Combination of cyclooxygenase-2 inhibitor and doxorubicin increases the growth inhibition and apoptosis in human hepatocellular carcinoma cells
Inhibition of cyclooxygenase (COX)-2 elicits therapeutic effects in solid tumors that are coupled with the inhibition of cell proliferation and induction of apoptosis in tumor cells. Aim: This study was designed to investigate the role of COX-2 inhibitor nimesulide in cell growth and apoptosis of th...
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| Опубліковано в: : | Experimental Oncology |
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| Дата: | 2007 |
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Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України
2007
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| Цитувати: | Combination of cyclooxygenase-2 inhibitor and doxorubicin increases the growth inhibition and apoptosis in human hepatocellular carcinoma cells / Y.Y. Pan, S.P. Xu, X.Y. Jia, H.Q. Xu, Y. Zhang, L.X. Rui, W. Wei // Experimental Oncology. — 2007. — Т. 29, № 1. — С. 23-29. — Бібліогр.: 45 назв. — англ. |
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Pan, Y.Y. Xu, S.P. Jia, X.Y. Xu, H.Q. Zhang, Y. Rui, L.X. Wei, W. 2018-06-19T09:28:43Z 2018-06-19T09:28:43Z 2007 Combination of cyclooxygenase-2 inhibitor and doxorubicin increases the growth inhibition and apoptosis in human hepatocellular carcinoma cells / Y.Y. Pan, S.P. Xu, X.Y. Jia, H.Q. Xu, Y. Zhang, L.X. Rui, W. Wei // Experimental Oncology. — 2007. — Т. 29, № 1. — С. 23-29. — Бібліогр.: 45 назв. — англ. 1812-9269 https://nasplib.isofts.kiev.ua/handle/123456789/138556 Inhibition of cyclooxygenase (COX)-2 elicits therapeutic effects in solid tumors that are coupled with the inhibition of cell proliferation and induction of apoptosis in tumor cells. Aim: This study was designed to investigate the role of COX-2 inhibitor nimesulide in cell growth and apoptosis of the cultured human hepatocellular carcinoma HepG2 cells. Methods: We performed the MTT assay, flow cytometric analysis and cell morphology study to evaluate growth inhibition and cell apoptosis upon the action of nimesulide alone or along with doxorubicin, a common agent for the treatment of human hepatocellular carcinoma. Results: Our results showed that the treatment of HepG2 cells with more than 50 µM of nimesulide suppressed COX-2 enzyme activity because of reduced PGE2 production, and then induced growth inhibition and cell apoptosis despite no alterations of COX-2 protein expression. Importantly, the combination of 50 µM or 100 µM of nimesulide and low concentrations (5 µM to 20 µM) of doxorubicin resulted in enhanced cell growth inhibition, apoptosis induction and reduced VEGF production. Conclusion: These data suggest synergistic and/or additive effects of COX-2 inhibitors and chemotherapeutic agents, and may provide the rational for clinical studies of COX-2 inhibitors on the treatment or chemoprevention of human hepatocellular carcinoma. Угнетение циклооксигеназы-2 (ЦОГ-2) оказывает терапевтический эффект при лечении больных с солидными опухолями и сопровождается снижением пролиферации опухолевых клеток и индукцией апоптоза. Цель: изучение роли ингибитора ЦОГ-2 — нимесулида в процессах роста и апоптоза культивированных клеток гепатокарциномы человека HepG2. Методы: для оценки апоптоза и угнетения роста клеток при применении нимесулида самостоятельно и в сочетании с доксорубицином применяли MTT-анализ, проточную цитометрию и стандартные морфологические методы. Результаты: установлено, что обработка клеток HepG2 cells нимесулидом в концентрации > 50 μM приводила к угнетению активности ЦОГ-2 за счет снижения продукции PGE2 , после чего отмечали подавление роста и апоптоз клеток при неизмененном уровне экспрессии ЦОГ-2. Комбинированное применение 50 μM или 100 μM нимесулида и доксорубицина в концентрации 5–20 μM обусловило усиленное угнетение роста клеток, индукции апоптоза и снижение продукции VEGF. Выводы: полученные данные свидетельствуют о синергическом и/или аддитивном эффекте при применении ингибиторов ЦОГ-2 и химиотерапевтических препаратов. We thank Hong-Mei Xu for excellent technical assistance. en Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України Experimental Oncology Original contributions Combination of cyclooxygenase-2 inhibitor and doxorubicin increases the growth inhibition and apoptosis in human hepatocellular carcinoma cells Комбинированное применение ингибитора циклооксигеназы-2 и доксорубицина приводит к угнетению роста и апоптозу клеток гепатокарциномы человека Article published earlier |
| institution |
Digital Library of Periodicals of National Academy of Sciences of Ukraine |
| collection |
DSpace DC |
| title |
Combination of cyclooxygenase-2 inhibitor and doxorubicin increases the growth inhibition and apoptosis in human hepatocellular carcinoma cells |
| spellingShingle |
Combination of cyclooxygenase-2 inhibitor and doxorubicin increases the growth inhibition and apoptosis in human hepatocellular carcinoma cells Pan, Y.Y. Xu, S.P. Jia, X.Y. Xu, H.Q. Zhang, Y. Rui, L.X. Wei, W. Original contributions |
| title_short |
Combination of cyclooxygenase-2 inhibitor and doxorubicin increases the growth inhibition and apoptosis in human hepatocellular carcinoma cells |
| title_full |
Combination of cyclooxygenase-2 inhibitor and doxorubicin increases the growth inhibition and apoptosis in human hepatocellular carcinoma cells |
| title_fullStr |
Combination of cyclooxygenase-2 inhibitor and doxorubicin increases the growth inhibition and apoptosis in human hepatocellular carcinoma cells |
| title_full_unstemmed |
Combination of cyclooxygenase-2 inhibitor and doxorubicin increases the growth inhibition and apoptosis in human hepatocellular carcinoma cells |
| title_sort |
combination of cyclooxygenase-2 inhibitor and doxorubicin increases the growth inhibition and apoptosis in human hepatocellular carcinoma cells |
| author |
Pan, Y.Y. Xu, S.P. Jia, X.Y. Xu, H.Q. Zhang, Y. Rui, L.X. Wei, W. |
| author_facet |
Pan, Y.Y. Xu, S.P. Jia, X.Y. Xu, H.Q. Zhang, Y. Rui, L.X. Wei, W. |
| topic |
Original contributions |
| topic_facet |
Original contributions |
| publishDate |
2007 |
| language |
English |
| container_title |
Experimental Oncology |
| publisher |
Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України |
| format |
Article |
| title_alt |
Комбинированное применение ингибитора циклооксигеназы-2 и доксорубицина приводит к угнетению роста и апоптозу клеток гепатокарциномы человека |
| description |
Inhibition of cyclooxygenase (COX)-2 elicits therapeutic effects in solid tumors that are coupled with the inhibition of cell proliferation and induction of apoptosis in tumor cells. Aim: This study was designed to investigate the role of COX-2 inhibitor nimesulide in cell growth and apoptosis of the cultured human hepatocellular carcinoma HepG2 cells. Methods: We performed the MTT assay, flow cytometric analysis and cell morphology study to evaluate growth inhibition and cell apoptosis upon the action of nimesulide alone or along with doxorubicin, a common agent for the treatment of human hepatocellular carcinoma. Results: Our results showed that the treatment of HepG2 cells with more than 50 µM of nimesulide suppressed COX-2 enzyme activity because of reduced PGE2 production, and then induced growth inhibition and cell apoptosis despite no alterations of COX-2 protein expression. Importantly, the combination of 50 µM or 100 µM of nimesulide and low concentrations (5 µM to 20 µM) of doxorubicin resulted in enhanced cell growth inhibition, apoptosis induction and reduced VEGF production. Conclusion: These data suggest synergistic and/or additive effects of COX-2 inhibitors and chemotherapeutic agents, and may provide the rational for clinical studies of COX-2 inhibitors on the treatment or chemoprevention of human hepatocellular carcinoma.
Угнетение циклооксигеназы-2 (ЦОГ-2) оказывает терапевтический эффект при лечении больных с солидными опухолями
и сопровождается снижением пролиферации опухолевых клеток и индукцией апоптоза. Цель: изучение роли ингибитора
ЦОГ-2 — нимесулида в процессах роста и апоптоза культивированных клеток гепатокарциномы человека HepG2.
Методы: для оценки апоптоза и угнетения роста клеток при применении нимесулида самостоятельно и в сочетании с
доксорубицином применяли MTT-анализ, проточную цитометрию и стандартные морфологические методы. Результаты:
установлено, что обработка клеток HepG2 cells нимесулидом в концентрации > 50 μM приводила к угнетению активности
ЦОГ-2 за счет снижения продукции PGE2
, после чего отмечали подавление роста и апоптоз клеток при неизмененном
уровне экспрессии ЦОГ-2. Комбинированное применение 50 μM или 100 μM нимесулида и доксорубицина в концентрации
5–20 μM обусловило усиленное угнетение роста клеток, индукции апоптоза и снижение продукции VEGF. Выводы:
полученные данные свидетельствуют о синергическом и/или аддитивном эффекте при применении ингибиторов ЦОГ-2
и химиотерапевтических препаратов.
|
| issn |
1812-9269 |
| url |
https://nasplib.isofts.kiev.ua/handle/123456789/138556 |
| citation_txt |
Combination of cyclooxygenase-2 inhibitor and doxorubicin increases the growth inhibition and apoptosis in human hepatocellular carcinoma cells / Y.Y. Pan, S.P. Xu, X.Y. Jia, H.Q. Xu, Y. Zhang, L.X. Rui, W. Wei // Experimental Oncology. — 2007. — Т. 29, № 1. — С. 23-29. — Бібліогр.: 45 назв. — англ. |
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| fulltext |
Experimental Oncology ���� ������� ����� ��arc��� ������ ������� ����� ��arc��� ���arc��� ���� �� ��
Hepatocellular carcinoma �HCC�� is t�e fift� most
common cancer and t�e fourt� leading cause of
cancer related mortality worldwide�� wit� t�e �ig�est
incidence in Asia�� w�ere �epatitis B or C is epidemic
[1]. Alt�oug� t�e clinical diagnosis and management
of early-stage HCC �as significantly improved�� HCC
prognosis remains poor. Only 1����% of patients
qualify for curative surgery [�]. T�e median survival
of patients w�o �ave unresectable tumors is only
4 mont�s [�]. Currently�� no effective systemic �c�e-
mot�erapeutic or c�emopreventive�� treatments are
available. Hence�� investigating HCC pat�ogenesis and
finding new treatment strategies is an urgent need.
T�e cyclooxygenase-� �COX-��� is �ig�ly expressed
in a variety of �uman cancers [4�� 5]. COX-� �as been
associated wit� tumor growt��� angiogenesis�� invasion��
and metastasis [6�� ��]. Overexpression of COX-� may
increase t�e resistance of cancer cells to apoptosis
[8]. T�us�� t�e reduction of t�e COX-� enzyme activity
or protein expression may in�ibit cell growt� in cancer
cells. Non-steroidal anti-inflammatory drugs �NSAIDs��
�ave been s�own to in�ibit COX enzymes and may be
employed for t�e c�emoprevention of cancer [��� 1�].
Some NSAIDs suc� as nimesulide ex�ibit �ig� selectiv-
ity for COX-� enzyme�� but �ave little effects on COX-1
enzyme [11]. T�e expression pattern of COX-� protein
in HCC is well correlated wit� t�e differentiation grade��
suggesting t�at abnormal COX-� expression plays an
important role in �epatocarcinogenesis [1��� 1�]. In�ibi-
tion of COX-� by several in�ibitors including celecoxib��
NS-��8 and nimesulide induces growt� in�ibition and
marked apoptosis in cultured HCC cells by various
mec�anisms [1��� 14���].
T�ere is also evidence suggesting an important
role of angiogenesis in COX-�-mediated �epatocar-
cinogenesis. COX-� �as been s�own to induce angio-
genesis via vascular endot�elial growt� factor �VEGF����
a well-studied regulator of pat�ological angiogenesis
[�1���]. Several recent studies s�ow t�at elevated
COX-� expression correlates wit� increased VEGF level
and microvascular density in �uman HCCs [16�� �4��
�5]. In cultured �epatocellular carcinoma cells�� VEGF
production is increased by overexpression of COX-�
or by treatment wit� prostaglandin E� �PGE����� and t�is
effect is blocked by in�ibition of COX-� [16]. A separate
study also s�ows a role for PGE� in t�e up-regulation
of VEGF in t�e �epatocellular carcinoma cells [16].
T�ese findings suggest t�at COX-� and COX-�-derived
PGE� signaling may promote �epatocarcinogenesis
in part t�roug� VEGF-induced angiogenesis. In t�is
regard�� targeting COX-�-derived prostaglandin sig-
naling represents a promising strategy to reduce t�e
tumor burden.
Doxorubicin is one of t�e effective agents for t�e
treatment of patients wit� unresectable HCCs [�6�� ���].
Single use of doxorubicin yields response rate of up to
��%�� �owever median survival is not prolonged. T�ere
is no convincing evidence from randomized trails t�at
t�e combination c�emot�erapy prolongs t�e survival
of patients wit� unresectable HCC better t�an single
agents. W�et�er COX-� in�ibitors are beneficial in t�e
treatment of HCC if given in combination wit� t�ese
COMBINATION OF CYCLOOXYGENASE-2 INHIBITOR
AND DOXORUBICIN INCREASES THE GROWTH INHIBITION
AND APOPTOSIS IN HUMAN HEPATOCELLULAR CARCINOMA CELLS
Y.Y. Pan1, *, S.P. Xu1, X.Y. Jia1, H.Q. Xu1, Y. Zhang, L.X. Rui2, W. Wei1
1Institute of Clinical Pharmacology of Anhui Medical University, Key Laboratory of Antiinflammatory and
Immunological Pharmacology in Anhui Province, Anhui Medical University, China
2Metabolism Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA
Inhibition of cyclooxygenase (COX)-2 elicits therapeutic effects in solid tumors that are coupled with the inhibition of cell proliferation
and induction of apoptosis in tumor cells. Aim: This study was designed to investigate the role of COX-2 inhibitor nimesulide in cell
growth and apoptosis of the cultured human hepatocellular carcinoma HepG2 cells. Methods: We performed the MTT assay, flow
cytometric analysis and cell morphology study to evaluate growth inhibition and cell apoptosis upon the action of nimesulide alone
or along with doxorubicin, a common agent for the treatment of human hepatocellular carcinoma. Results: Our results showed that
the treatment of HepG2 cells with more than 50 μM of nimesulide suppressed COX-2 enzyme activity because of reduced PGE2
production, and then induced growth inhibition and cell apoptosis despite no alterations of COX-2 protein expression. Importantly,
the combination of 50 μM or 100 μM of nimesulide and low concentrations (5 μM to 20 μM) of doxorubicin resulted in enhanced
cell growth inhibition, apoptosis induction and reduced VEGF production. Conclusion: These data suggest synergistic and/or addi-
tive effects of COX-2 inhibitors and chemotherapeutic agents, and may provide the rational for clinical studies of COX-2 inhibitors
on the treatment or chemoprevention of human hepatocellular carcinoma.
Key Words: hepatocellular carcinoma, nimesulide, COX-2, doxorubicin, apoptosis, VEGF.
Received: January 31, 2007.
*Correspondence: Fax: +86 551 2922826,
E-mail: yueyinpan@gmail.com
Abbreviation used: COX-2 – cyclooxygenase-2; ELISA — enzyme-
linked immunosorbent assay; HCC – hepatocellular carcinoma;
MTT — 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium
bromide; PGE2 – prostaglandin E2; PI – propidium iodide;
VEGF – vascular endothelial growth factor.
Exp Oncol �����
���� 1�� �����
�4 Experimental Oncology ���� ������� ����� ��arc���
effective agents suc� as doxorubicin wit� comple-
mentary mec�anisms remains unknown.
In t�is study�� we �ave c�aracterized t�e effects of
COX-� in�ibition on cell growt� and survival by treat-
ment wit� nimesulide�� a COX-� specific in�ibitor�� in
�epatocellular carcinoma cell line HepG�. Our results
s�owed t�at t�e treatment wit� nimesulide en�anced
doxorubicin-mediated cytotoxicity and reduced pro-
duction of PGE� and VEGF. Our results t�at s�ow addi-
tive in�ibitory effects of COX-� in�ibitor nimesulide and
doxorubicin on t�e growt� of �epatocellular carcinoma
cells may provide t�e rationale for clinical studies of
COX-� in�ibitors on t�e treatment or c�emopreven-
tion of HCC.
MATERIALS AND METHODS
Reagents. Human HepG� �epatocellular carci-
noma cell line was obtained from T�e Researc� Insti-
tute on Hepatoma of S�ang�ai. R�PI 164��� D�E�
and �E� culture medium was purc�ased from Gibco
BRL. Fetal bovine serum �FBS�� was provided by Sijiqing
Biological Engineering �aterial �Hangz�ou�� C�ina��.
Rabbit anti-COX-� was from Cayman. S�eep anti-
rabbit�� s�eep anti-mouse secondary antibodies were
purc�ased from Santa Cruz Biotec�nology�� Inc. �Santa
Cruz�� CA��. �-�4�� 5-Dimet�ylt�iazol-�-yl��-��� 5-dip�en-
yltetrazolium bromide ��TT���� nimesulide and acridine
orange �AO�� were purc�ased from Sigma C�emical Co.
�St. Louis�� �O��. Doxorubicin was provided by Wanle
Co. �S�enz�en�� C�ina��.
Cell culture. HepG� cells were maintained in
D�E� medium supplemented wit� 1�% fetal bovine
serum �FBS���� 1�� units/ml penicillin�� and 1�� mg/ml
streptomycin. HepG� cells were cultured at 5% CO�
and ���° C in a �umidified incubator �Nacpo-61����
DuPont Company�� USA��.
MTT assay. T�e number of viable cells was deter-
mined by �TT assay. Eac� cell sample was plated at
a density of 1�4 cells/well in �6-well cluster dis�es for
�4 � before treatment. T�en t�e medium was c�anged
and nimesulide and doxorubicin were added in various
concentrations and analyzed at indicated time points.
After addition of �TT solution �5 g/L�� in eac� well�� t�e
cells were incubated at ���° for 4 ��� and t�en 15� µL
D�SO was added to dissolve t�e dark blue crystals.
T�e absorbance was measured in an ELISA plate
reader �EL��1 Strip reader�� BIOTEK�� USA�� wit� a test
wavelengt� of 5��� nm�� and t�e relative percentage of
survival was calculated by dividing t�e absorbance of
treated cells by t�at of t�e control in eac� experiment.
Percent cytotoxicity was calculated using t�e formula:
percent cytotoxicity = [1-�absorbance of experimental
wells/absorbance of control wells��] × 1��%.
Cell morphology. After treatement wit� drugs��
cytological morp�ology c�anges were observed under
t�e Olympus optical microscope. Cells were subcul-
tured on coverslips in 6-well culture plates. After �4 ���
t�e coverslips were taken out and stained wit� acridine
orange. Cells were observed and p�otograp�s were
taken under fluorescence microscope.
Flow cytometry. HepG� cells were seeded in cul-
ture flasks. T�e culture bottles were divided into one
medium alone and t�ree drug-treated groups. Eac�
group was in triplicate. W�en t�e cells were anc�ored
to t�e plates�� various drugs were added and t�e cells
incubated at ���°�� 5% CO� for 48 �. T�en eac� group
of cells were was�ed wit� PBS�� trypsinized and t�en
stained wit� propidium iodide �PI�� Sigma�� USA��. T�e
red fluorescence of DNA-bound PI in eac� group was
measured at 488 nm by FACScan flow cytometry
�EPTCS XLL��CL Beckman Coulter�� UK��.
Western blot analysis. For immunoblot analysis
of COX-��� HepG� cells were lysed in a buffer consisting
of 5� m� Tris-HCl�� pH ��.4�� 1% Tween ���� � m� EDTA��
1 m� p�enylmet�ylsulfonyl fluoride�� 1� µg/mL aprotinin��
and 1� µg/mL leupeptin. Debris was removed by centrifu-
gation �Beckman GPKR mac�ine�� at ������ g for �� min
at 4 °C. A total of 6� µg of precipitated and denatured
protein was separated by 1�% sodium dodecyl sulfate-
polyacrylamide gel electrop�oresis �SDS-PAGE�� and
transferred electrop�oretically to polyvinylidene fuoride
membranes �Immun-Blot PVDF membrane�� �.� µm;
Bio-Rad��. �embranes were blocked wit� 5% nonfat milk
powder in �.5 % Tween ��-PBS for � �. Primary antibod-
ies to COX-� were used at a final dilution of 1 : 1��� for
overnig�t�� at 4 °C. T�e blot was was�ed several times wit�
�.�5% Tween ��-PBS and incubated wit� t�e appropri-
ate goat anti-rabbit antibody for � �. Immunodetection
was carried out using en�anced c�emiluminescence
reagent following t�e manufacturer’s instructions �Pierce
Biotec�nology Inc�� USA��.
Measurement of prostaglandin E2 and VEGF
production. HepG� carcinoma cells were plated at a
density of 5 × 1�5 cells / p6� Petri dis� in complete D�E�
medium for 1� �. HepG� cells were treated wit� �5�
1�� µ� nimesulide for �4 �. After drug treatment�� cul-
ture supernatant was collected and centrifuged briefly.
T�e amount of PGE� in t�e medium was measured using
a commercial RIA kit �Wu�an Boster Co�� Wu�an�� C�ina��
and VEGF level determined wit� an enzyme-linked im-
munosorbent assay �ELISA�� kit �Promega Corporation��
following t�e manufacturer’s instructions.
Statistical analysis. Data from t�e popula-
tion of cells treated wit� different conditions were
analyzed using paired Student’s t-test or ANOVA test
�a comparison of multiple groups���� and p value of
< �.�5 was considered statistically significant in t�e
experiments.
RESULTS
Nimesulide inhibits the COX-2 activity in HepG2
hepatocellular carcinoma cells. First�� we performed
t�e Western blot analysis to examine w�et�er COX-�
protein is expressed in HepG�cells. As s�own in Fig. 1�� a��
COX-� protein was �ig�ly expressed in HepG� cells. T�is
is consistent to a recent immuno�itoc�emistry study
t�at s�ows �ig� COX-� protein expression in HepG�
and Hu��� cells [�8]. As treatment wit� �5�1�� µ�
nimesulide for �4 � did not significantly alter expression
levels of COX-� protein in HepG� cells �Fig. 1�� a���� next
Experimental Oncology ���� ������� ����� ��arc��� �5���� ������� ����� ��arc��� �5�arc��� �5�� �5 �5
we tested w�et�er treatment wit� nimesulide in�ibits
t�e COX-� enzyme activity by measuring PGE� levels
of culture supernatant. T�e average amount of PGE� in
t�e medium alone controls from t�ree independent ex-
periments was �.65 ng/ml. Compared wit� t�e control��
t�e PGE� production was reduced by about 8�%�� 65%
and 4�% �4 � after t�e treatment wit� �5 µ��� 5� µ� or
1�� µ� of nimesulide�� respectively �Fig. 1�� b���� indicating
t�e dose-dependent in�ibition.
Fig.1. Effects of nimesulide on t�e COX-� protein expression
and PGE� production in �uman HepG� cells. �a�� HepG� cells
were treated wit� �5�1�� µ� nimesulide for �4 � as indicated.
T�e total protein extracts were subjected to Western blot analysis
using anti-COX-� antibody. Anti-ERK-� antibody was used as a
loading control. �b�� T�e HepG� cells were treated wit� �5�1�� µ�
nimesulide. After incubation for �4 ��� t�e PGE� level in t�e
culture medium was measured by a PGE� EIA kit following t�e
manufacturer’s protocol. T�e means ± S.E of t�ree independent
experiments in eac� treatment are s�own
Nimesulide inhibits cell growth and enhances
doxorubicin-mediated apoptosis of cultured
HepG2 cells. To determine w�et�er COX-� in�ibi-
tion by nimesulide contributes to growt� in�ibition of
HepG� cells we measured t�e number of apoptotic
cells after treatment wit� various doses of nimesulide
by �TT assay. As s�own in t�e left panel of Fig. ��� a��
t�e number of viable cells started to decline by treat-
ment wit� a low concentration of nimesulide ��5 µ���
and continued to drop by up to 4�% at 4�� µ� of
nimesulide. As expected�� cell apoptosis induced by
doxorubicin is more evident t�an t�at by nimesulide as
treatment wit� 8� µ� of doxorubicin induced deat� of
almost all cells in t�e culture �Fig. ��� a�� rig�t panel��. T�e
apoptosis triggered by nimesulide or doxorubicin was
s�own in a concentration-dependent manner.
Because nimesulide-induced cell deat� is t�roug�
COX-� in�ibition and doxorubicin acts as cell cycle
in�ibitor on triggering cell apoptosis�� we �ypot�esize
t�at t�e in�ibition of COX-� by nimesulide may en-
�ance t�e doxorubicin-mediated cytotoxity. To test t�is
possibility�� we performed t�e �TT assay by co-treat-
ment of HepG� cells wit� doxorubicin and nimesulide.
As s�own in Fig. ��� b�� compared to cell treated wit�
5 µ� of doxorubicin alone�� addition of �5�� 5� or 1�� µ�
nimesulide yielded a � to �.5 fold increase in cell deat���
respectively. T�is synergistic effect of nimesulide
persisted even at �ig�er concentrations of doxorubi-
cin�� 1� µ� or �� µ�. Since treatment wit� more t�an
4� µ� of doxorubicin alone already induced apoptosis
of most cells�� an increase in doxorubicin-mediated
cytotoxity of HepG� cells by addition of nimesulide
was limited �Fig. ��� b�� rig�t two panels��.
Fig. 2. Co-treatment of nimesulide and doxorubicin additively
in�ibited t�e growt� of HepG� cells. �a�� T�e cells were treated
wit� indicated �5�4�� µ� nimesulide or indicated 5�8� µ�
of doxorubicin for �4 �. T�e cell survival was measured by �TT
assay. Results were obtained from � independent experiments
and t�e bar represents S.E. Significant c�anges between un-
treated and treated samples are marked by asterisks: *p < �.�5
and **p < �.�1. �b�� T�e cells were co-treated wit� 5�8� µ�
doxorubicin alone or toget�er wit� �5�1�� µ� nimesulide for
�4 �. T�en t�e cell survival was measured by �TT assay. Results
were obtained from 4 independent experiments and t�e bar
represents t�e S.E. Significant c�anges between doxorubicin
alone and co-treatment wit� nimesulide samples are marked
by asterisks: *p < �.�5
To furt�er confirm t�e cytotoxic synergy between
nimesulide and doxorubicin we performed morp�ol-
ogy study by staining cells wit� acridine orange. We
observed t�e normal morp�ology of drug-untreated
HepG� cells: s�ape of tri- or multi-angles wit� abundant
�6 Experimental Oncology ���� ������� ����� ��arc���
cytoplasma�� and large oval nuclei wit� dispersed c�ro-
matin �Fig. ��� a�� left panel��. �4 � after treatment wit� 1��
µ� of nimesulide and 1� µ� of doxorubicin�� HepG�
cells ex�ibited t�e typical morp�ology of apoptotic
cells�� including cell s�rinkage�� deep-dyed pyknotic
nuclei�� margination of nuclear c�romatin�� cytoplasmic
blebbing and clusters of apoptotic bodies �Fig. ��� a��
rig�t panel��. Also�� we measured t�e DNA content by
propidium iodide �PI�� staining�� and cell apoptosis was
reflected by t�e appearance of a cell population wit�
subdiploid and pre-G1p�ase t�roug� flow cytometric
analysis. Consistent wit� �TT assay�� addition of 1�� µ�
of nimesulide significantly increased a portion of sub-
diploid and pre-G1 p�ase ��1.��%�� compared to t�at of
doxorubicin alone group �1�.6%�� �Fig. ��� b�� bottom two
panels��. By contrast�� a muc� smaller portion of pre-G1
p�ase was s�own in t�e cells treated wit� nimesulide
alone �Fig. ��� b�� rig�t top panel��.
Fig. 3. Co-treatment of nimesulide and doxorubicin en�anced
apoptosis induction of HepG� cells. �a�� T�e HepG� cells were
treated wit� or wit�out nimesulide 1�� µ� and doxorubicin 1� µ�
for �4 � and stained wit� acridine orange. T�e cell morp�ology
was observed under fluorescent microscope. �b�� Flow cytometry
analysis of DNA content of HepG� cells after treated wit� 1�� µ�
of nimesulide and/or �� µ� of doxorubicin for �4 �. Represen-
tative DNA �istograms were from � independent experiments.
T�e percentage of pre-G1 p�ase at various culture conditions
is indicated
Co-treatment with doxorubicin and nimesulide
reduces production of VEGF in HepG2 cells. VEGF��
one of t�e most potent angiogenic factors�� �as been
s�own to play a pivotal role in tumor angiogenesis�� in-
cluding HCC. A line of evidence reveals t�at t�e elevated
COX-� expression correlates wit� increased VEGF level
and microvascular density in �uman HCCs [16�� �4�� �5].
However�� it is not known w�et�er t�e in�ibition of COX-�
reduces VEGF production in �epatocellular carcinoma
cells. To test t�is possibility�� we treated HepG� cells
wit� �5 µ� or 5� µ� of nimesulide and measured VEGF
levels in t�e culture supernatants by an ELISA. As s�own
in Fig. 4�� treatment wit� 5� µ� of nimesulide alone led to
an about � fold reduction in VEGF levels and t�is effect
was largely augmented by addition of 5 µ� or 1� µ�
of doxorubicin. VEGF production was also significantly
reduced if t�e cells were co-treated wit� doxorubicin
and a lower concentration ��5 µ��� of nimesulide�� sug-
gesting t�e synergistic in�ibition of VEGF production in
HepG� cells by two agents.
Fig. 4. Co-treatment wit� nimesulide and doxorubicin reduced
VEGF production of cultured HepG� cells. T�e cells were co-
treated wit� 5�1� µ� doxorubicin and �5�5� µ� nimesulide for
48 �. T�e VEGF levels were determined in culture supernatants by
ELISA following t�e manufacturer’s protocol. VEGF secretion was
significantly reduced in t�e co-treatment group compared wit�
t�e single nimesulide or doxorubicin treated group �*p < �.�5��
DISCUSSION
Doxorubicin is one of t�e most often used drugs for
treatment of HCC and single use of doxorubicin yields
response rate of about ��% for unresectable tumors.
However�� t�ere is no convincing evidence t�at t�e use
of doxorubicin improves t�e prognosis of patients wit�
HCC�� t�erefore t�e effect of t�is drug on HCC is still
limited [�����]. NSAIDs�� suc� as aspirin�� indomet�a-
cin and sulindac�� may play a role in t�e in�ibition of
proliferation and induction of apoptosis in tumor cells
t�roug� t�e in�ibition of COX-� activity [����5]. T�e
anti-inflammatory and anti-angiogenic effects of
NSAIDs �ave been explored for t�e cancer t�erapy and
some of t�ese agents are currently under clinical trials
[�6�� ���]. Nimesulide�� a specific COX-� in�ibitor�� can
bind specifically to t�e large catalytic moiety of COX-���
wit� muc� less adverse effects on t�e gastrointestinal
tract compared to t�e non-specific NSAIDs. A selective
COX-� in�ibitor JTE-5�� �as been reported to en�ance
cytotoxity in bladder cancer wit� 5-fluorouracil [�8].
A combination of common antitumor drugs and COX-�
in�ibitors may en�ance c�emot�erapeutic efficacy��
reduce drug dose and adverse side effects. It �as been
s�own t�at nimesulide in�ibits t�e growt� of �uman
�epatoma cell line S��C-�����1 in vitro [1��]. However��
studies on t�e combination of selective COX-� in�ibi-
tors including nimesulide and common c�emot�erapy
agents against �uman �epatocellular carcinoma are
not documented. In t�is study�� we investigated t�e
effects of a combination of nimesulide and doxorubi-
cin on t�e growt� in�ibition in �uman �epatocellular
Experimental Oncology ���� ������� ����� ��arc��� ������� ������� ����� ��arc��� ����arc��� ����� ��� ���
carcinoma HepG� cells. Our results s�owed t�at treat-
ment of nimesulide significantly in�ibited cell growt� in
HepG� cells. �oreover�� t�e combination of doxorubi-
cin and nimesulide additively increased t�e cytotoxicity
and growt� in�ibition in HepG� cells. T�erefore�� our
results suggest t�at t�e use of COX-� in�ibitors may
be beneficial w�en combined wit� doxorubicin for t�e
treatment of patients wit� HCC.
Prostaglandins�� including PGE��� synt�esized by
COX enzymes�� are reported to increase cell growt�
and induce proliferation in t�e cultured rat �epatocytes
[���� 4�]. In �uman �epatocellular carcinoma cells��
treatment wit� prostaglandin E� �PGE��� increases VEGF
production and t�is effect is blocked by in�ibition of
COX-��� suggesting a link of COX-� wit� VEGF signaling
in �epatocarcinogenesis [16]. In support of t�ese find-
ings�� we �ave observed t�e �ig� expression of COX-�
protein in HepG� cells and in�ibition of COX-� by nime-
sulide led to reduced production of PGE� and VEGF��
and decreased viable cells in t�e culture. T�erefore��
t�e interplay between COX-�-derived prostaglandin
signaling and ot�er growt�-regulatory pat�ways suc�
as VEGF is expected to provide important t�erapeutic
implications.
Apoptosis is an important p�ysiological process
t�at prevents t�e formation of tumor clone and t�e
failures of apoptosis lead to t�e development of many
tumors including �epatocellular carcinoma [41]. T�e
recent reports �ave demonstrated t�at NSAIDs induce
apoptosis in different tumor cells [1��� 4�]. It is also
conceivable t�at apoptosis mig�t occur in t�e �uman
�epatocellular carcinoma cells in response to nime-
sulide. To evaluate t�e role of nimesulide in cell apop-
tosis�� we cultured HepG� cells and treated cells wit�
various concentrations of nimesulide or toget�er wit�
a low concentration of doxorubicin. Flow cytometry
analysis of sub-diploid peak of DNA content revealed
t�e ability of nimesulide to trigger apoptosis in HepG�
cells at t�ese concentrations t�at were sufficient to
in�ibit cell proliferation. In addition�� t�e nimesulide-in-
duced apoptosis is significantly increased by addition
of a low concentration of doxorubicin. T�us�� apoptosis
may be one of t�e mec�anisms for nimesulide to in�ibit
cell growt� in HepG� cells�� especially w�en nimesulide
is combined wit� doxorubicin.
In patients wit� �epatocellular carcinoma�� �y-
pervascularity correlated wit� t�e over-expression
of VEGF and significantly associated wit� t�e tumor
extension and s�orter median survival time [�5�� 4���
44]. Prostaglandin E� increases t�e expression and
t�e secretion of VEGF in �epatocellular carcinoma
cells [16�� 45]. We found t�at t�e in�ibition of COX-� by
nimesulide reduced VEGF production in HepG� cells.
Interestedly�� treatment of doxorubicin also in�ibited
VEGF secretion by HepG� cells�� and co-treatment of
doxorubicin and nimesulide markedly decreased t�e
levels of VEGF in cultured supernatants. Toget�er�� our
data suggest t�at co-treatment wit� doxorubicin and
nimesulide induces growt� in�ibition and cell deat�
in part t�roug� t�e in�ibition of VEGF production in
HepG� cells. However�� t�e precise mec�anism of t�e
in�ibition of VEGF production in t�is situation needs
furt�er investigation.
T�is study for t�e first time presents evidence
t�at a combination of COX-� in�ibitor nimesulide and
doxorubicin additively in�ibits growt� of t�e �uman
�epatocellular carcinoma cells. T�is effect was mainly
observed owing to increased apoptosis and reduced
VEGF production. T�e study provides a new strategy
t�at t�e combination of COX-� in�ibitors and doxo-
rubicin may be effective in t�e treatment of �uman
�epatocellular carcinoma.
ACKNOWLEDGEMENT
We t�ank Hong-�ei Xu for excellent tec�nical as-
sistance.
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Experimental Oncology ���� ������� ����� ��arc��� ������ ������� ����� ��arc��� ���arc��� ���� �� ��
КОМБИНИРОВАННОЕ ПРИМЕНЕНИЕ ИНГИБИТОРА
ЦИКЛООКСИГЕНАЗЫ-2 И ДОКСОРУБИЦИНА
ПРИВОДИТ К УГНЕТЕНИЮ РОСТА И АПОПТОЗУ КЛЕТОК
ГЕПАТОКАРЦИНОМЫ ЧЕЛОВЕКА
Угнетение циклооксигеназы-2 (ЦОГ-2) оказывает терапевтический эффект при лечении больных с солидными опухолями
и сопровождается снижением пролиферации опухолевых клеток и индукцией апоптоза. Цель: изучение роли ингибито-
ра ЦОГ-2 — нимесулида в процессах роста и апоптоза культивированных клеток гепатокарциномы человека HepG2.
Методы: для оценки апоптоза и угнетения роста клеток при применении нимесулида самостоятельно и в сочетании с
доксорубицином применяли MTT-анализ, проточную цитометрию и стандартные морфологические методы. Результаты:
установлено, что обработка клеток HepG2 cells нимесулидом в концентрации > 50 μM приводила к угнетению активности
ЦОГ-2 за счет снижения продукции PGE2, после чего отмечали подавление роста и апоптоз клеток при неизмененном
уровне экспрессии ЦОГ-2. Комбинированное применение 50 μM или 100 μM нимесулида и доксорубицина в концентра-
ции 5–20 μM обусловило усиленное угнетение роста клеток, индукции апоптоза и снижение продукции VEGF. Выводы:
полученные данные свидетельствуют о синергическом и/или аддитивном эффекте при применении ингибиторов ЦОГ-2
и химиотерапевтических препаратов.
Ключевые слова: гепатокарцинома, нимесулид, ЦОГ-2, доксорубицин, апоптоз, VEGF.
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