Apoptosis and content of mobile lipid domains in human leukemia K-562 cells induced to differentiate by quercetin or dimethyl sulfoxide
The apoptotic index, cell cycle progression and caspase-3 activation in K-562 cells induced to differentiate by DMSO or quercetin have been studied. Quercetin treatment of K-562 cells was accompanied by cell cycle arrest in G2 /M and apoptosis with caspase-3 activation. In contrast, DMSO-induced dif...
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| Date: | 2010 |
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Інститут біохімії ім. О.В. Палладіна НАН України
2010
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| Cite this: | Apoptosis and content of mobile lipid domains in human leukemia K-562 cells induced to differentiate by quercetin or dimethyl sulfoxide / A.A. Philchenkov, M.P. Zavelevich, V.M. Mikhailenko, L.M. Kuyava // Укр. біохім. журн. — 2010. — Т. 82, № 2. — С. 104-110. — Бібліогр.: 27 назв. — англ. |
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Digital Library of Periodicals of National Academy of Sciences of Ukraine| _version_ | 1860083737614614528 |
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| author | Philchenkov, A.A. Zavelevich, M.P. Mikhailenko, V.M. Kuyava, L.M. |
| author_facet | Philchenkov, A.A. Zavelevich, M.P. Mikhailenko, V.M. Kuyava, L.M. |
| citation_txt | Apoptosis and content of mobile lipid domains in human leukemia K-562 cells induced to differentiate by quercetin or dimethyl sulfoxide / A.A. Philchenkov, M.P. Zavelevich, V.M. Mikhailenko, L.M. Kuyava // Укр. біохім. журн. — 2010. — Т. 82, № 2. — С. 104-110. — Бібліогр.: 27 назв. — англ. |
| collection | DSpace DC |
| container_title | Український біохімічний журнал |
| description | The apoptotic index, cell cycle progression and caspase-3 activation in K-562 cells induced to differentiate by DMSO or quercetin have been studied. Quercetin treatment of K-562 cells was accompanied by cell cycle arrest in G2 /M and apoptosis with caspase-3 activation. In contrast, DMSO-induced differentiation was accompanied by the complete cell cycle arrest in G1 /G0 with negligible caspase-3 activation. In spite of the appearance of benzidine-positive cells and the decreased CD71 level in K-562 cells after exposure to quercetin, the analysis of 1H NMR spectra revealed the overall balance in favor of apoptosis, namely the increase in the content of NMR-visible mobile lipid domains and the decreased intensity of choline-containing metabolites.
Визначали апоптотичний індекс, проходження клітинного циклу та активацію каспази-3 в клітинах K-562 в разі диференціювання, індукованого кверцетином або диметилсульфоксидом (ДМСО). Обробка клітин кверцетином супроводжувалась зупинкою клітинного циклу в фазі G2/M та призводила до апоптозу і активації каспази-3. Навпаки, в разі диференціювання, індукованого ДМСО, спостерігали повну зупинку клітинного циклу у фазі G1/G0 без активації каспази-3. Незважаючи на появу бензидин-позитивних клітин та зниження вмісту CD71 після інкубації з кверцетином, аналіз спектрів 1H ЯМР виявив баланс змін у бік апоптозу, а саме збільшення вмісту ЯМР-візуалізованих мобільних ліпідних доменів та зниження інтенсивності сигналу холін-вмісних метаболітів.
Определяли апоптотический индекс, прохождение клеточного цикла и активацию каспазы-3 в клетках K-562 при индукции дифференцировки кверцетином или диметилсульфоксидом (ДМСО). Обработка клеток кверцетином сопровождалась остановкой клеточного цикла в фазе G2 /M и активацией каспазы-3. В противоположность этому при дифференцировке, индуцированной ДМСО, отмечали полную остановку клеточного цикла в фазе G1 /G0 без активации каспазы-3. Несмотря на появление бензидин-положительных клеток и снижение содержания CD71 после инкубации с кверцетином, анализ спектров 1H ЯМР выявил баланс изменений в сторону апоптоза, а именно, увеличение содержания ЯМР-визуализируемых мобильных липидных доменов и снижение интенсивности сигнала холин-содержащих метаболитов.
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ISSN 0201 — 8470. Укр. біохім. журн., 2010, т. 82, № 2104
УДК 577.152.3 + 576.38
apoptosis and content of mobile lipid
domains in human leukemia k-562 cells induced
to differentiate by quercetin or dimethyl sulfoxide
A. A. PhIlcheNkov, M. P. ZAvelevIch, v. M. MIkhAIleNko, l. M. kuyAvA
R.e. kavetsky Institute of experimental Pathology, oncology and Radiobiology,
National Academy of Sciences of ukraine, kyiv;
e-mail: a_philch@onconet.kiev.ua
The apoptotic index, cell cycle progression and caspase-3 activation in k-562 cells induced to differen-
tiate by DMSo or quercetin have been studied. Quercetin treatment of k-562 cells was accompanied by cell
cycle arrest in G2 /M and apoptosis with caspase-3 activation. In contrast, DMSo-induced differentiation was
accompanied by the complete cell cycle arrest in G1 /G0 with negligible caspase-3 activation. In spite of the ap-
pearance of benzidine-positive cells and the decreased cD71 level in k-562 cells after exposure to quercetin,
the analysis of 1h NMR spectra revealed the overall balance in favor of apoptosis, namely the increase in the
content of NMR-visible mobile lipid domains and the decreased intensity of choline-containing metabolites.
k e y w o r d s: apoptosis, caspase-3, cell cycle, chronic myelogenous leukemia, DMSo, flavonoids,
mobile lipid domains, quercetin.
The induction of leukemic cell differentia-
tion may provide an alternative approach
to the conventional cytotoxic chemotherapy
of leukemia [1]. Nevertheless, the application of
differentiation therapy in leukemia patients is cur-
rently limited only to several forms of leukemias.
Meanwhile, the precise mechanisms of pharmaco-
logically induced differentiation of leukemic cells
and their relations to apoptotic cell pathways are
not well understood.
Quercetin (3,3′,4′,5,7-pentahydroxy flavone),
which is one of the most widely distributed fla-
vonoids in nature, has been reported to possess
antiproliferative and proapoptotic activities in
leukemic cells [2–4]. These activities do not seem
to be related only to the conventional antioxidant
properties of this substance. Several mechanisms
of the apoptogenic effects of quercetin have been
suggested, with death receptor-5, anti-apoptotic
Bcl-2 and Bcl-xL proteins, caspases, Akt/PKB
kinase, Cu-Zn superoxide dismutase, fatty acid
synthase and heat shock proteins being among the
cellular targets [5–7]. Few reports have shown that
quercetin may also induce differentiation in hu-
man chronic myelogenous leukemia [8, 9]. Quer-
cetin was also shown to induce apoptosis in K562
and several other human leukemic cell lines [10].
Nevertheless, the relations between apoptosis and
differentiations in quercetin-treated cells as well as
the mechanisms of quercetin-triggered differentia-
tion have not been studied in detail.
Abundant experimental evidence has demon-
strated that besides their role in the initiation and
execution of apoptosis, caspases have been also
involved in such physiological processes as cell
survival, proliferation, differentiation, and inflam-
mation [11, 12]. Several data suggest the roles of
caspases in erythroid differentiation in the bone
marrow [13, 14].
The dual effect of quercetin as well as some
other flavonoids inducing both apoptosis and cell
differentiation makes it rather difficult to analyze
these effects separately in cell populations. The use
of the approaches allowing one to assess the overall
effects may be advantageous for characterizing the
balance of these two processes in cell populations.
While 1H NMR has been shown to detect the apop-
totic changes in cell membranes by the shifts in
the content of NMR-visible mobile lipid domains
and choline-containing metabolite intensity [15],
the changes in 1H NMR detectable parameters in
the systems of cell differentiation have not yet been
followed.
Therefore, the major objective of the study
was to compare the apoptosis induction, cell cyc-
le progression and caspase-3 activation in K-562
cells induced to differentiate by dimethyl sulfoxide
(DMSO) or quercetin. We also have attempted to
analyze the patterns of 1H NMR spectra, namely
the content of NMR-visible mobile lipid domains
and choline-containing metabolite intensity in
cells undergoing apoptosis or differentiation with
експериментальні роботи
ISSN 0201 — 8470. Укр. біохім. журн., 2010, т. 82, № 2 105
the aim of assessing the overall balance of apopto-
sis and differentiation in K-562 cells treated with
quercetin.
materials and methods
Chemicals. Quercetin purchased from Sigma
Chem. Co. (USA) was dissolved in 96% ethanol
and further diluted in culture medium. Vepeside
was purchased from Brystol-Myers Squibb SpA
(Italy).
Cell culture and treatment. Human K-562
cell line was obtained from the National Collec-
tion of Cell Lines of the Institute of Experimen-
tal Pathology, Oncology and Radiobiology (Kyiv,
Ukraine). Quercetin or DMSO was added to the
cells at the beginning of the exponential growth
phase. Cell growth and viability were assessed by
direct counting of trypan blue dye-excluding cells.
The percentage of hemoglobin-producing cells was
determined by a benzidine staining method as pre-
viously described [16].
Proton NMR-spectroscopy. The high-reso-
lution 1H NMR spectra were acquired using a
300 MHz Varian Mercury 300BB NMR spectrome-
ter (Varian, USA). The details of the technique
were described [17]. The areas of signals at 0.9 and
1.3 ppm arising from –CH3 and (–CH2–)n groups
of fatty acyl chains of lipids as well as signals at
3.2 ppm arising from choline-containing metabo-
lites (Cho), which include choline and phospho-
rylcholine, were integrated using VNMR software
(Varian, USA) and expressed in relative units.
Flow cytometric analysis. A) cell cycle distri-
bution and apoptosis. The cells were resuspended in
hypotonic buffer containing 0.1% sodium citrate,
0.1% Triton X-100, 5 μg/ml propidium iodide.
250 μg/ml of RNAse A was added to each sample,
and the cells were stained for 15 min at 37 °C.
Flow cytometry was performed on a FACScan
automated system (Becton Dickinson, USA), and
data were analyzed using CellQuest software packa-
ge and ModFit LT 2.0 program.
B) Immunostaining. The percentage of cells
with active form of caspase-3 was assessed using
FITC-conjugated MAb active caspase-3 kit (BD
Biosciences, USA) according to the manufacturer’s
recommendations. Expression of CD71 on the sur-
face of K-562 cells was evaluated by flow cytomet-
ry as described previously [18].
Statistical analysis. Each experiment was rep-
licated three times. The results of the experimental
and control groups were tested for statistical sig-
nificance by a one-tailed Student’s t test.
results and discussion
First, the effects of quercetin and DMSO
on cell growth, cell cycle and differentiation were
compared. K-562 cells were treated with quercetin
at concentrations up to 40 μM or 1.9% DMSO.
Quercetin slightly inhibited cell proliferation,
while DMSO suppressed completely cell growth
without increasing cell death. In cells treated with
quercetin, the increase in hypodiploid cell frac-
tion up to 15% was evident. Meanwhile, the cells
grown in the presence of DMSO have shown no
increase in apoptotic cell fraction (Fig. 1, A). As
shown in Fig. 1, B, quercetin treatment for 48 h re-
sulted in relatively moderate increase (about 25%)
in the percentage of cells with the active form of
caspase-3. In contrast, DMSO treatment has not
induced caspase-3 activation relative to baseline
level. In addition, the opposite effects of quercetin
and DMSO on cell cycle traverse have been shown.
Namely, DMSO induced complete arrest in G0/G1
phase, while quercetin suppressed the growth of K-
562 cells by blocking the cell cycle predominantly
in G2 /M phase (Fig. 2).
In contrast to DMSO-induced differentia-
tion of K-562 cells involving practically the whole
cell population, morphological and cytochemical
analysis of quercetin-treated cells reveal only par-
tial features of erythroid differentiation in 15–20%
of cells. Nevertheless, the differentiation effect of
quercetin was confirmed by the increasing percenta-
ge of hemoglobin-containing (benzidine-positive)
cells (38 ± 4% over the baseline level 16 ± 2%).
We next investigated the expression of cell sur-
face transferrin receptor (CD71) as a non-lineage
specific marker of early erythroid progenitor cells
in the course of DMSO- or quercetin-induced dif-
ferentiation of K-562 cells. CD71 is known to be
lost as these cells differentiate to mature eryth-
rocytes. Flow cytometric analysis demonstrated
strong decrease in the percentage of CD71-positive
cells upon DMSO treatment: in 72 h transferrin
receptor was practically undetectable (Fig. 3). In
quercetin-treated cells, slight reduction of the per-
centage of CD71-positive cells was observed (75%
at 48 h as compared with the basal level). The
differences between DMSO and quercetin treat-
ments in CD71 are quite in line with morphologi-
cal changes and assessment of benzidine-positive
cells – therefore indicating only partial differen-
tiation within cell population and in a different cell
cycle setting.
Since the findings above suggest that the
differentiation of K562 cells induced by querce-
tin but not DMSO is accompanied by apoptosis,
A. A. PhIlcheNkov, M. P. ZAvelevIch, v. M. MIkhAIleNko, l. M. kuyAvA
ISSN 0201 — 8470. Укр. біохім. журн., 2010, т. 82, № 2106
it would be of interest to assess the overall balan-
ce of differentiation and apoptosis in this system
by 1H NMR analysis. Some representative spec-
tra given in Fig. 4 showed 1.6-fold decrease of the
CH2/CH3 signal intensity ratio corresponding to
MLD content and 2.4-fold increase of the inten-
sity of Cho resonance in K-562 cells treated with
DMSO. In contrast, the CH2/CH3 signal intensity
ratio in the same cells treated with vepeside as a
typical apoptosis inducer increased about two-fold,
while the intensity of Cho resonance was thrice as
low as that in untreated cells. The analysis of 1H
NMR spectra in K-562 cells exposed to quercetin
revealed the overall balance of changes in favor of
apoptosis, namely the 1.6-fold increase in MLD
content and 1.8-fold decrease in Cho resonance
intensity as compared to the control values.
The human K562 cell line has been used
widely as a valuable model for erythroid differen-
tiation in vitro which is induced by various substan-
ces. The intrinsic commitment of at least part of
K562 cell population to differentiate has been also
Fig. 1. Induction of apoptosis (A) and activation of caspase-3 (B) in k-562 cells treated with quercetin or
DMSo. a – control; b – 40 μM quercetin, 48 h; c – 1.9% DMSo, 48 h. The figures show a representative
staining profile for 10,000 cells per experiment. M1 is the cell population defined as apoptotic (A) or caspase-
3-positive (B)
Fig. 1. Induction of apoptosis (A) and activation of caspase-3 (B) in K-562 cells treated with
quercetin or DMSO. Each experiment was done in triplicate. a – control; b – 40 M quercetin,
48 h; c – 1.9% DMSO, 48 h. The figures show a representative staining profile for 10,000 cells
per experiment. M1 is the cell population defined as apoptotic (A) or caspase-3-positive (B).
3.3% 4.0%15.2%
25.22% 4.21% 8.78%
A
B
Ev
en
ts
A c
Ev
en
ts
Ev
en
ts
Fig. 1. Induction of apoptosis (A) and activation of caspase-3 (B) in K-562 cells treated with
quercetin or DMSO. Each experiment was done in triplicate. a – control; b – 40 M quercetin,
48 h; c – 1.9% DMSO, 48 h. The figures show a representative staining profile for 10,000 cells
per experiment. M1 is the cell population defined as apoptotic (A) or caspase-3-positive (B).
3.3% 4.0%15.2%
25.22% 4.21% 8.78%
A
B
Fig. 1. Induction of apoptosis (A) and activation of caspase-3 (B) in K-562 cells treated with
quercetin or DMSO. Each experiment was done in triplicate. a – control; b – 40 M quercetin,
48 h; c – 1.9% DMSO, 48 h. The figures show a representative staining profile for 10,000 cells
per experiment. M1 is the cell population defined as apoptotic (A) or caspase-3-positive (B).
3.3% 4.0%15.2%
25.22% 4.21% 8.78%
A
B
FL2-H FL2-H FL2-H
100 101 102 103 104
M1
0
25
6
3.3%
a
12
8 25
6
0 0
M1
15.2% 4.0%
M1
b
100 101 102 103 104 100 101 102 103
B
Fig. 1. Induction of apoptosis (A) and activation of caspase-3 (B) in K-562 cells treated with
quercetin or DMSO. Each experiment was done in triplicate. a – control; b – 40 M quercetin,
48 h; c – 1.9% DMSO, 48 h. The figures show a representative staining profile for 10,000 cells
per experiment. M1 is the cell population defined as apoptotic (A) or caspase-3-positive (B).
3.3% 4.0%15.2%
25.22% 4.21% 8.78%
A
B
c
Ev
en
ts
FL1-H
Fig. 1. Induction of apoptosis (A) and activation of caspase-3 (B) in K-562 cells treated with
quercetin or DMSO. Each experiment was done in triplicate. a – control; b – 40 M quercetin,
48 h; c – 1.9% DMSO, 48 h. The figures show a representative staining profile for 10,000 cells
per experiment. M1 is the cell population defined as apoptotic (A) or caspase-3-positive (B).
3.3% 4.0%15.2%
25.22% 4.21% 8.78%
A
B
Ev
en
ts
FL1-H
100 101 102 103 104
M1
0
12
8
4.21%
a
12
8
0
6.78%
M1
100 101 102 103 104
Fig. 1. Induction of apoptosis (A) and activation of caspase-3 (B) in K-562 cells treated with
quercetin or DMSO. Each experiment was done in triplicate. a – control; b – 40 M quercetin,
48 h; c – 1.9% DMSO, 48 h. The figures show a representative staining profile for 10,000 cells
per experiment. M1 is the cell population defined as apoptotic (A) or caspase-3-positive (B).
3.3% 4.0%15.2%
25.22% 4.21% 8.78%
A
B
Ev
en
ts
FL1-H
12
8
0
M1
25.22%
b
100 101 102 103 104
експериментальні роботи
ISSN 0201 — 8470. Укр. біохім. журн., 2010, т. 82, № 2 107
Fig. 2. cell cycle analysis of k-562 cells after 48 h treatment with quercetin or DMSo: a – control; b – 40 μM
quercetin; c – 1.9% DMSo. cells were stained with PI for flow cytometry analysis and DNA histograms were
plotted with ModFit software
Treatment
Cell cycle phase distribution in %
G0 /G1 S G2 /M
Control 37.92 ± 0.87 57.78 ± 0.41 4.29 ± 0.29
Quercetin 40 μM 15.48 ± 0.73 60.30 ± 0.54 24.22 ± 0.25
DMSO 1.9% 77.24 ± 0.26 15.04 ± 0.39 7.73 ± 0.95
Fig. 2. Cell cycle analysis of K-562 cells after 48 h treatment with quercetin or DMSO. a –
control; b – 40 M quercetin; c – 1.9% DMSO. Cells were stained with PI for flow cytometry
analysis and DNA histograms were modeled with ModFit software.
Cell cycle phase distribution in % Treatment
G0/G1 S G2/M
Control 37.92 ± 0.87 57.78 ± 0.41 4.29 ± 0.29
Quercetin 40 M 15.48 ± 0.73 60.30 ± 0.54 24.22 ± 0.25
DMSO 1.9% 77.24 ± 0.26 15.04 ± 0.39 7.73 ± 0.95
Fig. 2. Cell cycle analysis of K-562 cells after 48 h treatment with quercetin or DMSO. a –
control; b – 40 M quercetin; c – 1.9% DMSO. Cells were stained with PI for flow cytometry
analysis and DNA histograms were modeled with ModFit software.
Cell cycle phase distribution in % Treatment
G0/G1 S G2/M
Control 37.92 ± 0.87 57.78 ± 0.41 4.29 ± 0.29
Quercetin 40 M 15.48 ± 0.73 60.30 ± 0.54 24.22 ± 0.25
DMSO 1.9% 77.24 ± 0.26 15.04 ± 0.39 7.73 ± 0.95
C
ou
nt
C
ou
nt
Channels
0 40 80 120 160 200
a
800
C
ou
nt
Fig. 2. Cell cycle analysis of K-562 cells after 48 h treatment with quercetin or DMSO. a –
control; b – 40 M quercetin; c – 1.9% DMSO. Cells were stained with PI for flow cytometry
analysis and DNA histograms were modeled with ModFit software.
Cell cycle phase distribution in % Treatment
G0/G1 S G2/M
Control 37.92 ± 0.87 57.78 ± 0.41 4.29 ± 0.29
Quercetin 40 M 15.48 ± 0.73 60.30 ± 0.54 24.22 ± 0.25
DMSO 1.9% 77.24 ± 0.26 15.04 ± 0.39 7.73 ± 0.95
Channels
0 40 80 120 160 200
Channels
0 40 80 120 160 200
cb
800
800
Fig. 3. cell surface transferrin receptor expression in k-562 cells treated with 1.9% DMSo (A) or 40 μM
quercetin (B): a – control; b – 48 h; c – 72 h. Black peaks (d) show negative control (anti-transferrin receptor
antibody is omitted). The figures show a representative staining profile for 10,000 cells per experiment. The log
fluorescence intensity is shown on the horizontal axis. on the vertical axis the cell number is plotted
Fig. 3. Cell surface transferrin receptor expression in K-562 cells treated with 1.9% DMSO (A)
or 40 M quercetin (B). a – control; b – 48 h; c – 72 h. Black peaks (d) show negative control
(anti-transferrin receptor antibody is omitted). The figures show a representative staining profile
for 10,000 cells per experiment. The log fluorescence intensity is shown on the horizontal axis.
On the vertical axis the cell number is plotted.
Fig. 3. Cell surface transferrin receptor expression in K-562 cells treated with 1.9% DMSO (A)
or 40 M quercetin (B). a – control; b – 48 h; c – 72 h. Black peaks (d) show negative control
(anti-transferrin receptor antibody is omitted). The figures show a representative staining profile
for 10,000 cells per experiment. The log fluorescence intensity is shown on the horizontal axis.
On the vertical axis the cell number is plotted.
A B
a
b
c
d
a
b
c
d
demonstrated [19]. Molecular mechanisms of such
differentiation induced by unrelated agents and
their relevance to the normal process of erythroid
differentiation have been still far from being elu-
cidated. The existence of various regulatory path-
ways involved in the final differentiation effects
has not been excluded. Among the agents capable
of inducing the differentiation of K562 cells are
cytotoxic chemotherapeutics drugs as well, which
induce differentiation at the subtoxic doses [20].
Therefore, the relation between the differentiation
and the involvement of the apoptothic pathway
mechanisms is still to be investigated. Nevertheless,
several authors believe that apoptosis and erythroid
differentiation of chronic myelogenous leukemia
cells represent the different processes, which may
be easily dissociated [21]. Although some inves-
tigators suggested that quercetin can trigger dif-
A. A. PhIlcheNkov, M. P. ZAvelevIch, v. M. MIkhAIleNko, l. M. kuyAvA
ISSN 0201 — 8470. Укр. біохім. журн., 2010, т. 82, № 2108
Fig. 4. Representative high-resolution 300 Mhz spectra of k-562 cells. 1 – cells treated with DMSo (1.9%,
18 h); 2 – control; 3 – cells treated with quercetin (40 μM, 48 h); 4 – cells treated with vepeside (20 μM,
18 h)
ferentiation in K-562 cells [8, 9], this could not be
confirmed by others [22]. Furthermore, in some
cases quercetin was shown to inhibit the environ-
mental stress-induced differentiation of K-562 cells
[23].
Our studies demonstrated some features of
erythroid differentiation of K562 cells induced by
quercetin. In particular, the exposure to quercetin
resulted in the increase of benzidine-positive cell
count in K-562 culture over the baseline level.
Treatment of those cells with either DMSO or
quercetin downregulated cell surface transferrin
receptors, which may be important in causing
cessation of transferrin-mediated iron transport
required for cell growth.
Nevertheless, the features of differentiation
of K562 cells induced by quercetin are distinct
from DMSO-induced differentiation. The most
important is that quercetin-induced differentiation
is accompanied by caspase-3 activation and
apoptosis in a fraction of cell population. This result
is in agreement with the data obtained in human
acute myelogenous or promyelocytic leukemia cells
treated with flavonoids [4, 24]. In contrast, DMSO-
induced differentiation was not accompanied by
Fig. 4. Representative high-resolution 300 MHz spectra of K-562 cells. 1 – cells treated with
DMSO (1.9%, 18 h); 2 – control; 3 – cells treated with quercetin (40 M, 48 h); 4 – cells treated
with vepeside (20 M, 18 h).
1
2
3
4
caspase-3 activation suggesting that at least in this
system caspase-3 is not involved in differentiation
pathways. While quercetin-treated K-562 cells are
arrested in G2/M transition phase, DMSO blocked
cell cycle progression in G0 /G1 phase. However, it
is not possible to demonstrate whether caspase-3
activation in quercetin-treated cells is associated
with the fraction of cells undergoing differentiation
or apoptosis.
It is known that flavonoids may induce both
apoptosis and differentiation in leukemic cells [25].
Therefore, we have attempted to assess the overall
balance of apoptosis and differentiation in cell
population with the aid of 1H NMR-spectroscopy.
An increased 1H NMR-visible MLD formation
has been reported as a peculiar feature of cells
undergoing apoptosis or proliferating cells [15].
Earlier, we have observed MLD accumulation and
Cho decrease in apoptotic malignant lymphoid cells
upon quercetin treatment [17]. In the present study,
the opposite spectral changes in cells undergoing
erythroid differentiation were identified. Namely,
DMSO-induced differentiation in K-562 cells was
accompanied by the decrease of the CH2/CH3 signal
intensity ratio and the marked increase of Cho
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ISSN 0201 — 8470. Укр. біохім. журн., 2010, т. 82, № 2 109
resonance intensity being evident as early as 18 h
after the onset of differentiation. These data are in
line with those of Agris and Campbell [26] who
found a dramatic increase of a signal at 3.2 ppm
in Friend leukemia cells induced to differentiate
by DMSO. Furthermore, it was proposed that
an increased phosphorylcholine level might be a
marker of differentiation in diverse cell systems
(see in [27]). According to our data, the decrease
in 1H NMR-visible MLD formation may be
regarded as one of the features characteristic of the
increased differentiation status of the cells, at least
in the system of DMSO-induced differentiation in
K-562 cells. As to quercetin treatment of K-562
cells, the data on MLD content suggest the overall
shift towards apoptosis.
To sum up, our results demonstrated that
quercetin in contrast to DMSO induced both apop-
tosis and erythroid differentiation in K-562 cells
with the apoptotic trend in the overall effects. The
potential role of the apoptotic effectors in the con-
trol of cell differentiation merits further investiga-
tion using other differentiating agents.
This work was partly supported by the grant
from the research program ‘‘Fundamentals of Ge-
nomics and Proteomics’’ No. 107U002244 of Na-
tional Academy of Sciences of Ukraine. We would
like to thank Dr. N. Khranovskaya for her help
with flow cytometry.
апоптоз та вміст мобільних
ліпідних доменів в клітинах
к-562 лейкемії людини при
їхньому диференціюванні,
індукованому кверцетином
або диметилсульфоксидом
о. о. Фільченков, м. п. Завелевич,
В. м. михайленко, л. м. куява
Інститут експериментальної
патології, онкології та радіобіології
ім. Р. Є. Кавецького НАН України, Київ;
e-mail: a_philch@onconet.kiev.ua
Визначали апоптотичний індекс, прохо-
дження клітинного циклу та активацію каспа-
зи-3 в клітинах K-562 в разі диференціювання,
індукованого кверцетином або диметилсуль-
фоксидом (ДМСО). Обробка клітин кверцети-
ном супроводжувалась зупинкою клітинного
циклу в фазі G2/M та призводила до апоптозу і
активації каспази-3. Навпаки, в разі диферен-
ціювання, індукованого ДМСО, спостерігали
повну зупинку клітинного циклу у фазі G1/G0
без активації каспази-3. Незважаючи на поя-
ву бензидин-позитивних клітин та зниження
вмісту CD71 після інкубації з кверцетином,
аналіз спектрів 1H ЯМР виявив баланс змін у
бік апоптозу, а саме збільшення вмісту ЯМР-
візуалізованих мобільних ліпідних доменів та
зниження інтенсивності сигналу холін-вміс-
них метаболітів.
К л ю ч о в і с л о в а: апоптоз, каспаза-3,
клітинний цикл, хронічна мієлоїдна лейкемія,
ДМСО, флавоноїди, мобільні ліпідні домени,
кверцетин.
апоптоз и содержание
мобильных липидных
доменов в клетках к-562
лейкоза человека при их
дифференцировке,
индуцированной кверцетином
или диметилсульфоксидом
а. а. Фильченков, м. п. Завелевич,
В. м. михайленко, л. м. куява
Институт экспериментальной
патологии, онкологии и радиобиологии
им. Р. Е. Кавецкого НАН Украины, Киев;
e-mail: a_philch@onconet.kiev.ua
Определяли апоптотический индекс,
прохождение клеточного цикла и активацию
каспазы-3 в клетках K-562 при индукции диф-
ференцировки кверцетином или диметилсуль-
фоксидом (ДМСО). Обработка клеток кверце-
тином сопровождалась остановкой клеточного
цикла в фазе G2 /M и активацией каспазы-3.
В противоположность этому при дифферен-
цировке, индуцированной ДМСО, отмечали
полную остановку клеточного цикла в фазе
G1 /G0 без активации каспазы-3. Несмотря на
появление бензидин-положительных клеток и
снижение содержания CD71 после инкубации
с кверцетином, анализ спектров 1H ЯМР вы-
явил баланс изменений в сторону апоптоза, а
именно, увеличение содержания ЯМР-визуа-
лизируемых мобильных липидных доменов и
снижение интенсивности сигнала холин-со-
держащих метаболитов.
К л ю ч е в ы е с л о в а: апоптоз, каспаза-3,
клеточный цикл, хронический миелолейкоз,
ДМСО, флавоноиды, мобильные липидные
домены, кверцетин.
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експериментальні роботи
|
| id | nasplib_isofts_kiev_ua-123456789-19047 |
| institution | Digital Library of Periodicals of National Academy of Sciences of Ukraine |
| issn | 0201-8470 |
| language | English |
| last_indexed | 2025-12-07T17:18:14Z |
| publishDate | 2010 |
| publisher | Інститут біохімії ім. О.В. Палладіна НАН України |
| record_format | dspace |
| spelling | Philchenkov, A.A. Zavelevich, M.P. Mikhailenko, V.M. Kuyava, L.M. 2011-04-16T18:09:12Z 2011-04-16T18:09:12Z 2010 Apoptosis and content of mobile lipid domains in human leukemia K-562 cells induced to differentiate by quercetin or dimethyl sulfoxide / A.A. Philchenkov, M.P. Zavelevich, V.M. Mikhailenko, L.M. Kuyava // Укр. біохім. журн. — 2010. — Т. 82, № 2. — С. 104-110. — Бібліогр.: 27 назв. — англ. 0201-8470 https://nasplib.isofts.kiev.ua/handle/123456789/19047 577.152.3 + 576.38 The apoptotic index, cell cycle progression and caspase-3 activation in K-562 cells induced to differentiate by DMSO or quercetin have been studied. Quercetin treatment of K-562 cells was accompanied by cell cycle arrest in G2 /M and apoptosis with caspase-3 activation. In contrast, DMSO-induced differentiation was accompanied by the complete cell cycle arrest in G1 /G0 with negligible caspase-3 activation. In spite of the appearance of benzidine-positive cells and the decreased CD71 level in K-562 cells after exposure to quercetin, the analysis of 1H NMR spectra revealed the overall balance in favor of apoptosis, namely the increase in the content of NMR-visible mobile lipid domains and the decreased intensity of choline-containing metabolites. Визначали апоптотичний індекс, проходження клітинного циклу та активацію каспази-3 в клітинах K-562 в разі диференціювання, індукованого кверцетином або диметилсульфоксидом (ДМСО). Обробка клітин кверцетином супроводжувалась зупинкою клітинного циклу в фазі G2/M та призводила до апоптозу і активації каспази-3. Навпаки, в разі диференціювання, індукованого ДМСО, спостерігали повну зупинку клітинного циклу у фазі G1/G0 без активації каспази-3. Незважаючи на появу бензидин-позитивних клітин та зниження вмісту CD71 після інкубації з кверцетином, аналіз спектрів 1H ЯМР виявив баланс змін у бік апоптозу, а саме збільшення вмісту ЯМР-візуалізованих мобільних ліпідних доменів та зниження інтенсивності сигналу холін-вмісних метаболітів. Определяли апоптотический индекс, прохождение клеточного цикла и активацию каспазы-3 в клетках K-562 при индукции дифференцировки кверцетином или диметилсульфоксидом (ДМСО). Обработка клеток кверцетином сопровождалась остановкой клеточного цикла в фазе G2 /M и активацией каспазы-3. В противоположность этому при дифференцировке, индуцированной ДМСО, отмечали полную остановку клеточного цикла в фазе G1 /G0 без активации каспазы-3. Несмотря на появление бензидин-положительных клеток и снижение содержания CD71 после инкубации с кверцетином, анализ спектров 1H ЯМР выявил баланс изменений в сторону апоптоза, а именно, увеличение содержания ЯМР-визуализируемых мобильных липидных доменов и снижение интенсивности сигнала холин-содержащих метаболитов. This work was partly supported by the grant from the research program ‘‘Fundamentals of Genomics and Proteomics’’ No. 107U002244 of National Academy of Sciences of Ukraine. We would like to thank Dr. N. Khranovskaya for her help with flow cytometry. en Інститут біохімії ім. О.В. Палладіна НАН України Український біохімічний журнал Експериментальні роботи Apoptosis and content of mobile lipid domains in human leukemia K-562 cells induced to differentiate by quercetin or dimethyl sulfoxide Апоптоз та вміст мобільних ліпідних доменів в клітинах К-562 лейкемії людини при їхньому диференціюванні, індукованому кверцетином або диметилсульфоксидом Апоптоз и содержание мобильных липидных доменов в клетках К‑562 лейкоза человека при их дифференцировке, индуцированной кверцетином или диметилсульфоксидом Article published earlier |
| spellingShingle | Apoptosis and content of mobile lipid domains in human leukemia K-562 cells induced to differentiate by quercetin or dimethyl sulfoxide Philchenkov, A.A. Zavelevich, M.P. Mikhailenko, V.M. Kuyava, L.M. Експериментальні роботи |
| title | Apoptosis and content of mobile lipid domains in human leukemia K-562 cells induced to differentiate by quercetin or dimethyl sulfoxide |
| title_alt | Апоптоз та вміст мобільних ліпідних доменів в клітинах К-562 лейкемії людини при їхньому диференціюванні, індукованому кверцетином або диметилсульфоксидом Апоптоз и содержание мобильных липидных доменов в клетках К‑562 лейкоза человека при их дифференцировке, индуцированной кверцетином или диметилсульфоксидом |
| title_full | Apoptosis and content of mobile lipid domains in human leukemia K-562 cells induced to differentiate by quercetin or dimethyl sulfoxide |
| title_fullStr | Apoptosis and content of mobile lipid domains in human leukemia K-562 cells induced to differentiate by quercetin or dimethyl sulfoxide |
| title_full_unstemmed | Apoptosis and content of mobile lipid domains in human leukemia K-562 cells induced to differentiate by quercetin or dimethyl sulfoxide |
| title_short | Apoptosis and content of mobile lipid domains in human leukemia K-562 cells induced to differentiate by quercetin or dimethyl sulfoxide |
| title_sort | apoptosis and content of mobile lipid domains in human leukemia k-562 cells induced to differentiate by quercetin or dimethyl sulfoxide |
| topic | Експериментальні роботи |
| topic_facet | Експериментальні роботи |
| url | https://nasplib.isofts.kiev.ua/handle/123456789/19047 |
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