Photocytotoxic effect of C₆₀ fullerene against L1210 leukemic cells is accomPanied by enhanced nitric oxide Production and p38 maPk activation
Aim: To estimate the combined action of C₆₀ fullerene and light irradiation on viability of L1210 leukemic cells, nitric oxide (NO) generation, p38 mitogen-activated protein kinase (MAPK) activity and cell cycle distribution. Methods: Cell viability was assessed by MTT test. Light-emitting diode lam...
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Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України
2016
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| Назва видання: | Experimental Oncology |
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| Цитувати: | Photocytotoxic effect of C₆₀ fullerene against L1210 leukemic cells is accomPanied by enhanced nitric oxide Production and p38 maPk activation / D.V. Franskevych, I.I. Grynyuk, S.V. Prylutska, G.V. Pasichnyk, D.M. Petukhov, L.B. Drobot, O.P. Matyshevska, U. Ritter // Experimental Oncology. — 2016 — Т. 38, № 2. — С. 89–93. — Бібліогр.: 26 назв. — англ. |
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nasplib_isofts_kiev_ua-123456789-1380022025-02-23T20:26:01Z Photocytotoxic effect of C₆₀ fullerene against L1210 leukemic cells is accomPanied by enhanced nitric oxide Production and p38 maPk activation Franskevych, D.V. Grynyuk, I.I. Prylutska, S.V. Pasichnyk, G.V. Petukhov, D.M. Drobot, L.B. Matyshevska, O.P. Ritter, U. Original contributions Aim: To estimate the combined action of C₆₀ fullerene and light irradiation on viability of L1210 leukemic cells, nitric oxide (NO) generation, p38 mitogen-activated protein kinase (MAPK) activity and cell cycle distribution. Methods: Cell viability was assessed by MTT test. Light-emitting diode lamp (λ = 410–700 nm, 2.45 J/cm²) was used for C₆₀ fullerene photoexcitation. Nitrite level and NO-synthase activity were measured by Griess reaction and by conversion of L-arginine to L-citrulline, respectively. p38 MAPK activity was assessed by Western blot analysis. Cell cycle distribution was determined by flow cytometry. Results: It was shown that light irradiation of C₆₀ fullerene-treated L1210 cells was accompanied by 55% decrease of their viability at 48 h of culture. Nitrite level measured as an index of reactive NO generation was increased at the early period after C₆₀ fullerene photoexcitation due to activation of both constitutive and inducible NO-synthase isoforms. The simultaneous activation of p38 MAPK was detected. Accumulation of L1210 cells in sub-G₁ phase of cell cycle was observed after C₆₀ fullerene photoexcitation. Conclusion: Photoexcited C₆₀ fullerene exerts cytotoxic effect, at least in part, through triggering production of reactive NO species and activation of p38 kinase apoptotic pathways in L1210 leukemic cells. 2016 Article Photocytotoxic effect of C₆₀ fullerene against L1210 leukemic cells is accomPanied by enhanced nitric oxide Production and p38 maPk activation / D.V. Franskevych, I.I. Grynyuk, S.V. Prylutska, G.V. Pasichnyk, D.M. Petukhov, L.B. Drobot, O.P. Matyshevska, U. Ritter // Experimental Oncology. — 2016 — Т. 38, № 2. — С. 89–93. — Бібліогр.: 26 назв. — англ. 1812-9269 https://nasplib.isofts.kiev.ua/handle/123456789/138002 en Experimental Oncology application/pdf Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України |
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Digital Library of Periodicals of National Academy of Sciences of Ukraine |
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DSpace DC |
| language |
English |
| topic |
Original contributions Original contributions |
| spellingShingle |
Original contributions Original contributions Franskevych, D.V. Grynyuk, I.I. Prylutska, S.V. Pasichnyk, G.V. Petukhov, D.M. Drobot, L.B. Matyshevska, O.P. Ritter, U. Photocytotoxic effect of C₆₀ fullerene against L1210 leukemic cells is accomPanied by enhanced nitric oxide Production and p38 maPk activation Experimental Oncology |
| description |
Aim: To estimate the combined action of C₆₀ fullerene and light irradiation on viability of L1210 leukemic cells, nitric oxide (NO) generation, p38 mitogen-activated protein kinase (MAPK) activity and cell cycle distribution. Methods: Cell viability was assessed by MTT test. Light-emitting diode lamp (λ = 410–700 nm, 2.45 J/cm²) was used for C₆₀ fullerene photoexcitation. Nitrite level and NO-synthase activity were measured by Griess reaction and by conversion of L-arginine to L-citrulline, respectively. p38 MAPK activity was assessed by Western blot analysis. Cell cycle distribution was determined by flow cytometry. Results: It was shown that light irradiation of C₆₀ fullerene-treated L1210 cells was accompanied by 55% decrease of their viability at 48 h of culture. Nitrite level measured as an index of reactive NO generation was increased at the early period after C₆₀ fullerene photoexcitation due to activation of both constitutive and inducible NO-synthase isoforms. The simultaneous activation of p38 MAPK was detected. Accumulation of L1210 cells in sub-G₁ phase of cell cycle was observed after C₆₀ fullerene photoexcitation. Conclusion: Photoexcited C₆₀ fullerene exerts cytotoxic effect, at least in part, through triggering production of reactive NO species and activation of p38 kinase apoptotic pathways in L1210 leukemic cells. |
| format |
Article |
| author |
Franskevych, D.V. Grynyuk, I.I. Prylutska, S.V. Pasichnyk, G.V. Petukhov, D.M. Drobot, L.B. Matyshevska, O.P. Ritter, U. |
| author_facet |
Franskevych, D.V. Grynyuk, I.I. Prylutska, S.V. Pasichnyk, G.V. Petukhov, D.M. Drobot, L.B. Matyshevska, O.P. Ritter, U. |
| author_sort |
Franskevych, D.V. |
| title |
Photocytotoxic effect of C₆₀ fullerene against L1210 leukemic cells is accomPanied by enhanced nitric oxide Production and p38 maPk activation |
| title_short |
Photocytotoxic effect of C₆₀ fullerene against L1210 leukemic cells is accomPanied by enhanced nitric oxide Production and p38 maPk activation |
| title_full |
Photocytotoxic effect of C₆₀ fullerene against L1210 leukemic cells is accomPanied by enhanced nitric oxide Production and p38 maPk activation |
| title_fullStr |
Photocytotoxic effect of C₆₀ fullerene against L1210 leukemic cells is accomPanied by enhanced nitric oxide Production and p38 maPk activation |
| title_full_unstemmed |
Photocytotoxic effect of C₆₀ fullerene against L1210 leukemic cells is accomPanied by enhanced nitric oxide Production and p38 maPk activation |
| title_sort |
photocytotoxic effect of c₆₀ fullerene against l1210 leukemic cells is accompanied by enhanced nitric oxide production and p38 mapk activation |
| publisher |
Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України |
| publishDate |
2016 |
| topic_facet |
Original contributions |
| url |
https://nasplib.isofts.kiev.ua/handle/123456789/138002 |
| citation_txt |
Photocytotoxic effect of C₆₀ fullerene against L1210 leukemic cells is accomPanied by enhanced nitric oxide Production and p38 maPk activation / D.V. Franskevych, I.I. Grynyuk, S.V. Prylutska, G.V. Pasichnyk, D.M. Petukhov, L.B. Drobot, O.P. Matyshevska, U. Ritter // Experimental Oncology. — 2016 — Т. 38, № 2. — С. 89–93. — Бібліогр.: 26 назв. — англ. |
| series |
Experimental Oncology |
| work_keys_str_mv |
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2025-11-25T04:38:18Z |
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2025-11-25T04:38:18Z |
| _version_ |
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| fulltext |
Experimental Oncology 38, 89–93, 2016 (June) 89
Photocytotoxic effect of c60 fullerene against
l1210 leukemic cells is accomPanied by enhanced nitric
oxide Production and p38 maPk activation
D.V. Franskevych1,*, I.I. Grynyuk1, S.V. Prylutska1, G.V. Pasichnyk2,
D.M. Petukhov2, L.B. Drobot2, O.P. Matyshevska1, U. Ritter3
1Taras Shevchenko National University of Kyiv, Kyiv 01601, Ukraine
2Palladin Institute of Biochemistry, NAS of Ukraine, Kyiv 01011, Ukraine
3Technical University of Ilmenau, Ilmenau 98693, Germany
Aim: To estimate the combined action of C60 fullerene and light irradiation on viability of L1210 leukemic cells, nitric oxide (NO)
generation, p38 mitogen-activated protein kinase (MAPK) activity and cell cycle distribution. Methods: Cell viability was assessed
by MTT test. Light-emitting diode lamp (λ = 410–700 nm, 2.45 J/cm2) was used for C60 fullerene photoexcitation. Nitrite level
and NO-synthase activity were measured by Griess reaction and by conversion of L-arginine to L-citrulline, respectively. p38 MAPK
activity was assessed by Western blot analysis. Cell cycle distribution was determined by flow cytometry. Results: It was shown that
light irradiation of C60 fullerene-treated L1210 cells was accompanied by 55% decrease of their viability at 48 h of culture. Nitrite
level measured as an index of reactive NO generation was increased at the early period after C60 fullerene photoexcitation due
to activation of both constitutive and inducible NO-synthase isoforms. The simultaneous activation of p38 MAPK was detected.
Accumulation of L1210 cells in sub-G1 phase of cell cycle was observed after C60 fullerene photoexcitation. Conclusion: Photoex-
cited C60 fullerene exerts cytotoxic effect, at least in part, through triggering production of reactive NO species and activation
of p38 kinase apoptotic pathways in L1210 leukemic cells.
Key Words: С60 fullerene, photoexcitation, L1210 cells, NO radicals, p38 MAPK, cell cycle.
The direct influence on the signalling pathways
involved in coordinated control of cells proliferation
and apoptosis is considered to be promising in tumor
growth inhibition. It is assumed that intense reac
tive oxygen species (ROS) production due to imba
lance of prooxidant — antioxidant equilibrium, which
precedes caspase activation could be the inductor
of receptorindependent apoptotic pathway in can
cer cells. Carbon nanostructure C60 fullerene and its
derivatives are shown to be the effective regulators
of cell oxidant status and the perspective compounds
for photodynamic killing of cancer cells [1–3].
Due to the extended πconjugated system of mo
lecular orbitals, C60 effectively absorbs UV/visible light
and shifts to triplet state. In the presence of electron
donors in the medium the excited C60 is reduced and
converted into anion radical C∙–
60 with further transfer
ring of electron to molecular oxygen and producing
singlet oxygen, superoxide radical anion, hydroxyl
radical, which play an important role as regulators
of cell death signalling [1, 4].
The studies of biological activity of C60 fullerenes were
focused mainly on watersoluble chemically modified
derivatives. But substantial modification of fullerene core
appears to cause perturbation of its electronic structure
and hence to reduce the level of its photoexcitation [1,
5]. Therefore, photodynamic potential of pristine (non
modified) C60 fullerene needs detailed investigation.
Using fluorescentlabeled C60 fullerene, obtained
by covalent conjunction of C60 with rhodamine B iso
thiocyanate we have demonstrated accumulation
of carbon nanostructure inside the leukemic cells [6].
We have shown previously apoptosis induction
in human leukemic cells treated with pristine C60 and
irradiated in UVvisible range [7]. But detailed ROS
dependent mechanisms involved in C60induced pho
tocytotoxic effect are still to be elucidated.
Cytotoxic effect of ROS could be amplified by its
interaction with reactive nitric species, nitric oxide
(NO) and NO radicals, that is followed by the formation
of peroxynitrite ions (ONOO–), irreversible modifica
tion of proteins tyrosine and cysteine residues and
oxidative/nitrative stress [8, 9]. One of the important
redoxsensitive and NOdependent components
of mitogenactivated protein kinases (MAPK) signal
ling pathways is p38 MAPK, which is involved in the
control of cell cycle and apoptosis [10–12].
The aim of the study was to estimate the combined
action of C60 fullerene and light irradiation on NO gene
ration, p38 MAPK activity and cell cycle distribution
in order to elucidate the possible mechanisms involved
in photocytotoxic effect of C60 fullerene against murine
L1210 leukemic cells.
materials and methods
Water colloid solution of C60 fullerene (10–4 M,
purity > 99.5%, nanoparticle average size 50 nm,
stabi lity — 12 months) was synthesized and charac
terized in Technical University of Ilmenau (Germany)
as described in [13].
The murine L1210 leukemic cell line was obtained
from the Bank of Cell Lines from Human and Animal
Submitted: November 25, 2015.
*Correspondence: E-mail: dashaqq@gmail.com
Abbreviation used: cNOS – constitutive NOS isoform; iNOS – in-
ducible NOS; MAPK – mitogen-activated protein kinase; NO – ni-
tric oxide; NOS – NO-synthases; ROS – reactive oxygen species.
Exp Oncol 2016
38, 2, 89–93
90 Experimental Oncology 38, 89–93, 2016 (June)
Tissues of R.E. Kavetsky Institute of Experimental Pa
thology, Oncology and Radiobiology, NAS of Ukraine.
Cells were incubated in RPMI 1640 medium with 5%
FBS. The cells of the control group were incubated for
2 h without treatment, of second group were irradi
ated (100 mW, during 2 min), of the third group were
incubated with C60 (10–5 M), of the fourth group were
incubated with C60 fullerene with further irradiation.
Efficiency of C60 fullerene photoactivation strongly
depends on optical absorption of the molecule, which
is highest in UV region, but the tail stretches into red
region. To exclude UV irradiation, which is damaging
to cell and to maximize the efficiency of C60 fullerene
photoexcitation by visible light we use the range
of 410–700 nm.
Cell viability was assessed by the MTT [3(4,5di
methylthiazol2yl)2,5diphenyl tetrazolium bromide]
reduction assay. At indicated time points of incuba
tion 200 µl aliquots (1•105 cells) were placed into the
96well microplates, 20 μl of MTT solution (4 mg/ml)
was added to each well and the plates were incubated
for 2 h. The culture medium was then replaced with
100 μl of DMSO; diformazan formation was determined
by measuring absorption at 570 nm with a μQuant
microplate reader (BioTek, USA).
Nitrite level was measured by Griess reaction [14].
An aliquot (0.5 ml) of the cell suspension (106 cells/ml)
was mixed with an equal volume of Griess reagent
(sulfanilamide 1% w/v, naphthylethylenediamine
dihydrochloride 0.1% w/v and orthophosphoric acid
2.5% w/v) and incubated at room temperature for
10 min prior to measurement of absorbance at 546 nm.
The amount of nitrite formed was compared to those
of known concentrations of sodium nitrite and norma
lized to the protein content.
NOsynthase (NOS) activity was measured spec
trophotometrically by the conversion of Larginine to L
citrulline [15, 16]. Cells (106 cells per probe) were incu
bated in buffer containing 50 mM KH2PO4, 1 mM MgCl2,
2 mM CaCl2, 1 mM NADPH, 2 mM Larginine, pH 7.0 for
60 min at 37°C. The reaction was stopped by adding
2N HClO4. The content of Lcitrulline was determined
in the protein free supernatant. To determine the activity
of Ca2+independent inducible NOS (iNOS) 2 mM EDTA
was added instead of CaCl2. Activity of Ca2+dependent
constitutive NOS isoform (cNOS) was calculated as the
difference between total NOS activity and iNOS activity.
The level of Lcitrulline was determined spectropho
tometrically [17]. Protein free aliquot was mixed with
reagent (59 mM diacetyl monooxime, 32 mM antipyrine
and 55 mM iron sulfate (ΙΙ) in 6N H2SO4) and boiled du
ring 15 min. After cooling the extinction at 465 nm was
measured. Amount of Lcitrulline was determined using
a calibration curve for Lcitrulline.
After irradiation of cells treated with C60 fullerene
the aliquots of suspension (5•106 cells) were taken
at 30; 60 and 120 min for Western blotting. The cells
were centrifuged at 600 g, washed with PBS, lysed
with icecold lysis buffer (50 mM ТrisHCl, pH 7.5,
150 mM NaCl, 1% Triton X100, 1 mM ovanadate,
50 mM NaF, 2 mM ЕDTA, 1 mM PMSF, complete pro
tease inhibitor cocktail) and centrifuged at 12 000 g for
20 min at 4 °C. Protein content in supernatants was de
termined using PierceTMBCA Protein Assay kit (Thermo
Scientific, USA). Proteins (30 μg per sample) were
separated by electrophoresis on 10% polyacrylamide
gels and transferred to nitrocellulose membranes.
Membranes were incubated with antiphosphop38 ki
nase antibodies (Cell Signaling, USA) and antiβactin
antibodies (Sigma, USA) overnight at 4 °C. As second
ary antibodies peroxidaseconjugated antirabbit
or antimouse IgG were used, respectively. The im
munoreactive bands were visualized using enhanced
chemoluminescence detection reagent (Amersham
Pharmacia Biotech, USA). Densitometric analysis was
performed using the GelPro analyzer software (Media
Cybernetics, Silver Spring, USA).
For cell cycle analysis cells (1•106) were resus
pended in 0.1 ml PBS (pH 7.4), fixed by adding
0.9 ml of 90% ethanol at –20 °C overnight and centri
fuged at 13 000 g for 1 min. The fixed cells were rinsed
twice with PBS and resuspended in propidium iodide
(10 μg/ml) solution containing RNase A (100 μg/ml,
Sigma, USA) in PBS without calcium and magnesium.
The stained cells were analyzed by a COULTER EPICS
XLTM (Beckman Coulter, USA) and FCS Express 3 Flow
Cytometry Software (DeNovo Software, USA).
Data processing and plotting were performed
by IBM PC using specialized applications Excel 2010.
Statistical analysis was performed using Statis-
tica 6.0 computer program (StatSoft Inc., USA). Paired
Student’s ttests were performed. Difference values
p < 0.05 were considered to be statistically significant.
results and discussion
To evaluate photocytotoxic effect of C60 fullerene,
nanostructuretreated L1210 cells were incubated for
a longterm period after irradiation (Fig. 1). Cells survival
after light irradiation per se was not less than 80% when
the time of incubation was extended to 48 h. C60 fulle
rene in concentration 10–5 М didn’t change the viability
of L1210 cells. This effect is harmonized with data about
lack of C60 fullerene cytotoxicity in low concentration
range [18–20]. In contrast, cells responded to the
combined action of C60 fullerene and irradiation by time
dependent decrease of cell viability. After 48 h of culture,
viability of leukemic cells declined to 55% as compared
to control.
Free radical NO and its various forms are known
to be involved in the early phases of cell death. NO can
either suppress apoptosis or activate cell death path
ways depending on its concentration and cell redox
potential [8, 9, 21]. Direct evaluation of the NO level
in cell population is complicated by its short lifetime
and quick metabolism. At the same time, the level of its
stable metabolite nitrite anion is proved to be an ade
quate index of NO generation [22]. No changes in NO2
–
level were detected in cells treated with C60 alone,
while after C60 fullerene photoexcitation investigated
index was increased at 1 and 3 h and was substantially
Experimental Oncology 38, 89–93, 2016 (June) 91
higher than in control or in irradiated cells (Fig. 2, a).
These data indicated that photoexcited C60 fullerene
potentiates NO generation in leukemic cells.
40
50
60
70
80
90
100
0 12 24 36 48
Ce
ll
via
bi
lit
y,
%
Control
Irradiation
C60 + irradiation
Time of incubation, h
fig. 1. Viability of L1210 cells after combined action of C60 fulle
rene (10–5 М) and light irradiation. M ± m, n = 8
NO is generated due to oxidation of Larginine
to Lcitrulline by NADPHdependent NOS fa mily, con
sisting of two main isoforms. cNOS could be quickly
activated in a Ca2+dependent manner and is asso
ciated with plasmalemma, while iNOS is cytokine
dependent and needs several hours to reach maximum
activity and is located predominantly in the inner
mitochondrial membrane [23]. The data presented
at Fig. 2, b, c show that C60 per se had no effect on ac
tivation of NOS isoforms, whereas photoexcitation
of accumulated C60 fullerene was followed by early
activation of both cNOS and iNOS. Enzymes’ ac
tivity was increased already at 1 h and was further
enhanced at 3 h after C60 fullerene photoexcitation.
This observation is in a good agreement with the data
presented in Fig. 2, a concerning enhanced NO2
– level
and NO generation induced by photoexcited C60 fulle
rene in the leukemic cells.
Taking into account that NO generation coincides
with increased ROS production and disturbance
of antioxidant system (inhibition of glutathione peroxi
dase against superoxide dismutase activation, which
was detected in L1210 cells at 3 h after combined
action of C60 fullerene and irradiation) [24] we sug
gested the possibility of oxidative modification of the
components of MAPK signalling pathways. Previous
investigations indicate that p38 kinase is stress
activated, redoxsensitive, NOdependent and could
promote cell death [10–12]. We examined p38 MAPK
activity by estimation the level of its active phosphory
lated form (pp38 MAPK) using Western blot analysis.
As shown in Fig. 3, irradiation alone or in combination
with C60 fullerene activated p38 MAPK at 1 h while
C60 fullerene treatment was ineffective. Whereas the
phosphorylation level of p38 MAPK in cells irradiated
without C60 fullerene was normalized at 2 h, in cells
treated with photoexcited C60 fullerene a pronounced
2.5 fold increase in pp38 MAPK level was observed
at this time point (Fig. 3, b, c). There are data that
p38 MAPK pathway is engaged in caspase3, 8 and
9 activation during NOdependent apoptosis of diffe
rent cell types [10, 12]. It is also shown that C60 fullerene
exert photocytotoxicity in the MCF7 cancer cell line
through modulation of ROS and p38 MAPK activa
tion [25]. Our results allow to suggest that activation
of p38 MAPK can be involved in redoxdependent cyto
toxic effect of photoexcited C60 fullerene in L1210 cells.
0
5
10
15
20
Control C60 Irradiation C60 + irradiation
µm
ol
e/
m
g
pr
ot
ei
n
NO2
–
*
0
5
10
15
20
25
30
35
Control C60 Irradiation C60 + irradiation
Constitutive NOS1 h
3 h
*
*
0
2
4
6
8
10
12
14
16
18
20
Control C60 Irradiation C60 + irradiation
Inducible NOS
*#
*#
*
nm
ol
L
-c
itr
ul
lin
e*
m
g
pr
ot
ei
n/
m
in
*m
l
nm
ol
L
-c
itr
ul
lin
e*
m
g
pr
ot
ei
n/
m
in
*m
l
*#
*#
*# b
c
a
fig. 2. The level of NO2
– (a) and NOS activity (b, c) in L1210 cells
after combined action of C60 fullerene and light irradiation. M ± m,
n = 6; *p < 0.05 compared to control cells; #p < 0.05 compared
to irradiated cells
Because transcription factors and cyclindepen
dent kinases are among the substrates of activated
p38 kinase we examined the cell cycle distribution
as the possible longterm effect of C60 fullerene
in L1210 cells. Flow cytometric analysis showed that
treatment with C60 fullerene or irradiation alone had lit
tle effect on cell cycle profile at 24 h of incubation. Pho
toexcited C60 fullerene was proved to influence accu
mulation of cells in different cell cycle phases (Fig. 4).
A decreased number of cells in G2/M phase (12.1%
vs 20.6% in control) was accompanied by an increased
number in subG1 phase (7.3% vs 3.1% in control).
Since increase of subG1 cell fraction is shown to cor
relate with apoptotic DNA fragmentation [26], the data
obtained indicate that photoexcitation of C60 fullerene
92 Experimental Oncology 38, 89–93, 2016 (June)
is followed by cell cycle redistribution of L1210 cells
with accumulation of apoptotic cells.
0
1000
2000
3000
4000
5000
6000
0' 30' 60' 120' 0' 30' 60' 120'
r.u
.
control C60
control C60
0
1000
2000
3000
4000
5000
6000
0' 30' 60' 120' 0' 30' 60' 120'
r.u
. irradiation
C60 + irradiation d
b
c
a
0' 30' 60' 120' 0' 30' 60' 120'
irradiation C60 + irradiation
0' 30' 60' 120' 0' 30' 60' 120'
pp38
Actin
pp38
Actin
fig. 3. Activation of p38 MAPK in leukemic cells after C60 fullerene
photoexcitation: a, c — Western blot analysis of the pp38 MAPK
level (typical blotogram); b, d — quantitative analysis of the fold
increase of pp38 MAPK level. Actin was used as loading control
The results of this study demonstrate that the
cytotoxic effect of photoexcited C60 fullerene against
L1210 leukemic cells is realized through a number
of apoptosisassociated pathways. It was shown that
visible light irradiation of cells treated with C60 fullerene
was accompanied by activation of both constitutive
and inducible forms of NOS and generation of reactive
NO species. The simultaneous activation of p38 MAPK
was also detected. The L1210 cell cycle is shown
to be disturbed after C60 fullerene photoexcitation
with cell accumulation in subG1, which is the marker
of apoptosis.
conclusion
In summary, our results suggest that photoexci
tated C60 fullerene induces cytotoxic effect at least
in part through triggering production of reactive
NO species and activation of p38 MAPKdependent
apoptotic pathways in leukemic cells. The cytotoxic
effect of photoexcited C60 fullerene could be used for
designing and development of complex approached
in anticancer therapy.
0
5
10
15
20
25
30
35
40
45
50
Control C60 Irradiation C60 + irradiation
Di
st
rib
ut
io
n
in
c
el
l c
yc
le
,%
sub-G1 G0/G1 S G2/M
*
*
fig. 4. The effect of C60 fullerene photoexcited on cell cycle
phase distribution in L1210 cells (M ± m, n = 4; *p < 0.05 com
pared to control cells)
references
1. Mroz P, Pawlak A, Satti M, et al. Functionalized fulle
renes mediate photodynamic killing of cancer cells: Type I ver
sus Type II photochemical mechanism. Free Radic Biol Med
2007; 43: 711–9.
2. Otake E, Sakuma S, Torii K, et al. Effect and mechanism
of a new photodynamic therapy with glycoconjugated fullerene.
Photochem Photobiol 2010; 86: 1356–63.
3. Li WT. Nanotechologybased strategies to enhance the
efficacy of photodynamic therapy for cancers. Curr Drug Met
2009; 10: 851–60.
4. Kamat J, Devasagayam T, Priyadarsini K, et al. Reac
tive oxygen species mediated membrane damage induced
by fullerene derivatives and its possible biological implications.
Toxicology 2000; 155: 55–61.
5. Guldi DM, Prato M. Excitedstate properties of C(60)
fullerene derivatives. Acc Chem Res 2000; 33: 695–703.
6. Matyshevska OP, Palyvoda KO, Prylutska SV, et al. In
duction of apoptotic leukemic cells death with the use of fulle
rene C60. In: Nanoscale Systems and Nanomaterials Research
in Ukraine. Kyiv: Akademperiodiс 2014: 524–9 (in Russian).
7. Palyvoda KO, Grynyuk II, Prylutska SV, et al. Apoptosis
photoinduction by C60 fullerene in human leukemic T cells.
Ukr Biokhim Zh 2010; 82: 121–7.
8. Nicotera P, Bernassola F, Melino G. Nitric oxide (NO),
a signaling molecule with a killer soul. Cell Death Differ 1999;
6: 931–3.
9. Hsieh HJ, Liu CA, Huang B, et al. Shearinduced
endothelial mechanotransduction: the interplay between
reactive oxygen species (ROS) and nitric oxide (NO) and the
pathophysiological implications. J Biomed Sc 2014; 21: 3–17.
10. Lee JH, Lee SW, Choi SH, et al. p38 MAP kinase and
ERK play an important role in nitric oxideinduced apoptosis
of the mouse embryonic stem cells. Toxicol in Vitro 2013;
27: 492–8.
11. Son Y, Cheong YK, Kim NH, et al. Mitogenactivated
protein kinases and reactive oxygen species: how can ROS acti
vate MAPK pathways. J Signal Transduct 2011: 2011: 792639.
12. Iwaoka S, Nakamura T, Takano S, et al. Cationic
liposomes induce apoptosis through p38 MAP kinasecaspase
8Bid pathway in macrophagelike RAW264.7 cells. J Leukoc
Biol 2006; 79: 184–91.
Experimental Oncology 38, 89–93, 2016 (June) 93
13. Scharff P, CartaAbelmann L, Siegmund C, et al. Ef
fect of XRay and UV irradiation of the C60 fullerene aqueous
solution on biological samples. Carbon 2004; 42: 1199–201.
14. Green LC, Wagner DA, Glogowski J, et al. Analysis
of nitrate, nitrite and [15N] nitrite in biological fluids. Anal
Biochem 1982; 126: 131–8.
15. Salter M, Knowles R, Moncada S. Widespread tis
sue distribution, species distribution and changes in activity
of Ca2+dependent and Ca2+independent nitric oxide syn
thases. FEBS Lett 1991; 291: 145–9.
16. Chin SY, Pandey KN, Shi SJ, et al. Increased activity
and expression of Ca2+dependent NOS in renal cortex of ANG
IIinfused hypertensive rats. Am J Physiol Renal Physiol 1999;
277: 797–804.
17. Boyde TR, Rahmatullah M. Optimization of condi
tions for the colorimetric determination of citrulline, using
diacetyl monoxime. Anal Biochem 1980; 107: 424–31.
18. Levi N, Hantgan RR, Lively MO, et al. C60Fulle
renes: detection of intracellular photoluminescence and lack
of cytotoxic effects. J Nanobiotechnology 2006; 14: 4–14.
19. Sayes CM, Fortner JD, Guo W, et al. The differential
cytotoxicity of watersoluble fullerenes. Nano Lett 2004;
4: 1881–7.
20. Liu S, Liu H, Yin Z, et al. Cytotoxicity of pristine
C60 fullerene on baby hamster kidney cells in solution. J Bio
mater Nanobiotechnol 2012; 3: 385–90.
21. Masri F. Role of nitric oxide and its metabolites as po
tential markers in lung cancer. Ann Thorac Med 2010; 5: 123–7.
22. Murad F. Discovery of some of the biological effects
of nitric oxide and its role in cell signaling. Biosci Rep 2004;
24: 452–74.
23. Forstermann U, Sessa WC. Nitric oxide syntha
ses: regu lation and function. Eur Heart J 2012; 33: 829–37.
24. Grynyuk II, Grebinyk SM, Prylutska SV, et al. Photoex
cited fullerene C60 disturbs prooxidantantioxidant balance in leu
kemic L1210 cells. Matwiss u Werkstofftech 2013; 44: 139–43.
25. Li Z, Zhang FL, Wang Z, et al. Fullerene (C60)
nanoparticles exert photocytotoxicity through modulation
of reactive oxygen species and p38 mitogenactivated protein
kinase activation in the MCF7 cancer cell line. J Nanopart
Res 2013; 15: 2102–12.
26. Jantova S, Repicky A, Letasiova S, et al. 4Amino
3acetylquinolineinduced apoptosis of murine L1210 leu
kemia cells involves ROSmitochondrialmediated death
signaling and activation of p38 MAPK. Cell Biochem Funct
2008; 26: 609–19.
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