Journal of Receptors and Signal Transduction

ISSN: 1079-9893 (Print) 1532-4281 (Online) Journal homepage: http://www.tandfonline.com/loi/irst20

Curcumin inhibits apoptosis by regulating

intracellular calcium release, reactive oxygen
species and mitochondrial depolarization levels in
SH-SY5Y neuronal cells

Abdülhadi Cihangir Uğuz, Ahmi Öz & Mustafa Nazıroğlu

To cite this article: Abdülhadi Cihangir Uğuz, Ahmi Öz & Mustafa Nazıroğlu (2015): Curcumin
inhibits apoptosis by regulating intracellular calcium release, reactive oxygen species and
mitochondrial depolarization levels in SH-SY5Y neuronal cells, Journal of Receptors and Signal
Transduction, DOI: 10.3109/10799893.2015.1108337

To link to this article: http://dx.doi.org/10.3109/10799893.2015.1108337

Published online: 25 Nov 2015.

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 http://informahealthcare.com/rst
ISSN: 1079-9893 (print), 1532-4281 (electronic)

J Recept Signal Transduct, Early Online: 1–7

! 2015 Taylor & Francis. DOI: 10.3109/10799893.2015.1108337

RESEARCH ARTICLE

Curcumin inhibits apoptosis by regulating intracellular calcium release,

reactive oxygen species and mitochondrial depolarization levels in SH-
SY5Y neuronal cells
Abdülhadi Cihangir Uğuz1,2, Ahmi Öz1, and Mustafa Nazıroğlu1,2
Downloaded by [University of California, San Diego] at 22:43 31 December 2015

1
Department of Biophysics, School of Medicine, Süleyman Demirel University, Isparta, Turkey and 2Neuroscience Research Center, Süleyman Demirel
University, Isparta, Turkey

Abstract Keywords
Neurological diseases such as Alzheimer’s and Parkinson’s diseases are incurable progressive Apoptosis, calcium signaling, curcumin,
neurological disorders caused by the degeneration of neuronal cells and characterized by oxidative stress, SH-SY5Y neuronal cells
motor and non-motor symptoms. Curcumin, a turmeric product, is an anti-inflammatory agent
and an effective reactive oxygen and nitrogen species scavenging molecule. Hydrogen History
peroxide (H2O2) is the main source of oxidative stress, which is claimed to be the major source
of neurological disorders. Hence, in this study we aimed to investigate the effect of curcumin Received 10 September 2015
on Ca2+ signaling, oxidative stress parameters, mitochondrial depolarization levels and caspase- Accepted 28 September 2015
3 and -9 activities that are induced by the H2O2 model of oxidative stress in SH-SY5Y neuronal Published online 24 November 2015
cells. SH-SY5Y neuronal cells were divided into four groups namely, the control, curcumin,
H2O2, and curcumin + H2O2 groups. The dose and duration of curcumin and H2O2 were
determined from published data. The cells in the curcumin, H2O2, and curcumin + H2O2 groups
were incubated for 24 h with 5 mM curcumin and 100 mM H2O2. Lipid peroxidation and cytosolic
free Ca2+ concentrations were higher in the H2O2 group than in the control group; however,
their levels were lower in the curcumin and curcumin + H2O2 groups than in the H2O2 group
alone. Reduced glutathione (GSH) and glutathione peroxidase (GSH-Px) values were lower in
the H2O2 group although they were higher in the curcumin and curcumin + H2O2 groups than
in the H2O2 group. Caspase-3 activity was lower in the curcumin group than in the H2O2 group.
In conclusion, curcumin strongly induced modulator effects on oxidative stress, intracellular
Ca2+ levels, and the caspase-3 and -9 values in an experimental oxidative stress model in SH-
SY5Y cells.

Abbreviations: CaMKII: Ca2+/calmodulin-dependent protein kinase II; GSH: glutathione; GSH-

Px: glutathione peroxidase; H2O2: hydrogen peroxide; METC: mitochondrial electron transport
chain; MPP: 1-methyl-4-phenylpyridinium ion; O
2 superoxide radical; TH: tyrosine hydroxylase

Introduction literature research, curcumin can protect SH-SY5Y cells

against oxidative stress, but the detailed mechanism remains
Curcumin, a polyphenolic bioflavonoid, is an active compo-
unclear.
nent of the dietary spice turmeric and it is one of the main
SH-SY5Y is a human-derived neuroblastoma cell line
components in curry powders (1). It has been previously
oftenly used as an in vitro neuronal model. SH-SY5Y cells are
reported that curcumin has antioxidant (2), anti-inflammatory
known to be adrenergic in phenotype. Cells that have a similar
(3,4), antiproliferative (5), and other therapeutic properties.
morphology to neuroblasts are positive for dopamine-
Curcumin has also been shown to have protective effects in
b-hydroxylase and tyrosine hydroxylase (TH) and are a
neurodegenerative disease by reducing either inflammation or
feature of catecholaminergic neurons (7). They also express
oxidative damage in neurological disorders (6). Due to its
dopaminergic markers and they have been widely used to
strong antioxidant properties, curcumin has gained the
study Alzheimer’s and Parkinson’s disease models in vitro (8).
attention of specialists for various treatments. Based on the
Calcium (Ca2+) ions are vitally important in many cellular
functions of the nervous system. Ca2+ has a wide range of
Address for correspondence: Assist. Prof. Dr. A. Cihangir Uğuz, responsibilities ranging from releasing neurotransmitters to
Department of Biophysics, School of Medicine, Süleyman Demirel
University, TR-32260, Isparta, Turkey. Tel: +90 246 211 36 60. Fax: intracellular signal transduction. The high concentration
+90 246 237 11 65. E-mail: cihangiruguz@sdu.edu.tr differences between intracellular and extracellular Ca2+
 2 A. Cihangir Uğuz et al.
concentration ([Ca2+]i) highlights the crucial functions and
control mechanisms of Ca2+ influx and efflux. Any sudden
changes and failures in the regulatory mechanisms of this
cation and the subsequent increased [Ca2+]i levels may be
responsible for pathological events in the central nervous
system including stroke, brain trauma, epilepsy, and neuro-
logical diseases (9,10).
Caspases are a group of cysteine proteases that play crucial
critical roles in apoptosis, necrosis, and inflammatory
process. Caspases are play a crucial role in cellsular for
apoptosis, or in another term, programmed cell death, during
both in development and most other stages of mature living
periods. They are also called ‘‘executioner’’ proteins for their
roles in the cell.
Oxidative stress is associated with a wide range of
neurological disorders (11). Oxidative stress is described as
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an imbalanced situation between anti-oxidant and pro-oxidant
species, in favor of oxidant molecules. The deregulation
theory addresses the changeable roles played by the super-
oxide radical (O 2 ) and hydrogen peroxide (H2O2) in neuro-
logic cells in both the cell’s basal state and diseased state.
The main objective of the current study was to investigate
the molecular mechanism of curcumin in SH-SH5Y neuronal
cells. To investigate the molecular effects of curcumin, we
examined some potential intracellular signaling cascades.
Furthermore we hypothesized the way which curcumin’s
action is related to the inhibition of reactive oxygen species
(ROS) generation, as well as caspase activation and intracel-
lular calcium signaling ([Ca2+]i) in SH-SY5Y neuronal cells.
Moreover, we examined the role of the mitochondrial depolar-
ization levels in curcumin administered cytoprotection using
intracellular fluorescent dye.
Materials and methods
Chemicals
All chemicals (cumenehydroperoxide, KOH, NaOH, thiobar-
bituric acid, 1,1,3,3-tetraethoxypropane, 5,5-dithiobis-2 nitro-
benzoic acid, tris-hydroxymethyl-aminomethane, glutathione,
butyl hydroxyl toluol, Triton X-100, and ethylene glycol-bis
[2-aminoethyl-ether]-N,N,N,N-tetraacetic acid [EGTA]) were
obtained from Sigma-Aldrich (St. Louis, MO, USA) and all
organic solvents (n-hexane, ethyl alcohol) were purchased
from Merck (Darmstadt, Germany). Fura-2 AM (acetoxy-
methyl ester) was purchased from Invitrogen (Carlsbad, CA,
USA). All reagents were of analytical grade. All reagents
except the phosphate buffers were prepared daily and stored at
+4  C. Reagents were equilibrated at room temperature for
half an hour before an analysis was initiated or reagent
containers were refilled. Phosphate buffers were stable at
+4  C for 1 month. APOPercentageÔ assay kit was purchased
from Biocolor (Belfast, Northern Ireland).
Study groups
 Group I was Control group and SH-SY5Y cells were
incubated for 24 h with their medium (37  C and 5% CO2).
 Group II was Curcumin group and SH-SY5Y cells were
incubated with 5 mM curcumin during 24 h (37  C and 5%
CO2) Curcumin was solved in DMSO (12).
J Recept Signal Transduct, Early Online: 1–7
 Group III was H2O2 group and SH-SY5Y cells were
incubated with 100 mM H2O2 for 24 h (37  C and 5%
CO2) (13).
 Group IV was Curcumin + H2O2 group and SH-SY5Y
cells were incubated with 5 mM curcumin during 24 h and
100 mM H2O2 for 24 h (37  C and 5% CO2).
Cell culture
SH-SY5Y, human-derived cell line was purchased from (ATCC,
VA, USA) (14). They grown in a mixture of a medium
containing 1:1 ratio of Dulbecco’s modified Eagle’s medium
(DMEM) and Ham’s F12 medium supplemented with 10% fetal
bovine serum (Biochrom, Berlin, Germany) and 1% penicillin–
streptomycin combination (Biochrom, Germany) according
to procedure of supplier’s introduction. Cells were used at
passages 4 to 11.
Calcium ([Ca2+]i) Determination by fluorescent dye
Cells were loaded with Fura-2 by incubation with 4 mM Fura-
2 acetoxymethyl ester (Fura-2/AM) for 30 min at room
temperature according to a procedure published elsewhere
(16). Once loaded, the cells were washed and gently re-
suspended in Na-HEPES solution containing (in mM): NaCl,
140; KCl, 4.7; CaCl2, 1.2; MgCl2, 1.1; glucose, 10; and
HEPES, 10 (pH 7.4). The four groups were exposed to H2O2
for stimulating ([Ca2+]i) release. Fluorescence was recorded
from 2 ml aliquots of magnetically stirred cellular suspension
(2  106 cells/ml) at 37  C by using a spectrofluorometer
(Cary Eclipse, Varian Inc, Sydney, Australia) with excitation
wavelengths of 340 and 380 nM and emission at 505 nM.
Changes in [Ca2+]i were monitored by using the fura-2 340/
380 nM fluorescence ratio and were calibrated according to
the method of Grynkiewicz et al.. Ca2+ release was estimated
using the integral of the rise in [Ca2+]i for 40 seconds after
addition of H2O2. Ca2+ release is expressed nM taking a
sample every second (nM / s) as previously described (16).
Measurement of ROS-sensitive fluorescence
Cells were loaded with 2 mM dihydrorhodamine-123 (DHR-
123) by incubation at 37  C for 30 min as previously described
(15). This probe is a nonfluorescent cell-permeable compound.
Once inside the cell, it turns fluorescent upon oxidation to yield
rhodamine–123 (Rh-123), fluorescence being proportional to
ROS generation. The fluorescence intensity of Rh-123 was
measured in an automatic microplate reader (Tecan Infinite
M200, Grodig, Austria). Excitation was set at 488 nm and
emission at 543 nm. Treatments were carried out in triplicate.
Data are presented as fold increase over the pretreatment level
(experimental/control).
Measurement of mitochondrial membrane potential
(MMP)
SH-SY5Y cells were incubated with 1 mM JC-1 for 15 min at
37  C as previously described (15). The cationic dye, JC-1,
exhibits potential-dependent accumulation in mitochondria.
It indicates mitochondria depolarization by a decrease in the
red-to-green fluorescence intensity ratio. After incubation
with JC-1, the dye was removed, and the cells were washed in
PBS. The green JC-1 signal was measured at the excitation
 DOI: 10.3109/10799893.2015.1108337
wavelength of 485 nm and the emission wavelength of
535 nm, and the red signal, at the excitation wavelength of
540 nm and the emission wavelength of 590 nm. Fluorescence
changes were analyzed using a fluorescence spectrophotom-
eter (Tecan Infinite Pro200). Treatments were carried out in
triplicate. Data are presented as emission ratios (590/535).
Changes in mitochondrial membrane potential were quanti-
fied as the integral of the decrease in JC-1 fluorescence ratio.
Apoptosis assay
The APOPercentageÔ assay (Biocolor Ltd., Belfast, Northern
Ireland) was performed according to the instructions provided
by Biocolor Ltd. and elsewhere (10). The APOPercentageÔ
assay is a dye-uptake assay, which stains only the apoptotic
cells with a red dye. When the membrane of apoptotic cell
loses its asymmetry, the APOPercentage dye is actively
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transported into cells, staining apoptotic cells red, thus
allowing detection of apoptosis by spectrophotometer (10).
Assay for caspase activities
To determine caspase-3 and -9 activities, SH-SY5Y cells were
sonicated and cell lysates were incubated with 2 ml of
substrate solution (20 mM HEPES [pH 7.4], 2 mM EDTA,
0.1 % CHAPS, 5 mM DTT, and 8.25 mM of caspase substrate)
for 1 h at 37  C as previously described (16). The activities of
caspase-3 and -9 were calculated from the cleavage of the
respective specific fluorogenic substrate (AC-DEVD-AMC
for caspase-3 and AC-LEHDAMC for caspase-9). Substrate
cleavage was measured with a fluorescence spectrophotom-
eter with an excitation wavelength of 360 nm and an emission
wavelength of 460 nm. Preliminary experiments confirmed
that caspase-3 or -9 substrate cleaving was not detected in the
presence of the inhibitors of caspase-3 or -9, DEVD-CMK,
or z-LEHDFMK, respectively. The data were calculated as
fluorescence units per milligram of protein (16).
Measurement of Lipid Peroxidation (LP) Level
Lipid peroxidation levels in the SH-SY5Y cell lines were
measured with the thiobarbituric acid reaction by the method
of published elsewhere (17). The quantification of thiobarbi-
turic acid reactive substances was determined by comparing
the absorption to the standard curve of malondialdehyde
(MDA) equivalents generated by acid catalyzed hydrolysis of
1,1,3,3-tetramethoxypropane.
Reduced glutathione (GSH), glutathione peroxidase
(GSH-Px), and protein assay
The GSH content of the SH-SY5Y cells was measured at
412 nm using the method of published elsewhere (18). GSH-
Px activities of SH-SY5Y cells were measured spectrophoto-
metrically at 37  C and 412 nm according to the method
published elsewhere (19). The protein content in the SH-
SY5Y cells was measured by method of Lowry et al. with
bovine serum albumin as the standard (20).
Statistical analysis
Data are expressed as means ± SEM of the number of
determinations. Statistical significance was analyzed by using
Curcumin inhibits apoptosis in neuronal cells 3
4 a, b
3.5
Apoptosis Level (Fold Increase)
3
a, b, c
2.5
2
1.5
1
0.5
0
Control Cur H2O2 Cur+H2O2
Figure 1. Effects of curcumin (Cur) and hydrogen peroxide (H2O2) on
apoptosis levels in SH-SY5Y cells (mean ± SD; n ¼ 8). The values are
expressed as fold increase over the control level (experimental/control).
a: p50.001 versus the control group; b: p50.001 versus the curcumin
group; c: p50.05 versus the H2O2 group.
SPSS packet program (9.05, SPSS Inc., Chicago, IL). To
compare the effects of different treatments, statistical signifi-
cance was calculated by the Mann–Whitney U-test. p50.05
was considered to indicate a statistically significant difference.
Results
Effects of curcumin and hydrogen peroxide (H2O2) on
apoptosis levels in SH-SY5Y neuronal cells
The effects of curcumin and H2O2 on apoptosis levels in SH-
SY5Y cells are shown in Figure 1. The mean apoptosis levels
determined as fold increase in, curcumin, H2O2, and
curcumin + H2O2 compared to control group. The apoptosis
levels were significantly lower in the control and curcumin
(p50.001) groups compared to the H2O2 group samples.
However, apoptosis levels were significantly decreased in
curcumin + H2O2 group (p50.001) compared to H2O2 group.
Hence, we observed the protective effects of curcumin on
apoptosis levels of H2O2 triggered oxidative stress models in
SH-SY5Y.
Effects of curcumin and hydrogen peroxide (H2O2)
on lipid peroxidation (LP), reduced glutathione
(GSH), and glutathione peroxidase (GSH-Px) values
in SH-SY5Y neuronal cells
LP, GSH, and GSH-Px values obtained from the SH-SY5Y
cells in the four groups are shown in Table 1. The results
indicated that LP levels were markedly (p50.001) higher in
H2O2 group compared to the control group samples.
Moreover, GSH-Px activity and GSH levels were statistically
(p50.001) lower in the H2O2 group compared to the
control group. However, the LP levels were significantly
lower in the curcumin (p50.001) and curcumin + H2O2
(p50.001) groups compared to the H2O2 group alone.
Effects of curcumin and hydrogen peroxide on
reactive oxygen species (ROS) levels in SH-SY5Y
neuronal cells
ROS levels were determined with fluorescent dye. The ROS
levels obtained from the study groups are shown in Figure 2.
 4 A. Cihangir Uğuz et al. J Recept Signal Transduct, Early Online: 1–7

Table 1. The effects of curcumin (Cur) and hydrogen peroxide (H2O2) on lipid peroxidation (LP), glutathione peroxidase (GSH-Px), reduced
glutathione (GSH), caspase-3 (Casp-3), and caspase-9 (Casp-9) values in H2O2 incubated SH-SY5Y neuronal cells (mean ± SD) (optical density, O.D.).

Groups/parameters Control Curcumin (Cur) Hydrogen Peroxide (H2O2) Cur + H2O2

a,b
LP (mmol/g of protein) 27.69 ± 1.91 29.19 ± 2.42 33.90 ± 1.45 28,57 ± 0.82a,c
GSH (mmol/g of protein) 24.54 ± 2.72 32.23 ± 8.86a 10.95 ± 1.72a,b 23.96 ± 0.52a,b,c
GSH-Px (IU/g of protein) 32.43 ± 2.08 29.16 ± 2.27a 22.09 ± 2.84a,b 28.52 ± 2.63a,b,c

a: p50.001 versus control; b: p50.001 versus curcumin; c: p50.001 versus hydrogen peroxide (H2O2) group.

1.6 a, b (A) 1.8

DHR-123 Fluorescent (Fold Increase)

b, c

Caspase-3 Acvity (Fold Increase)

1.4 1.6
a, b, c 1.4
1.2 a, c, d
1.2
1
1 a
0.8
0.8
0.6 a
Downloaded by [University of California, San Diego] at 22:43 31 December 2015

0.6
0.4 0.4
0.2 0.2
0 0
Control Curcumin H2O2 Cur+H2O2 Control Curcumin H2O2 Cur+H2O2

Figure 2. Effects of curcumin (Cur) and hydrogen peroxide (H2O2) on (B) 2 b, c

reactive oxygen species levels in SH-SY5Y cells (mean ± SD; n ¼ 8).
The values are expressed as fold increase over the control level Caspase-9 Acvity (Fold Increase) 1.8
(experimental/control). a: p50.001 versus the control group; b: 1.6
p50.001 versus the curcumin group; c: p50.001 versus the H2O2 group. a, c, d
1.4
1.2
1
a
0.8
1.8 a, b
0.6
1.6
JC-1 Fluorescent (Fold Increase)

1.4
1.2
1 Control
a
0.8
0.6
0.4
0.2
0
Control Curcumin
Figure 3. Effects of curcumin (Cur) and hydrogen peroxide (H2O2) on
mitochondrial depolarization levels in SH-SY5Y cells (mean ± SD;
n ¼ 8). The values are expressed as fold increase over the control level
(experimental/control). a: p50.001 versus the control group; b:
p50.001 versus the curcumin group; c: p50.001 versus the H2O2 group.
The results indicated that ROS levels were significantly
(p50.001) higher in H2O2 group compared to the control
group samples. Moreover, ROS levels were statistically
(p50.001) lower in the curcumin group compared to the
H2O2 group.
Effects of curcumin and hydrogen peroxide (H2O2)
on mitochondrial depolarization levels in SH-SY5Y
neuronal cells
The mitochondrial depolarization levels in the curcumin and
curcumin + H2O2 groups were significantly lower than those
0.4
a, b, c 0.2
0
Curcumin H2O2 Cur+H2O2
Figure 4. Effects of curcumin (CUR) and hydrogen peroxide (H2O2) on
caspase-3 (A) and caspase-9 (B) in SH-SY5Y cells (mean ± SD; n ¼ 8).
The values are expressed as fold increase over the control level
(experimental/control). a: p50.05 versus the control group; b: p50.001
versus the control group; c: p50.001 versus the curcumin group; d:
p50.05 versus the H2O2 group.
H2O2 Cur+H2O2
in H2O2 group (P50.001). Moreover, it was determined that
the mitochondrial depolarization levels in the curcumin group
was significantly lower than that in the control and curcumin
groups (p50.001) (Figure 3).
Effects of curcumin and hydrogen peroxide (H2O2) on
caspase-3 and -9 levels in SH-SY5Y neuronal cells
The caspase-3 levels in Control and H2O2 groups were
significantly higher than Curcumin group (P50.05, P50.001
respectively). Curcumin incubation decreased caspase-3
activation in Curcumin+ H2O2 group. It was clearly under-
stood that Caspase-3 level in Curcumin + H2O2 group was
significantly lower than H2O2 group (P50.001) (Figure 4A).
The Caspase-9 levels in Curcumin and Curcumin+ H2O2
groups were significantly lower than H2O2 groups
(P50.001). Furthermore it was determined that Caspase-9
level in Curcumin+ H2O2 group was significantly lower than
H2O2 group (P50.001) (Figure 4B).
 DOI: 10.3109/10799893.2015.1108337 Curcumin inhibits apoptosis in neuronal cells 5
(A) 1800 Cur H2O2 induced oxidative stress triggered apoptosis and intra-
1600 Control cellular calcium release in rats.
H2O2+Cur Oxidative stress plays a major role in several neurodegen-
1400
H2O2 erative diseases including Parkinson’s and Alzheimer’s
1200 diseases (24). Increased ROS levels accompany the degener-
ation of dopaminergic neurons (25). (Ca2+)i levels have
[Ca2+]i (nM)

1000
crucial functions in cellular viability and a sudden and
800 unbalanced elevation in (Ca2+)i levels is strongly related to
600 intracellular ROS levels. Excessive ROS levels cause a
depolarizaiton in the cell membrane. Following this depolar-
400
ization, cations flows into the cytoplasm and if the buffering
200 systems are insufficient, then apoptosis will be triggered.
0 Moreover, excessive (Ca2+)i levels also generate elevations in
ROS levels and cause oxidative stress to the cells. Excessive
1
10
19
28
37
46
55
64
73
82
91
100
109
118
127
136
145
154
163
172
181
190
199
Time (second)
ROS levels lead to a disruption of free radical generation,
redox homeostasis, lipid peroxidation, and cholesterol and
(B) 180000
Downloaded by [University of California, San Diego] at 22:43 31 December 2015

a, b, c protein oxidation (24). Excessive ROS levels cause plasma

160000
a, b
membrane damage, defects in glutathione peroxidase expres-
140000 sion and a reduction in GSH levels, and mitochondrial
120000 dysfunction. These findings strongly indicate that these
[Ca2+]i (nM x s)

100000 results render the neurons more susceptible to oxidative

a
80000
60000
40000
20000
0 oxidative damage to the SH-SY5Y cell observed in previous
Control Curcumin H2O2 Cur+H2O2
Figure 5. (A) Calcium release from SH-SY5Y cells exposed to curcumin
(Cur) and hydrogen peroxide (H2O2) following stimulation with H2O2.
Original time course chart recordings showing [Ca2+]i transients in SH-
SY5Y cells. (B) Bar charts showing mean ± SD data for [Ca2+]i from
H2O2-stimulated SH-SY5Y cells (n ¼ 6 for each). Note: The significant
elevation in [Ca2+]i for SH-SY5Y cells compared to control. a: p50.001
versus control group; b: p50.001 versus curcumin group; c: p50.001
versus H2O2 group.
Effects of curcumin and hydrogen peroxide on [Ca2+]i
release in SH-SY5Y neuronal cells (H2O2)
Effects of curcumin, H2O2 and their combination on [Ca2+]i
release in the SH-SY5Y cells are shown in Figure 5A. The
[Ca2+]i release values were significantly (p50.001) lower in
curcumin group samples than in control samples. Incubation
with H2O2 markedly increased [Ca2+]i levels compared to
control and curcumin group samples. The area under curves,
at the same time bar charts (Figure 5B), reflects [Ca2+]i
concentrations from H2O2-stimulated SH-SY5Y neuronal
cells. H2O2 incubation increased [Ca2+]i levels. Curcumin
incubation significantly decreased [Ca2+]i levels (p50.001).
Discussion
Despite the fact that curcumin was firstly described as an
antioxidant molecule more than half a decade ago, the
molecular mechanisms by which curcumin induces/inhibits
cellular proliferation, survival, and apoptosis are still not well
understood. Curcumin has been showed to have specialties
such as being a strong antioxidant, being immunomodulatory,
and having anti-inflammatory properties (21–23). Hence, the
current study focused on the role of curcumin in minimizing
stress (6,16,23). In the current study, we determined that the
extracellular addition of H2O2 onto SH-SY5Y neuronal cells
significantly increased ROS levels. The H2O2 induced
elevations in ROS levels in the current study showed similar
MPP+ induced cytotoxicity and Parkinson’s diseases cell
models, where MPP+ selectively targets and degenerates
dopaminergic neurons due to the excess generation of ROS
(26).
In addition to its antioxidant effect, curcumin can prevent
cyclo-oxygenase, lipoxygenase, and xanthine oxidase from
generating free radicals (27). Curcumin can also prevent the
production of nitric oxide, arachidonic acid metabolism, and
block tumor necrosis factor-a with interleukin-12 synthesis
(28). Long-term exposure to oxidative stress products and
neurotoxins has previously been determined to trigger
caspase-3 activation (29,30). Caspase-3 is one of the most
important effector caspases in the final apoptotic cascade
leading to cell death (6). In another study, Yin et al. (2005)
transfected APPswe plasmids into SH-SY5Y neuronal cells to
induce an Alzheimer’s disease model and determine the
protective effects of curcumin against ROS. They reported
that curcumin significantly decreased the ROS levels (31).
Curcumin-induced apoptosis was also associated with the
proteolytic activation of caspase-3 and caspase-9 (28).
If oxidative stress triggers the etiology of neurodegenera-
tive diseases, then agents that can simultaneously attenuate
ROS damage and suppress caspase-3 activation may hold
promise for the treatment of neurodegenerative diseases. Here
we have demonstrated that curcumin, effectively inhibits
caspase-3 and caspase-9 activation and also ameliorates
apoptosis induced by extracellular stimulation of H2O2 to
SH-SY5Y cells. Caspase activation is assumed to be an
important step in regulating apoptosis (32). Moreover,
oxidative stress triggers an apoptotic cascade of neurons
(33) by forming a disturbance in calcium homeostasis (10).
Caspase-3 plays a pivotal role in neuronal apoptosis and
triggers cleavage as well as an activation of caspase-9 (16).
Enhanced expression of caspase-3 with H2O2 was suppressed
 6 A. Cihangir Uğuz et al.
with curcumin. The current data that we obtained from our
experiments suggest that the apoptotic cascade activation is
prevented, thereby reducing the risk of neuronal cell damage
(34). Here we have determined that H2O2 increased levels of
caspase-3 and -9 levels in SH-SY5Y cells. However,
curcumin reversed the situation and decreased caspase-3
and -9 levels.
It has been previously reported that curcumin may have
regulatory effects on mitochondrial complex I by increasing
the levels of thiol-rich glutathione. It was previously
mentioned that a sudden and strong depletion of GSH can
cause oxidative damage to mitochondrial complex I (35).
H2O2 treatment decreased GSH-Px levels resulting in
increased oxidative damage. Curcumin alone was found to
markedly elevate the GSH-Px levels. Thus, the combination
of curcumin with H2O2 was also determined to be increased,
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when compared to the H2O2 alone group, due to curcumin
antioxidant effects (36).
It has been reported that curcumin is an effective agent
against apoptosis (28). The antiapoptotic effects of curcumin
are related to the suppression of NF-kB, TNF-a, and
interleukins (37,38). Li et al. (2015) determined that long-
term treatment (12 weeks) with curcumin attenuated apop-
tosis by reducing the glutamate levels and down regulating
Ca2+/calmodulin-dependent protein kinase II (CaMKII), in
retinal neurons of diabetes-induced rats. Li et al. (2015) also
determined there is a protective effects of curcumin against
neurotoxicity in SK-N-MC cells by protecting them from
apoptotic cascade (39). Apoptosis levels were significantly
decreased in the current study. We also determined that Ca2+
release from the intracellular stores was significantly higher
in the H2O2 group. However, curcumin incubation reversed
the situation and decreased the Ca2+ release. This could be
related to the increased ROS levels and membrane depolar-
ization levels. If the elevation in ROS levels cannot be
controlled, membrane will depolarize in an increasing manner
and cations will flow into the membrane through ion channels
(16). The relationship between Ca2+ overload and apoptosis is
also well documented (40).
The mitochondrial electron transport chain (METC)
is implicated in cellular energy generation. Alternatively,
mitochondria serve as a main source of free radical produc-
tion. This has led to the perception that mitochondria
are responsible for a large range of pathological diseases,
especially neurological diseases (36). With neurodegenerative
disorders, mitochondrial components are susceptible to
lipid peroxidation and mitochondrial membrane depolariza-
tion as one of the key players in cell survival. We determined
there were decreased MMP levels in SH-SH5Y neuronal
cells. As we mentioned above, curcumin was effective
in improving the activity of mitochondrial complexes I, II
and III (41).
According to a full literature search, it can be observed
that, there are some manuscripts that are controversial to our
findings. For example Khan et al. found that curcumin
increases ROS levels in T-cell lymphoma (Hu T-78). We
believe this finding occurred because of the cell type, dosage
and incubation period (42). Hollborn et al. also determined
that curcumin accelerates chemotaxis with a 50 mM dosage
but it also triggers proliferation in 10 mM dosages (12).
J Recept Signal Transduct, Early Online: 1–7
In conclusion, we concluded that curcumin at concentra-
tions, written above shown previously, were found to be
effective in neuronal cell protection. When we checked our
results, we can easily see that, curcumin cannot fullu reversed
the H2O2 triggered cell death, but protects the cells. This
could be due to the scavenging capacity of curcumin.
According to the our literature search, the dosage and also
time period must be well designed. Since curcumin is a
natural food pigment that can cross the blood-brain barrier
and has widespread medicinal uses, it has a potential
therapeutic value for treating treatments against some various
neurological diseases such as including Alzheimer’s diseases,
Parkinson’s diseases and other neurodegenerative disorders.
However, additional studies are needed to further elucidate
the mechanisms of curcumin’s cytoprotective and/or cyto-
toxicity effects and its subsequent pathogenesis and progres-
sion in neurological diseases.
Acknowledgements
A.C.U. and A.Ö. formulated the hypothesis. A.Ö. was responsible for
experiments. A.C.U. was responsible writing the manuscript. M.N. made
critical revision to the manuscript.
Declaration of interest
None of the authors have any conflicts to disclose. All authors approved
the final version of the manuscript.
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