Glioma-associated protein CHI3L2 suppresses cells viability and induces G1/S transition arrest
Aim. To analyze the effect of the CHI3L2 protein on malignant and non-malignant cell viability, and determined the CHI3L2 impact on the cell cycle and signaling pathways involved in the cell cycle regulation. Methods. MTT-based cell proliferation assay, FACS, western blot analysis. Results. The CHI3...
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irk-123456789-1526122019-06-13T01:25:13Z Glioma-associated protein CHI3L2 suppresses cells viability and induces G1/S transition arrest Avdieiev, S.S. Gera, L. Hodges, R. Dmytrenko, V.V. Short Communications Aim. To analyze the effect of the CHI3L2 protein on malignant and non-malignant cell viability, and determined the CHI3L2 impact on the cell cycle and signaling pathways involved in the cell cycle regulation. Methods. MTT-based cell proliferation assay, FACS, western blot analysis. Results. The CHI3L2 protein inhibits the glioma cells viability and potentiates the effect of anti-cancer cytotoxic agents. The CHI3L2 treatment results in the G1/S transition arrest. CHI3L2 provoked a dramatic reduction of pRB phosphorylation and a significant decrease in the cyclin D1 expression, whereas the p53 and p21 expression levels were substantially increased. Conclusions. The CHI3L2 protein, which is overexpressed in human gliomas, is a negative regulator of the glioma cells viability. The reduced cell viability after the CHI3L2 treatment could be due to the activation of pRB and p53 and the downregulation of cyclin D. Мета. Проаналізувати життєздатність злоякісних та незлоякісних клітин за дії протеїна CHI3L2, а також визначити вплив CHI3L2 на клітинний цикл і сигнальні шляхи, залучені до його регуляції. Методи. МТТ–тест, проточна цитофлуориметрія, вестерн блот аналіз. Результати. CHI3L2 пригнічує життєздатність клітин гліоми людини і підсилює дію протиракових цитотоксичних агентів. CHI3L2 інгібує перехід клітин із G1- до S-фази клітинного циклу. CHI3L2 зумовлює зниження кількості фосфорильованої форми pRB, зменшення вмісту цикліну D1 та зростання вмісту p53 і p21. Висновки. CHI3L2, що надекспресується в гліомах людини, є негативним регулятором життєздатності клітин гліоми. Цитотоксичний вплив CHI3L2 може бути пов'язаним з активацією pRB та р53, а також зниженням вмісту цикліну D. Цель. Проанализировать жизнеспособность злокачественных и незлокачественных клеток при воздействии протеина CHI3L2, а также определить влияние CHI3L2 на клеточный цикл и сигнальные пути, вовлеченные в его регуляцию. Методы. МТТ-тест, проточная цитофлуориметрия, вестерн блот анализ. Результаты. CHI3L2 подавляет жизнеспособность клеток глиомы человека и усиливает действие противораковых цитотоксических агентов. CHI3L2 ингибирует переход клеток с G1- в S-фазу клеточного цикла. Влияние CHI3L2 приводит к снижению количества фосфорилированной формы pRB, уменьшению содержания циклина D1 и увеличению содержания p53 и p21. Выводы. CHI3L2, оверэкспрессия которого характерна для глиом человека, является негативным регулятором жизнеспособности клеток глиомы. Цитотоксическое влияние CHI3L2 может быть связано с активацией pRB и р53, а также снижением содержания циклина D. 2015 Article Glioma-associated protein CHI3L2 suppresses cells viability and induces G1/S transition arrest / S.S. Avdieiev, L. Gera, R. Hodges, V.V. Dmytrenko // Вiopolymers and Cell. — 2015. — Т. 31, № 4. — С. 316-320. — Бібліогр.: 8 назв. — англ. 0233-7657 DOI: http://dx.doi.org/10.7124/bc.0008F1 http://dspace.nbuv.gov.ua/handle/123456789/152612 576.322 577.22 en Вiopolymers and Cell Інститут молекулярної біології і генетики НАН України |
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Short Communications Short Communications |
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Short Communications Short Communications Avdieiev, S.S. Gera, L. Hodges, R. Dmytrenko, V.V. Glioma-associated protein CHI3L2 suppresses cells viability and induces G1/S transition arrest Вiopolymers and Cell |
| description |
Aim. To analyze the effect of the CHI3L2 protein on malignant and non-malignant cell viability, and determined the CHI3L2 impact on the cell cycle and signaling pathways involved in the cell cycle regulation. Methods. MTT-based cell proliferation assay, FACS, western blot analysis. Results. The CHI3L2 protein inhibits the glioma cells viability and potentiates the effect of anti-cancer cytotoxic agents. The CHI3L2 treatment results in the G1/S transition arrest. CHI3L2 provoked a dramatic reduction of pRB phosphorylation and a significant decrease in the cyclin D1 expression, whereas the p53 and p21 expression levels were substantially increased. Conclusions. The CHI3L2 protein, which is overexpressed in human gliomas, is a negative regulator of the glioma cells viability. The reduced cell viability after the CHI3L2 treatment could be due to the activation of pRB and p53 and the downregulation of cyclin D. |
| format |
Article |
| author |
Avdieiev, S.S. Gera, L. Hodges, R. Dmytrenko, V.V. |
| author_facet |
Avdieiev, S.S. Gera, L. Hodges, R. Dmytrenko, V.V. |
| author_sort |
Avdieiev, S.S. |
| title |
Glioma-associated protein CHI3L2 suppresses cells viability and induces G1/S transition arrest |
| title_short |
Glioma-associated protein CHI3L2 suppresses cells viability and induces G1/S transition arrest |
| title_full |
Glioma-associated protein CHI3L2 suppresses cells viability and induces G1/S transition arrest |
| title_fullStr |
Glioma-associated protein CHI3L2 suppresses cells viability and induces G1/S transition arrest |
| title_full_unstemmed |
Glioma-associated protein CHI3L2 suppresses cells viability and induces G1/S transition arrest |
| title_sort |
glioma-associated protein chi3l2 suppresses cells viability and induces g1/s transition arrest |
| publisher |
Інститут молекулярної біології і генетики НАН України |
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2015 |
| topic_facet |
Short Communications |
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http://dspace.nbuv.gov.ua/handle/123456789/152612 |
| citation_txt |
Glioma-associated protein CHI3L2 suppresses cells viability and induces G1/S transition arrest / S.S. Avdieiev, L. Gera, R. Hodges, V.V. Dmytrenko // Вiopolymers and Cell. — 2015. — Т. 31, № 4. — С. 316-320. — Бібліогр.: 8 назв. — англ. |
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Вiopolymers and Cell |
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316
ISSN 0233-7657
Biopolymers and Cell. 2015. Vol. 31. N 4. P. 316–320
doi: http://dx.doi.org/10.7124/bc.0008F1
Short Communication
UDC 576.322 577.22
Glioma-associated protein CHI3L2 suppresses cells viability
and induces G1/S transition arrest
S. S. Avdieiev1, L. Gera 2, R. Hodges2 , V. V. Dmytrenko1
1 Institute of Molecular Biology and Genetics, NAS of Ukraine
150, Akademika Zabolotnoho Str., Kyiv, Ukraine, 03680
2 Department of Biochemistry and Molecular Genetics, University of Colorado Denver,
Anschutz Medical Campus, Aurora CO 80045, USA
stasavdieiev@gmail.com
Aim. To analyze the effect of the CHI3L2 protein on malignant and non-malignant cell viability, and deter-
mined the CHI3L2 impact on the cell cycle and signaling pathways involved in the cell cycle regulation.
Methods. MTT-based cell proliferation assay, FACS, western blot analysis. Results. The CHI3L2 protein
inhibits the glioma cells viability and potentiates the effect of anti-cancer cytotoxic agents. The CHI3L2
treatment results in the G1/S transition arrest. CHI3L2 provoked a dramatic reduction of pRB phosphoryla-
tion and a signifi cant decrease in the cyclin D1 expression, whereas the p53 and p21 expression levels were
substantially increased. Conclusions. The CHI3L2 protein, which is overexpressed in human gliomas, is a
negative regulator of the glioma cells viability. The reduced cell viability after the CHI3L2 treatment could
be due to the activation of pRB and p53 and the downregulation of cyclin D.
K e y w o r d s: chitinase-like proteins, glioma, bradykinin antagonists, cell cycle regulation signaling cas-
cades.
© 2015 Avdieiev S . S. et al.; Published by the Institute of Molecular Biology and Genetics, NAS of Ukraine on behalf of Biopolymers and Cell.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/),
which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited
Introduction
Almost half of the intracranial tumors are gliomas,
the majority of which are astrocytic gliomas. They
are considered to be the most lethal among all types
of tumors. We have previously compared the gene
expression in glioblastoma, the most common and
aggressive malignant brain tumor, and normal adult
human brain by Serial Analysis of Gene Expression
(SAGE) to identify those involved in gliomagenesis
and found that that 44 genes were expressed at a sig-
nifi cantly higher level in the tumors compared to the
normal brain cells [1]. Chitinase 3-like 2 (CHI3L2)
is one of the genes expressed in glyoblastoma; it en-
codes a 39 kDa secreted chitinase-like protein.
CHI3L2 is a member of the 18 glycosyl hydrolase
family [1]. It is closely related to another mammali-
an chitinase-like protein (CLP) CHI3L1. CHI3L1 is
overexpressed in glioblastoma and possesses onco-
genic properties [2]. CHI2L1 and 2 may have differ-
ent functions since the molecular structures of these
homologous proteins have signifi cant differences
[3]. We have previously demonstrated that the acti-
vation of ERK1/2 phosphorylation by CHI3L2 in-
hibits the cell mitogenesis and proliferation. In con-
trast, the activation of ERK1/2 phosphorylation by
CHI3L1 leads to the proliferation [4]. Here we have
reported that CHI3L2 downregulates the glioma
cells viability and potentiates the effect of cytotoxic
agents; the CHI3L2-mediated growth suppression is
mediated by G1/S transition arrest.
Materials and Methods
Cell proliferation assay (MTT-assay) was performed
as described in [4]. The CHI3L2 protein was ob-
tained using Bac-toBac expression system (Invitro-
317
Glioma-associated protein CHI3L2 suppresses cells viability and induces G1/S transition arrest
gen, USA). Bradykinin (BK) antagonist BKM-570
was kindly provided by Dr. Lajos Gera (University
of Colorado Denver). Temozolomide was purchased
from Abcam (UK).
Cell cycle analysis. 293 cells were incubated with
the CHI3L2 protein or transfected with pcDNA4/
TO_CHI3L2. After 48 hours, the cells were fi xed
with cold 70 % ethanol, and resuspended in 1 ml of
hypotonic buffer (0.1 % sodium citrate, 0.1 % Triton
X-100, 5 mg/ml PI (Sigma, USA), 20 mg/ml RNase
A (Thermo Scientifi c, USA) and 40 mg/ml propidi-
um iodide (Sigma) for 1 hour. The cells were then
analyzed using the BD Accuri C6 machine (Becton
Dickinson, USA) according to the manufacturer’s
instructions. The data were analyzed using the soft-
ware package CFlow (Becton Dickinson) and Flow-
Jo (De Novo Software, USA).
Western-blot analysis was performed as described
earlier [4]. 293 cells were incubated with the CHI3L2
protein or transfected with pcDNA4/TO_CHI3L2.
After 48 hours, the cells were lyzed and probed with
the following antibodies: anti-pRB (Abcam), anti-
CyclinD1 (Cell Signaling Tech, USA) anti-p53 (Mil-
lipore, USA), anti-p21 (Santa Cruz Biotech., USA),
and anti-beta-actin (Sigma). Secondary polyclonal
HRP-conjugated anti-rabbit IgG or anti-mouse IgG
Abs (AbD Serotec, UK) and the ECL Western blot-
ting detection system (Amersham, USA) were used
to reveal immunoreactivity.
Results and Discussion
Although the investigation of human CLPs was
mainly focused on the expression patterns in a
number of pathological conditions [5], CHI3L2 re-
mains poorly characterized. To explore an effect of
the CHI3L2 protein on the cells viability, we per-
formed the MTT test with the human glioma cells
U251 and non-transformed 293 cells. The CHI3L2
protein exhibits cytotoxic properties in both U251
and 293 cells: an addition of 100 ng/ml CHI3L2 to
the cell culture medium led to ~ 40% reduction of
the cell viability as compared to the control group
(0 ng/ml) (Fig. 1 a). The ability of CHI3L2 to in-
hibit the viability of malignant and non-transformed
cells lines may suggest that a mode of its action is
cell-type independent.
Fig. 1. Reduction of cells viability after CHI3L2 treatment. a) U251 or 293 cells were treated with CHI3L2 for 48 h in DMEM with
2,5 % FBS. b) U251 cells were treated with 100 μM temozolomide, 1 μM BKM-570, 100 ng/ml CHI3L2, or their combinations for
48 h in DMEM with 2,5 % FBS. Data are expressed as a percentage of growth compared to the vehicle control (100 %). Values are
represented as means ± SD (n = 9). *p < 0.05 and **p < 0.01 vs control group
318
S. S. Avdieiev, L. Gera, R. Hodges, V. V. Dmytrenko
The proteins with cytotoxic properties could increase
the effi cacy of anticancer chemotherapy when applied
in combinations [6]. Taking into account that CHI3L2
is overexpressed in glioblastoma, we aimed to see the
outcome of the combination of CHI3L2 with the fi rst-
line anti-gliomic drug temozolomide, as well as with a
highly potent anti-cancer agent, bradykinin antagonist
BKM-570, possessing a signifi cant cytotoxic activity
against the glioma cell lines [7]. We did not observe any
potentiation of the temozolomide activity but the com-
bination of CHI3L2 with BKM-570 led to an increased
cytotoxic effect (Fig. 1 b), suggesting that CHI3L2
could potentially be used as a component of a combo
approach for the glioma treatment.
Fig. 2. Impact of CHI3L2 on cell cycle. 293 cells were treated with 100 ng/ml CHI3L2 or transfected with pcDNA4/TO_CHI3L2 in
DMEM with 2,5 % FBS. Cell cycle analysis was performed 48h after tretment
Fig. 3. CHI3L2 affects cell cycle regulation cascades. 293 cells were treated with 100 ng/ml CHI3L2 or transfected with pcDNA4/
TO_CHI3L2 in DMEM with 2,5 % FBS and lyzed after 48 h. Western blot analysis was performed with appropriate antibodies (a)
and immunoreactive bands were analyzed by densitometry (b)
319
Glioma-associated protein CHI3L2 suppresses cells viability and induces G1/S transition arrest
To elucidate the molecular mechanisms of the ob-
served cell viability reduction, we studied the impact
of CHI3L2 on cell cycle. While no apoptotic action
of CHI3L2 was observed, CHI3L2 induced the G1/S
transition arrest. The proportion of cells in G1 phase
increased after treatment by exogenous CHI3L2 as
well as after its ectopic expression by 19 % and 13,7
%, correspondingly (Fig. 2). To explore how CHI3L2
affects the specifi c signal cascades involved in the
cell cycle regulation, we have studied the state of
some proteins essential for the G1/S cell cycle tran-
sition. The CHI2L2 treatment or ectopic expression
leads to a reduction of pRB phosphorylation, as well
as to a decrease of the cyclin D1 expression level
(Fig. 3). Furthermore, the expression levels of p53
and its transcriptional target p21 were signifi cantly
upregulated. Thus, the G1/S transition arrest through
changes in the cell cycle regulation cascades is a pu-
tative component of the molecular mechanism un-
derlying the cytotoxic effect of CHI3L2 protein. The
blockage of cell cycle in a certain phase is the mech-
anism of action described for a set of cytotoxic
agents [8], however, it has never been reported for
the chitinase-like proteins. CHI3L1, the most inves-
tigated protein in this family, is also overexpressed
in gliomas and was shown to be an oncogen [2].
CHI3L2 has a high level of homology to CHI3L1,
however, a set of structural differences exists be-
tween them giving a ground to distinct functions [3].
CHI3L2 is not a glycoprotein and does not bind hep-
arin, a component of the important receptor com-
plexes [3]. Nevertheless nothing is know about a
function of CHI3L2 in brain issue, the results de-
scribed in this paper is a step forward shedding a
new light on molecular pathways involved by these
similar proteins to realize their function.
The complex application of chemotherapeutic
agents and cytotoxic proteins is a promising ap-
proach to treat cancer [6]. Taking into account the
cytotoxic properties of CHI3L2 and its overexpres-
sion in glioblastomas, one could speculate that at
least in a certain part of patients the CHI3L2 protein
could potentiates the action of chemotherapeutics.
Further investigations are needed to clarify a role of
the chitinase-like proteins in the human gliomas.
Conclusions
In summary, we have demonstrated that CHI3L2
acts as a negative regulator of the cell growth and
affects the cell cycle regulation machinery, resulting
in the G1/S transition blockage.
Acknowledgements
This work was supported in part by a travel grant of
the Boehringer Ingelheim Fonds ‘Complex app ro a-
ches for the treatment of chemoresistant gliomas and
lymphomas’ and The ERA-WIDE Project 294932
‘COMBIOM’.
REFERENCES
1. Kavsan V, Shostak K, Dmitrenko V, Zozulya Y, Rozumenko V,
Demotes-Mainard J. Characterization of genes with in-
creased expression in human glioblastomas. Tsitol Genet.
2005;39(6):37–49.
2. Kavsan VM, Baklaushev VP, Balynska OV, Iershov AV,
Areshkov PO, Yusubalieva GM, Grinenko NP, Victorov IV,
Rymar VI, Sanson M, Chekhonin VP. Gene encoding Chi-
tinase 3-Like 1 Protein (CHI3L1) is a putative oncogene. Int
J Biomed Sci. 2011;7(3):230–7.
3. Iershov A, Odynets K, Kornelyuk A, Kavsan V. Homology
modeling of 3D structure of human chitinase-like protein
CHI3L2. Cent Eur J Biol. 2010; 5(4):407–20.
4. Areshkov PO, Avdieiev SS, Balynska OV, Leroith D, Kavsan
VM. Two closely related human members of chitinase-like
family, CHI3L1 and CHI3L2, activate ERK1/2 in 293 and
U373 cells but have the different infl uence on cell prolifera-
tion. Int J Biol Sci. 2012;8(1):39–48.
5. Johansen JS. Studies on serum YKL-40 as a biomarker in
diseases with infl ammation, tissue remodelling, fi broses and
cancer. Dan Med Bull. 2006;53(2):172–209.
6. Asmana Ningrum R. Human interferon alpha-2b: a thera-
peutic protein for cancer treatment. Scientifi ca (Cairo).
2014;2014:970315.
7. Avdieiev S, Gera L, Havrylyuk D, Hodges RS, Lesyk R,
Ribrag V, Vassetzky Y, Kavsan V. Bradykinin antagonists and
thiazolidinone derivatives as new potential anti-cancer com-
pounds. Bioorg Med Chem. 2014;22(15):3815–23.
8. Shapiro GI, Harper JW. Anticancer drug targets: cell cy-
cle and checkpoint control. J Clin Invest. 1999;104(12):
1645–53.
320
S. S. Avdieiev, L. Gera, R. Hodges, V. V. Dmytrenko
Гліома-асоційований протеїн
CHI3L2 пригнічує життєздатність клітин
та блокує G1/S перехід
С. С. Авдєєв, Л. Гера, Р. Ходжес, В. В. Дмитренко
Мета. Проаналізувати життєздатність злоякісних та незлоя-
кісних клітин за дії протеїна CHI3L2, а також визначити
вплив CHI3L2 на клітинний цикл і сигнальні шляхи, залучені
до його регуляції. Методи. МТТ–тест, проточна цитофлуори-
метрія, вестерн блот аналіз. Результати. CHI3L2 пригнічує
життєздатність клітин гліоми людини і підсилює дію проти-
ракових цитотоксичних агентів. CHI3L2 інгібує перехід клі-
тин із G1- до S-фази клітинного циклу. CHI3L2 зумовлює
зниження кількості фосфорильованої форми pRB, зменшен-
ня вмісту цикліну D1 та зростання вмісту p53 і p21. Висно-
вки. CHI3L2, що надекспресується в гліомах людини, є нега-
тивним регулятором життєздатності клітин гліоми. Цитоток-
сичний вплив CHI3L2 може бути пов'язаним з активацією
pRB та р53, а також зниженням вмісту цикліну D.
Ключов і слова: хітиназо-подібні білки, гліоми, антаго-
ністи брадикініну, сигнальні каскади, що регулюють клітин-
ний цикл.
Глиома-ассоциированный протеин CHI3L2 подавляет
жизнеспособность клеток и блокирует G1 / S переход
С. С. Авдеев, Л. Гера, Р. Ходжес, В. В. Дмитренко
Цель. Проанализировать жизнеспособность злокачествен-
ных и незлокачественных клеток при воздействии протеина
CHI3L2, а также определить влияние CHI3L2 на клеточный
цикл и сигнальные пути, вовлеченные в его регуляцию.
Методы. МТТ-тест, проточная цитофлуориметрия, вестерн
блот анализ. Результаты. CHI3L2 подавляет жизнеспособ-
ность клеток глиомы человека и усиливает действие противо-
раковых цитотоксических агентов. CHI3L2 ингибирует пере-
ход клеток с G1- в S-фазу клеточного цикла. Влияние CHI3L2
приводит к снижению количества фосфорилированной фор-
мы pRB, уменьшению содержания циклина D1 и увеличению
содержания p53 и p21. Выводы. CHI3L2, оверэкспрессия ко-
торого характерна для глиом человека, является негативным
регулятором жизнеспособности клеток глиомы. Цито ток си-
чес кое влияние CHI3L2 может быть связано с активацией
pRB и р53, а также снижением содержания циклина D.
Ключевые слова: хитиназа-подобные белки, глиомы,
антагонисты брадикинина, сигнальные каскады, регулиру-
ющие клеточный цикл.
Received 03.06.2015
|