/ml-cat), melatonin (15 M-mel), AK1 (15 M) or AK2 (30 M) for 30 min and then exposed to 50 M curcumin for 4 h. Nuclear extracts (25 g) were subjected to SDS–
l
l
l
l
l
l
PAGE and immunoblotted with an antibody for phosphorylated c-Jun (A, C: top panels). Equal protein loading was verified by immunoblotting identical samples with a
specific antibody detecting total levels of c-Jun (A, C: bottom panels). The respective protein bands are indicated on the right of the panels. Bands were quantified by laser
scanning densitometry and plotted (B, D). Western blots are representative of at least three independent experiments with overlapping results and data are means sem for
at least three independent experiments. Statistical analyses were performed using one way ANOVA with Bonferroni post-test ( P <0.001 vs control and ⁄P <0.01 vs identically
treated cells in the absence of the antioxidants).

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M. Peleli et al. / Bioorg. Med. Chem. 23 (2015) 390–400
A
B
C
PARP fragment
PARP fragment
β-actin
β-actin
Ctr
AK1 AK2 AS
curcumin (50μM)
Ctr 0.5
1
2
4
Time (hours)
D
Ctr
AK1
AK2
AS
Time (hours)
curcumin (50μM)
Figure 8. Time-dependent profile of PARP cleavage in response to curcumin in C2 skeletal myoblasts; effect of antioxidants and a JNKs inhibitor. (A) C2 cells were left
untreated (Ctr) or treated with 50 M curcumin for increasing time periods ranging from 0.5 to 4 h. (C) C2 cells were left untreated (Ctr) or were pre-incubated with AK1
(15 M), AK2 (30 M) or AS601245 (2.5 M) for 30 min and then exposed to 50 M curcumin for 4 h. Extracts from cells lysed with buffer G (30 g) were subjected to SDS–
l
l
l
l
l
l
PAGE and immunoblotted with an antibody that detects endogenous levels of full length PARP as well as the large 89 kDa PARP fragment (A, C: top panels). Equal protein
loading was verified by immunoblotting identical samples with a specific anti-actin antibody (A, C: bottom panels). The respective protein bands are indicated on the right of
the panels. Bands were quantified by laser scanning densitometry and plotted (B, D). Western blots are representative of at least three independent experiments with
overlapping results and data are means sem for at least three independent experiments. Statistical analyses were performed using one way ANOVA with Bonferroni post-
test ( P <0.001 vs control and ⁄P <0.01 vs identically treated cells in the absence of AK2 or AS601245, respectively).
3.7. Treatment of C2 skeletal myoblasts with curcumin induces
apoptosis as evidenced by PARP cleavage; involvement of JNKs
of oxidative stress on skeletal muscle physiology and performance
is expanding rapidly. In the present study, we have demonstrated
the diverse effects of two novel antioxidants (AK1 and AK2) on cell
viability of H2O2- and curcumin-treated C2 skeletal myoblasts
and have monitored the signaling mechanisms mediating the
responses triggered.
In an attempt to elucidate the molecular mechanisms triggered
by curcumin causing cell death, we investigated the potential acti-
vation of apoptosis. Thus, an effort was made to identify cleavage
of PARP, a routinely used apoptotic marker.42 As illustrated in
Figure 8A, treatment with curcumin for 4 h, induced PARP proteol-
ysis, with protein levels of the 89 kDa PARP fragment reaching a
3.5 0.3 fold increase, relative to control (P <0.001 and Fig. 8B).
In order to examine whether the apoptosis conferred by curcumin
could be attributed to its pro-oxidative properties, we next tested
the effect of our newly synthesized antioxidants: AK1 and AK2.
Intriguingly, only AK2 abolished PARP proteolysis (Fig. 8C: 4th lane
and D), while AK1 had no impact (Fig. 8C: 3rd lane and D). Based
on the observed inhibition of JNKs activation only by AK2 versus
AK1, we next looked into the potential impact of AS601245
Initially, both compounds tested, showed considerable DPPH
radical-scavenging capacity with AK1 exhibiting an IC50 value
(119
under the same experimental conditions as a reference antioxi-
dant, while AK2 had a higher IC50 value (271 M) (Fig. 3). Interest-
lM) similar to that of resveratrol (IC50 value: 96 lM), used
l
ingly, AK1 was observed to exhibit its radical scavenging activity
more rapidly than AK2. These findings are consistent with a previ-
ous report marking the significant antioxidant properties of both
compounds in vitro as evidenced by inhibition of lipid peroxidation
and LDL-oxidation. Furthermore, AK1 and AK2 also suppressed
total cholesterol, LDL-cholesterol and MDA levels in hyperlipi-
demic rats, exhibiting their antidyslipidemic (through squalene
synthase inhibition) and antioxidant properties in vivo.29
(2.5 lM), a selective pharmacological inhibitor of JNKs, on PARP
fragmentation. Our results revealed that AS601245 abolished cur-
cumin-induced proteolytic cleavage of PARP (Fig. 8C last lane and
D). Therefore, JNKs appear to mediate curcumin-induced PARP pro-
teolysis, since hindering their activity resulted in the attenuation of
PARP cleavage. AK1, AK2 as well as AS601245 alone, had no effect
on PARP fragment protein levels (data not shown). Equal protein
loading was verified by immunoblotting identical samples with a
specific anti-actin antibody (Fig. 8A and C: bottom panels).
Correlating with the aforementioned results, the MTT analysis
carried out in the present study, showed that AK1 repressed the
deleterious effect of H2O2 on C2 skeletal myoblasts viability at a
lower concentration (15
lM-Table 1) compared to that of AK2
(30 M-Table 2). This was also the case when looking into the anti-
l
oxidant potential of these compounds in vitro as well as in vivo.
Specifically, in both cases, AK1 exhibited a better antioxidant activ-
ity than AK2, inhibiting the lipid peroxidation in vitro with an IC50
4. Discussion
value of 9 lM (IC50 = 83 lM for AK2) and decreasing the MDA
content in the plasma of hyperlipidemic rats by 63% (31% decrease
for AK2).29 The salutary effects of AK1 and AK2 were similar to
those of established antioxidants tested in parallel: (a) curcumin
Research on the synthesis and evaluation of antioxidant com-
pounds that could counteract and alleviate the detrimental effects

M. Peleli et al. / Bioorg. Med. Chem. 23 (2015) 390–400
399
H2O2 1mM
AK1 (15μΜ)
AK2 (30μΜ)
multiple pathways activated (i.e. ERKs, JNKs)
?
cell death by
necrosis
Figure 9. Schematic diagram of a model depicting the inhibitory effect of AK1 as well as AK2 on the signaling effectors activated by H2O2 in C2 skeletal myoblasts. All these
compounds also suppress necrosis conferred by H2O2 in C2 cells. ? activation, inhibition.
thereby leading to induction of ROS levels via NADPH oxidase
activation.51
Therefore, we next decided to assess the effect of our antioxi-
dants against the potential pro-oxidative action of curcumin in
C2 skeletal myoblasts. To our surprise, while AK2 (30 M) was
found to repress the deleterious effect of curcumin (50 M) on
C2 viability, along with catalase and melatonin, AK1 (15 M) dra-
curcumin 50μM
ROS
phospho-JNKs
phospho-c-Jun
phospho-ERKs
l
l
l
AK2 (30μΜ)
matically enhanced cell death (Table 4). The differential effects
observed, could be associated with the chemical structure and
mechanism of action of the compounds; a minimal structural
change, the replacement of O in the benzoxazine ring of AK1 with
its respective isostere (S) in the resulting benzothiazine (AK2), led
to a completely different antioxidant mode of action, with AK1
exhibiting its antioxidant activity more rapidly versus the more
delayed and sustained activity of AK2 (Fig. 3). Consequently, AK1
could preferentially block activation of signaling effectors
‘recruited’ at an early stage, while AK2 could more effectively inhi-
bit downstream signaling mediators which are activated at a later
stage, respectively, modulating the final impact on cell fate.
In order to test this hypothesis, we decided to investigate the
effect of AK1 and AK2 on signaling pathways triggered by curcu-
min in C2 skeletal myoblasts. Given the central role of MAPKs in
fundamental biological processes, we focused on the response of
ERKs and JNKs, two major MAPK subfamilies. In this context, cur-
cumin was found to cause the immediate upregulation of ERKs
phosphorylation, while JNKs exhibited a more delayed activation
profile (Fig. 4). Accordingly, ERKs activation by curcumin has been
certified in human leukemia THP-1 cells,52 multi-potent neural
progenitor cells47 and nasopharyngeal carcinoma cells,53 with
reports marking JNKs activation in gastric AGS and colon HT-29
carcinoma cells.54
AK2 (30μΜ),
AS601245
PARP fragmentation
cell death by
apoptosis
AK2 (30μΜ)
AK1 (15μΜ)
Figure 10. Diagram of a representative scheme depicting the inhibitory effect of:
(a) AK2 on curcumin-induced JNKs pathway, (b) AK2 or AS601245 (AS) on
curcumin-conferred PARP fragmentation hence apoptosis in C2 skeletal myoblasts.
AK1 exerts a different effect. ? activation,
inhibition, – versus.
(15
properties,43 (b) catalase (50 U/ml), an enzyme depleting H2O2
and (c) melatonin (15 M), the principal hormone of the pineal
lM), a polyphenol with antioxidant and anti-inflammatory
44
l
gland, known for its potent antioxidant and free radical-scavenging
properties.45
Emphasizing on the effect of our novel antioxidant agents on
the signaling responses conferred by curcumin, we observed both
AK1 and AK2 to effectively attenuate ERKs activation by H2O2
and curcumin (Figs. 5 and 6A and B) as well as JNKs activation
by H2O2 (Fig. 5C and D). However, while AK2 equally inhibited
JNKs activation by curcumin, AK1 was unable to do so (Fig. 6C
and D). In accordance, AK1 did not block phosphorylation of c-
Jun, an established JNKs substrate, which was found to be activated
by curcumin (Fig. 7). This finding prompted us to look further into
the molecular mechanisms inducing cell death after treatment
with curcumin, since it is in agreement with the MTT analysis,
showing AK1 to enhance rather than abrogate the cytotoxic effect
of curcumin on C2 skeletal myoblasts, in contrast to the effect of
AK2 (Table 4).
On the other hand, curcumin at a higher dose (50 lM) was dem-
onstrated to decrease C2 skeletal myoblasts viability and to have a
profound detrimental effect when combined with H2O2. In accor-
dance with our findings, Chen et al. have also marked curcumin
to moderately but significantly promote cell death of HepG2 cells
in a dose-dependent manner (at P10 lM) and to enhance H2O2-
induced cytotoxicity.46 Further corroborating our findings, while
low concentrations of curcumin have been shown to stimulate pro-
liferation of embryonic neural progenitor cells, high concentrations
were found to be cytotoxic.47 Curcumin has attracted intense
scientific interest, due to its potential role in the treatment of
malignant, metabolic, neuro- and muscle-degenerative diseases.
However, studies establishing its cytoprotective, antioxidant
activity48,49 have been challenged by reports underlying its pro-
apoptotic, ROS-generating capacity mainly in tumor cells.39,50 In
particular, curcumin has been demonstrated to favor superoxide
anion generation39 and to inhibit thioredoxin reductase activity
The detected cleavage of PARP shown in Figure 8A indicates
that curcumin stimulates apoptosis in C2 skeletal myoblasts. The
pro-apoptotic activity of curcumin has also been reported in

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M. Peleli et al. / Bioorg. Med. Chem. 23 (2015) 390–400
human monocytic leukemia THP-152 and lung adenocarcinoma
cells.55 Liang et al. have also marked curcumin to induce apoptosis
of gastric cancer BGC-823 cells via ROS production and JNKs activa-
tion,38 in line with a previous study of ours, showing curcumin to
induce apoptosis in H9c2 cells, also via ROS and JNKs upregula-
tion.37 JNKs major role in mediating curcumin-conferred apoptosis
was also demonstrated in the present study, since AK2 (found to
inhibit curcumin-induced JNKs phosphorylation) and AS601245
(known to suppress JNKs activity), were observed to also abolish
PARP fragmentation in curcumin-treated C2 cells in contrast to
AK1 (Fig. 8C and D). Thus, as depicted in Figures 9 and 10, while
both AK1 and AK2 were found to exert a beneficial impact abolish-
ing necrotic death caused by H2O2 in C2 skeletal myoblasts, only
AK2 was effective in blocking apoptosis conferred by the pro-
oxidant activity of curcumin, by also inhibiting JNKs activation.
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Collectively, this study disclosed the different effects of two
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Acknowledgements
This work was supported by the Special Research Account of the
University of Athens (project number: 70/4/11242). The funding
body had no involvement in the design of the present study, nei-
ther in the experiments performed nor in the analysis of the data
or the writing of the report.
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