Celecoxib enhances anticancer effect of cisplatin and induces anoikis in osteosarcoma via PI3K/Akt pathway
Liu et al. Cancer Cell Int
Celecoxib enhances anticancer effect of cisplatin and induces anoikis in osteosarcoma via PI3K/Akt pathway
Bing Liu 1
Shigui Yan 1
Jian Zhu 1
0 88 Jie Fang Road , Hangzhou 310009, Zhejiang , People's Republic of China
1 Department of Orthopedics, 2nd Affiliated Hospital, School of Medicine, Zhejiang University
Background: COX-2, an inducible enzyme, is associated with inflammatory diseases and carcinogenesis. Overexpression of COX-2 occurs in many human malignancies, including osteosarcoma. COX-2 positivity is form 67 to 92% in osteosarcoma, and COX-2 expresses 141-fold more in cancer stem cell spheres than daughter adherent cells. In our study, we have reported that celecoxib, a cyclooxygenase-2 inhibitor, induces apoptosis in human osteosarcoma cell line MG-63 via down-regulation of PI3K/Akt. It has been confirmed that celecoxib enhances apoptosis and cytotoxic effect of cisplatin, although the mechanism remains unclear. Methods: We have attempted to identify the anti-proliferation of celecoxib, a selective COX-2 inhibitor, and the combination of celecoxib and cisplatin in MG-63 cells, and to explore the potential molecular mechanisms involved. MG-63 cells were treated with the combination of celecoxib and cisplatin or either agent alone for 48 h in serumsupplemented medium. Results: MDR1, MRP1, BCRP and Trkb, E-cadherin, β-catenin were significantly downregulated in cells treated with the combination of celecoxib and cisplatin, and decreased β-catenin level was found in cells with wortmannin, a specific PI3K inhibitor. Conclusion: Therefore, celecoxib enhances anticancer effect of cisplatin and induces anoikis in osteosarcoma, which may be PI3K/Akt-dependent, and MDR and β-catenin-related. PI3K may be at the center of the celecoxib effects, which play an essential role in the regulation of MDR and anoikis.
COX-2; MDR; Anoikis; PI3K/Akt; Osteosarcoma
The cyclooxygenase (COX) isoenzymes, known as
prostaglandin (PG) rate-limiting synthase. COX-2, an
inducible enzyme, is associated with inflammatory
diseases and carcinogenesis, which is suspected to
promote angiogenesis and tissue invasion of tumours [1, 2]
and the overexpression of COX-2 has been mentioned
in connection with resistance to apoptosis [3, 4].
Nonsteroidal anti-inflammatory drugs (NSAIDs) have been
shown to induce apoptosis as well as potentiate the effect
of chemotherapeutic agents including cisplatin [5, 6].
Celecoxib is a NSAIDs that is a specific inhibitor of
2. Although the precise mechanisms for the chemopre
ventive effects of celecoxib are not yet known, the ability
of the inhibition of cell proliferation, and induction of
apoptosis has been well-known [7, 8]. COX-2 is also
involved in drug resistance and poor prognosis of tumor
Osteosarcoma is the most common primary bone
tumor in children and young adults, which has been
reported to express COX-2 constitutively [10–12].
COX-2 positivity is form 67 to 92% in osteosarcoma
[13, 14], and COX-2 expresses 141-fold more in
cancer stem cell (CSC) spheres than daughter adherent
cells . Therefore, we can exert the drug to affect the
© The Author(s) 2017. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License
(http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium,
provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license,
and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/
publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
over-expressed genes and achieve the therapies of human
In our study, we have reported that celecoxib, a
cyclooxygenase-2 inhibitor, induces apoptosis in human
osteosarcoma cell line MG-63 via down-regulation of
PI3K/Akt . It has been confirmed that celecoxib
enhances the apoptosis induction and cytotoxic effect
of cisplatin, although the mechanism remains unclear.
COX-inhibitors may sensitize cancer cells to
chemotherapeutic drugs via inhibiting P-gp, MRP1 and BCRP, and
enhance the effect of anticancer drugs. PI3K/Akt plays
an essential role in the cell/extracellar matrix (ECM) and
cell/cell adhesion. Lack of the correct adhesion, the
adhesion-dependent signals will be interrupted, which will
result in adhesion-related apoptosis: anoikis. We have
attempted to identify the anti-proliferation of celecoxib,
a selective COX-2 inhibitor, and the combination of
celecoxib and cisplatin in MG-63 cells, and to explore the
potential molecular mechanisms involved.
Celecoxib was generously provided by Pharmacia Cor.
(New York, NY, USA) and dissolved in DMSO (40 mM)
as a stock solution in 4 °C. Cisplatin was purchased
from Qilu Pharmaceutical Co., Ltd. (ShanDong
province, China) and dissolved in normal saline (2 mg/ml)
as a stock solution in 4 °C. Celecoxib and cisplatin were
added at various concentrations to cells in 10% fetal
bovine serum (FBS)-containing DMEM. The final DMSO
concentration never exceeded 0.1% (v/v) after addition
to medium. Mouse monoclonal antibodies specific for
PI3K p110 (sc-8010), β-catenin (sc-7963); rabbit
polyclonal antibodies specific for E-cadherin (sc-7870), Trkb
(sc-8316), β-actin (sc-1616R);secondary antibodies were
obtained from Santa Cruz Biotechnology, Inc.(Santa
Cruz, CA, USA) were purchased from Calbiochem (San
Diego, CA, USA).
Cells and cell culture
The human OS cell line MG-63 used in this study were
obtained from American Type Culture Collection
(Manassas, VA), and the U2OS cells were purchased
from the ATCC (HTB-96; Rockville, Maryland, USA),
HOS (CRL-1547TM, ATCC) was obtained from Cell
Bank of Shanghai Institute of Biochemistry & Cell
Biology, Chinese Academy of Sciences (Shanghai, China).
MG-63 Cells were grown in DMEM medium (Gibco Life
Sciences) supplemented with 10% (v/v) heat activated
fetal bovine serum (Gibco Life Sciences) in a humidified
atmosphere of 5% CO2 at 37 °C. U2OS cells were grown
in RPMI1640 medium and HOS cells were grown in
Eagle’s Minimum Essential medium containing
supplements as above.
Real time quantitative RT‑PCR analysis
RNA was isolated from the cells using Trizol (Invitrogen)
according to the manufacturer’s instructions. cDNA was
generated from total denatured RNA (2 µg) by using 1 µl
oligo(dT)18 primer, 25 units RNase inhibitor, 2 µl dNTPs
(10 mM), and a Moloney Murine Leukemia Virus reverse
transcriptase cDNA synthesis kit (Promega, Madison,
WI, USA). Quantitative real-time PCR was performed
using Applied Biosystems 7500 Real-Time PCR
tem and SYBR® Premix Ex Taq™ kit (Perfect Real Time)
(TaKaRa, Dalian, China). Human 18s rRNA was used as
internal control. The Cycle threshold (CT) values for each
gene were corrected using the mean CT value. Real-time
PCR data were quantified using the ΔCT method with
the formula: n = 100 × 2−(ΔCT targeted gene − ΔCT 18s rRNA).
Targeted genes were amplified by real time PCR with
the following primers: 18s rRNA forward: 5′GACTC
AACACGGGAAACCTCAC3′ and reverse: 5′CCAG
ACAAATCGCTCCACCAAC3′; Human BCRP,
forward 5′GCAGCAGGTCAGAGTGTGGTTT3′, reverse
5′GCTGCAAAGCCGTAAATCCATA3′; Human MRP1
forward: 5′CGCTGAGTTCCTGCGTACCTAT′ and
reverse: 5′CCATTCTCCATTTGCTTTGCTT3′, Human
MDR1 forward: 5′CCGTGGGGCAAGTCAGTTCAT3′
and reverse: 5′CCTTCCAATGTGTTCGGCATTAG3′.
Western blot analysis
After the exposure for 48 h, floating and adherent cells
were harvested and rinsed twice with cold PBS and lysed
in lysis buffer [50 mM Tris (pH 7.4), 400 mM NaCl,
50 mM NaF, 30 mM sodium PPi, 1 mM sodium
pyrovendidate, 1% SDS, and 0.5% NP40]. Protein concentrations
were determined with bicinchoninic acid protein assay
(Pierce, Rockford, IL, USA). Cellular proteins, at a
concentration of 40 µg, were fractionated on 12% SDS-PAGE
(Invitrogen/Novex, Carlsbad, CA, USA). Proteins were
then transferred to a polyvinylidene difluoride
membrane (Immobilon-P; Millipore, Bedford, MA, USA). The
membrane was blocked with 5% nonfat milk in TBST and
incubated overnight with antibody at 4 °C. After
washing three times with TBST, the membrane was incubated
at room temperature for 1 h with horseradish
peroxidase-conjugated secondary antibody diluted with TBST
(1:1000). The detected protein signals were visualized by
an enhanced chemiluminescence reaction system
(Amersham, Arlington Heights, IL, USA), and the signal was
visualized with X-ray film (Hyperfilm; Amersham).
Electrophoretic mobility shift assay (EMSA) for β‑catenin
Cells were treated with celecoxib (50 and 100 µmol/l),
cisplatin (10 µg/ml), and the combined administration
for 48 h. Nuclear proteins were prepared using nuclear
and cytoplasmic protein extraction reagents according to
the manufacturer’s protocols (Pierce, Rockford, IL, USA).
The crude nuclear pellet was suspended in 200 µL buffer
B (20 mmol/l HEPES pH 7.9; 25% glycerol, 1.5 mmol/l
MgCl2; 420 mmol/l NaCl, 0.5 mmol/l DTT; 0.2 mmol/l
EDTA; 0.5 mmol/l PMSF; and 4 µmol/l leupeptidin) and
incubated on ice for 30 min. The suspension was
centrifuged at 16,000×g at 4 °C for 30 min. The supernatant
was collected as nuclear proteins. A non-radioactive
EMSA was performed using an EMSA kit (Pierce,
Rockford, IL, USA) according to the manufacturer’s
instructions. Nuclear protein (4 µg) extracted from the cells or
tissues was incubated with biotinylated oligonucleotides
containing the β-catenin site for 30 min at room
temperature. The samples were separated in a nondenaturing
polyacrylamide gel (6%, with 2.5% glycerol) and
blotted on a Biodyne B (0.45 mm) positively charged nylon
membrane (Pall Schweiz AG, Basel, Switzerland). The
biotin was labeled with alkaline phosphatase-conjugated
streptavidin and alkaline phosphatase was detected with
Enhanced Chemiluminescense detection system (Santa
Cruz, CA, USA).
The entire experiment was done in triplicate and the data
are presented as mean ± SD. The statistical significance
of differences was determined by Student’s two-tailed
t test in two groups and one-way ANOVA in multiple
groups. P < 0.05 was considered statistically significant.
All data were analyzed with SPSS 16.0 software.
Down‑regulation of MDR1, MRP1 and BCRP correlated
with increased apoptosis
We have reported that celecoxib caused G1 phase arrest
and significantly inhibited cell growth, as well as
potentiating cisplatin-induced apoptosis. MG-63 cells were
exposed to celecoxib (50 and 100 µmol/l), cisplatin
(10 µg/ml), and the combined administration for 48 h,
and the apoptosis rate was 1.39, 4.06, 5.98, 5.93, 6.66,
37.15%, respectively, which may be result from
COX2-related drug resistance. The activity of
COX-2-PGE2Prostaglandin E Receptors signal pathway can upregulate
the expression of all three ABC transporters, MDR1/P-gp
(multidrug resistance/P-glycoprotein), MRP1 (multidrug
resistance protein 1) and BCRP (breast cancer resistance
protein), which encode efflux pumps, play important
roles in the development of multidrug resistance .
The potential mechanism of MDR1, MRP1 and BCRP
in celecoxib and/or cisplatin treated cells were therefore
investigated. Figure 1 shows that the downregulation of
MDR1, MRP1 and BCRP mRNA expression was found
in cells treated with celecoxib (50 and 100 µmol/l),
cisplatin (10 µg/ml), and the combined administration for
48 h. Moreover, significant downregulation of MDR1,
MRP1 and BCRP was detected in cells treated with the
combination of celecoxib (100 µmol/l) and cisplatin
compared with either agent alone, with apoptosis being
strongly increased. However, no potentiation of celecoxib
(50 µmol/l) and cisplatin was found. The results of treated
MG-63 cells were similar in Western blot analysis (Fig. 2).
Modulation of Trkb, E‑cadherin and β‑catenin expression
in MG‑63 cells treated with celecoxib and cisplatin
We have reported that celecoxib, a cyclooxygenase-2
inhibitor, induces apoptosis in human osteosarcoma cell
line MG-63 via down-regulation of PI3K/Akt. PI3K/
Akt plays an essential role in the cell/extracellar matrix
(ECM) and cell/cell adhesion. Lack of the correct
adhesion, the adhesion-dependent signals will be interrupted,
which will result in adhesion-related apoptosis: anoikis
. Trkb, E-cadherin and β-catenin play an important
role in the cell/cell adhesion . In this study we found
the down-regulations of E-cadherin and β-catenin in
MG-63, U2OS and HOS cells, as shown in Fig. 3.
Significant down-regulations of Trkb, E-cadherin and β-catenin
activation were observed in MG-63 cells treated with
cisplatin and celecoxib (100 µmol/l).
The effect of celecoxib and cisplatin on nuclear expression
As show in Fig. 4, constitutively active nuclear
expression of β-catenin binding activity was observed in nuclear
extracts from MG-63, U2OS and HOS cells. In all cells,
compared with untreated control, both celecoxib and
cisplatin treatment obviously decreased β-catenin
binding activity, meanwhile, significant down-regulation of
β-catenin binding activity was displayed in MG-63 and
U2OS cells. These results suggest that celecoxib not
only down-regulates β-catenin binding activity but also
sensitizes cisplatin effect, which may be responsible for
enhanced inhibition of cell viability and inducing
apoptosis by combination treatment.
PI3K inhibition by wortmannin causing to the decease
of pAkt and β‑catenin
To investigate the role of PI3K in cells treated with
celecoxib, we gave wortmannin (1 mmol/l) to cells for
48 h, but did not see downregulation of total PI3K and
Akt, although downregulation of pAkt (Thr308) and
β-catenin was detected (Fig. 5).
Fig. 1 Regulation of MDR1, MRP1 and BCRP expression. RT-PCR analysis MDR1, MRP1 and BCRP expression of MG-63 cells untreated or treated with
celecoxib (50, 100 µmol/l), cisplatin (10 µg/ml), and the combination for 48 h. Significant down-regulation of MDR1, MRP1 and BCRP expression was
observed in cells treated with cisplatin
Fig. 2 Regulation of MDR1, MRP1 and BCRP expression. Western blot
analysis MDR1, MRP1 and BCRP expression of MG-63 cells untreated
or treated with celecoxib (50, 100 µmol/l), cisplatin (10 µg/ml), and
the combination for 48 h. Significant down-regulation of MDR1, MRP1
and BCRP expression was observed in cells treated with cisplatin
A hypothetical model for PI3K and COX‑2 pathway
Celecoxib down-regulates the expression of PI3K/Akt,
which plays an essential role in the regulation of MDR1,
MRP1, BCRP and Trkb, E-cadherin, β-catenin, eventually
causing anoikis. PI3K negatively regulates the expression
of COX-2. It demonstrate that celecoxib enhances the
anoikis induction and cytotoxic effect of cisplatin (Fig. 6).
COX-2 is an inducible enzyme and associated with
inflammatory diseases and carcinogenesis, which is
suspected to promote angiogenesis and tissue invasion
of tumors and resistance to apoptosis. COX-2 is also
involved in drug resistance and poor prognosis of tumor.
In the present study, it reveals that celecoxib and
cisplatin inhibit cell growth via induction of apoptosis, and
celecoxib potentiate the growth inhibition of cisplatin
[5, 6]. In our study, a significant reduction in cell viability
and apoptosis in MG-63 cells treated with the
combination of celecoxib and cisplatin as compared with either
celecoxib or cisplatin alone was observed.
Recently, a causal link between COX-2 and MDR-1
gene expression, implicated in cancer chemoresistance,
has been demonstrated. Patel et al.  showed that the
overexpression of COX-2 leads to increased P-gp
expression and activity, and this effect is dependent on COX-2
activity. In addition, COX-2 inhibitor was able to block
the COX-2-mediated increase in MDR1 expression and
activity . It has revealed that the expression of COX-2
and the downstream enzyme involved in PGE2
biosynthesis, mPGES1, was correlated with P-gp and Bcl-xL
. COX-2 overexpression induced increased MRP-1
expression resulting in chemoresistance to cisplatin
. Strong positive correlation between the
expression of COX-2 and MDR1/P-gp was also observed in
hepatocellular carcinoma, breast cancer and ovarian
cancer [23–25]. In addition, COX-2 may be a factor that
can regulate the expression of all three ABC transporters:
MDR1/P-gp, MRP1 and BCRP .
Nonsteroidal anti-inflammatory drugs (NSAIDs) have
been shown to induce apoptosis as well as potentiate
the effect of chemotherapeutic agents including
cisplatin. Celecoxib is a NSAIDs which is a specific inhibitor
of COX-2. NSAIDs and COX-2 selective inhibitors have
been demonstrated to overcome MDR in many cancers.
It has been suggested that COX-inhibitors may
sensitize cancer cells to chemotherapeutic drugs via
inhibiting P-gp, MRP1 and BCRP, and enhance the effect of
anticancer drugs . In imatinib-resistant K562 cells,
celecoxib can inhibit COX-2 and down-regulate MDR-1
expression through Akt/p-Akt signaling pathway .
Akt/protein kinase B (PKB) belongs to the downstream
molecules of PI3K, which plays an important role in
PGE2-EP4 pathway, as shown above. COX-2 inhibitors
are known to inhibit the PI3K/Akt pathway . We have
confirmed that celecoxib induces apoptosis in human
osteosarcoma cell line MG-63 via down-regulation of
PI3K/Akt, as well as potentiates the effect of cisplatin
. In breast cancer, COX-2 inhibitors can also inhibit
P-gp expression and function . Celecoxib
down-regulated the expression of MRP1 protein in human lung
cancer, which was accompanied by increased accumulation
and enhanced cytotoxicity of doxorubicin . Ko et al.
 demonstrated that celecoxib reverses BCRP- and
MRP1-related drug resistance via the down-regulation
of MRP1 and BCRP mRNA in squamous cell carcinoma.
Therefore, COX-2 may play an important role in
upregulation the expression of MDR1/P-gp, MRP1 and BCRP
via the COX-2-PGE2-EP4-PI3K pathway. In addition,
COX-2 inhibitors can reverse the effects of COX-2. And
the mechanisms of COX-2 inhibitors regulate the
transcription of the MDR still do further research.
PI3K/Akt plays a central role in regulation of adhesion
. In cell/cell attachment, β-catenin serves as a
component of the cytoskeleton . β-catenin associates with
the intracellular tail of the intercellular adhesion
molecule E-cadherin . Through this association, β-catenin
plays an important role in strong cell–cell adhesion as it
links E-cadherin to the actin cytoskeleton through the
protein α-catenin . Another function of β-catenin is
that maintaining cell-to-cell adhesion and mediating the
Wnt/β-catenin signal transduction pathway, which plays
pivotal roles in embryogenesis and in malignant
transformation of cells . It has reported that E-cadherin is lost
from cell–cell contacts before the execution of apoptosis
. Activation of PI3K/Akt signaling has been shown to
mediate survival signals triggered by the engagement of
E-cadherin  and other classical cadherins [38, 39].
Fig. 3 The down-regulations of Trkb, E-cadherin and β-catenin activation were detected in MG-63 cells treated with celecoxib (50, 100 µmol/l),
cisplatin (10 µg/ml), and the combination for 48 h. Significant down-regulations of Trkb, E-cadherin and β-catenin activation were observed in MG-63
cells treated with cisplatin and celecoxib (100 µmol/l). However, the potentialization of celecoxib (50 µmol/l) and cisplatin was not demonstrated.
β-actin was used as an equal loading control
Fig. 4 Constitutively active β-catenin binding activity was observed
in nuclear extracts from MG-63, U2OS and HOS cells. In all cells,
compared with untreated control, both celecoxib and cisplatin treatment
obviously decreased β-catenin binding activity, meanwhile,
significant down-regulation of β-catenin binding activity was displayed in
MG-63 and U2OS cells
Fig. 5 MG-63 cells were cultured in the presence (+) or absence
(−) of 1 µmol/l Wortmannin for 48 h. The down-regulations of pAkt
(Thr308) and β-catenin were detected, which were similar to cells
treated with celecoxib
The present report suggests that β-catenin may also
lie downstream of integrins. Several
integrin-stimulated signaling pathways might lead to the induction of
β-catenin signaling. One possible connection between
integrins and β-catenin is the integrin-activated,
antiapoptotic kinase PKB/Akt. PKB is known to inhibit the
activity of glycogen synthase kinase 3-β, a serine kinase
that functions directly to reduce β-catenin protein and
signaling [40, 41]. It is possible that the result of these two
inhibitory interactions is that activation of PKB by
integrin signaling functions to positively activate β-catenin
signaling. TrkB is a receptor tyrosine kinase that has
brain-derived neurotrophic factor (BDNF) as one of its
primary ligands. It has been established that BDNF
binding to TrkB receptors results in a highly specific receptor
Fig. 6 A hypothetical model for PI3K and COX-2 pathway
interaction. Celecoxib down-regulates the expression of PI3K/Akt, which
plays an essential role in the regulation of MDR1, MRP1, BCRP and
Trkb, E-cadherin, β-catenin, eventually causing anoikis. PI3K activity
negatively regulates expression of COX-2
autophosphorylation  and in turn activates the PI3K/
Akt pathway .
In conclusion, MDR1, MRP1, BCRP and Trkb,
E-cadherin, β-catenin were significantly downregulated in
cells treated with the combination of celecoxib and
cisplatin, and decreased β-catenin level was found in cells
with wortmannin, a specific PI3K inhibitor. Therefore,
celecoxib enhances anticancer effect of cisplatin and
induces anoikis in osteosarcoma, which may be PI3K/
Akt-dependent, and MDR and β-catenin-related. PI3K
may be at the center of the celecoxib effects, which play
an essential role in the regulation of MDR and anoikis.
COX: cyclooxygenase; PG: prostaglandin; NSAIDs: nonsteroidal
anti-inflammatory drugs; CSC: cancer stem cell; ECM: extracellar matrix; MDR1/P-gp:
multidrug resistance/P-glycoprotein; MRP1: multidrug resistance protein 1;
BCRP: breast cancer resistance protein.
1. Tsujii M , Kawano S , DuBois RN. Cyclooxygenase-2 expression in human colon cancer cells increases metastatic potential . Proc Natl Acad Sci . 1997 ; 94 : 3336 - 40 .
2. Tsujii M , Kawano S , Tsuji S , Saeaoka H , Hori M , DuBois RN . Cyclooxygenase regulates angiogenesis induced by colon cancer cells . Cell . 1998 ; 93 : 705 - 16 .
3. Tsujii M , DuBois RN . Alterations in cellular adhesions and apoptosis in epithelial cells overexpressing prostaglandin endoperoxide synthase 2 . Cell . 1995 ; 83 : 493 - 501 .
4. Nzeako UC , Guicciardi ME , Yoon JH , Bronk SF , Gores GJ . COX-2 inhibits Fas-mediate apoptosis in cholangiocarcinoma cells . Hepatology . 2002 ; 35 : 552 - 9 .
5. Lin J , Hsiao PW , Chiu TH , Chao JI . Combination of cyclooxygenase-2 inhibitors and oxaliplatin increases the growth inhibition and death in human colon cancer cells . Biochem Pharmacol . 2005 ; 70 ( 5 ): 658 - 67 .
6. Mohammed SI , Craig BA , Mutsaers AJ , et al. Effects of the cyclooxygenase inhibitor, piroxicam, in combination with chemotherapy on tumor response, apoptosis, and angiogenesis in a canine model of human invasive urinary bladder cancer . Mol Cancer Ther . 2003 ; 2 : 183 - 8 .
7. Gupta RA , Dubois RN . Colorectal cancer prevention and treatment by inhibition of cyclooxygenase-2 . Nat Rev Cancer . 2001 ; 1 : 11 - 21 .
8. Williams CS , Watson AJM , Sheng H , Helou R , Shao J , DuBois RN . Celecoxib prevents tumor growth in vivo without toxicity to normal gut: lack of correlation between in vitro and in vivo models . Cancer Res . 2000 ; 60 : 6045 - 51 .
9. Fantappiè O , Solazzo M , Lasagna N , et al. P-glycoprotein mediates celecoxib-induced apoptosis in multiple drug-resistant cell lines . Cancer Res . 2007 ; 67 : 4915 - 23 .
10. Dickens DS , Kozielski R , Khan J , Forus A , Cripe TP . Cyclooxygenase-2 expression in pediatric sarcomas . Pediatr Dev Pathol . 2002 ; 5 : 356 - 64 .
11. Dickens DS , Kozielski R , Leavey PJ , Timmons C , Cripe TP . Cyclooxygenase-2 expression does not correlate with outcome in osteosarcoma or rhabdomyosarcoma . Pediatr Hematol Oncol . 2003 ; 25 : 282 - 5 .
12. Naruse T , Nishida Y , Hosono K , Ishiguro N. Meloxicam inhibits osteosarcoma growth, invasiveness and metastasis by COX-2-dependent and independent routes . Carcinogenesis . 2006 ; 27 : 584 - 92 .
13. Masi L , Recenti R , Silvestri S , Pinzani P , Pepi M , Paglierani M , Brandi ML , Franchi A. Expression of cyclooxygenase-2 in osteosarcoma of bone . Appl Immunohistochem Mol Morphol . 2007 ; 15 : 70 - 6 .
14. Rodriguez NI , Hoots WK , Koshkina NV , Morales-Arias JA , Arndt CA , Inwards CY , Hawkins DS , Munsell MF , Kleinerman ES . COX-2 expression correlates with survival in patients with osteosarcoma lung metastases . J Pediatr Hematol Oncol . 2008 ; 30 : 507 - 12 .
15. Pang LY , Gatenby EL , Kamida A , Whitelaw BA , Hupp TR , Argyle DJ . Global gene expression analysis of canine osteosarcoma stem cells reveals a novel role for COX-2 in tumour initiation . PLoS ONE . 2014 ; 9 ( 1 ): e83144 .
16. Liu B , Shi ZL , Feng J , Tao HM . Celecoxib, a cyclooxygenase-2 inhibitor, induces apoptosis in human osteosarcoma cell line MG-63 via downregulation of PI3K/Akt . Cell Biol Int . 2008 ; 32 : 494 - 501 .
17. Liu B , Qu L , Tao H. Cyclo-oxygenase 2 up-regulates the effect of multidrug resistance . Cell Biol Int . 2009 ; 34 ( 1 ): 21 - 5 .
18. Ruoslahti E , Reed JC. Anchorage dependence, integrins, and apoptosis. Cell . 1994 ; 77 : 477 - 8 .
19. Polakis P , Hart M , Rubinfeld B. Defects in the regulation of β-catenin in colon cancer . Adv Exp Med Biol . 1999 ; 470 : 23 - 32 .
20. Patel VA , Dunn MJ , Sorokin A. Regulation of MDR1 (P-glycoprotein) by cyclooxygenase-2 . J Biol Chem. 2002 ; 277 : 38915 - 20 .
21. Nardone G , Rocco A , Vaira D , et al. Expression of COX-2 , mPGE-synthase1, MDR-1 (P-gp), and Bcl-xL: a molecular pathway of H pylori-related gastric carcinogenesis . J Pathol . 2004 ; 202 : 305 - 12 .
22. Saikawa Y , Sugiura T , Toriumi F , et al. Cyclooxygenase-2 gene induction causes CDDP resistance in colon cancer cell line , HCT-15. Anticancer Res . 2004 ; 24 : 2723 - 8 .
23. Ziemann C , Schäfer D , Rüdell G , et al. The cyclooxygenase system participates in functional Mdr1b overexpression in primary rat hepatocyte cultures . Hepatology . 2002 ; 35 : 579 - 88 .
24. Surowiak P , Materna V , Denkert C , et al. Significance of cyclooxygenase 2 and MDR1/P-glycoprotein coexpression in ovarian cancers . Cancer Lett . 2006 ; 235 : 272 - 80 .
25. Surowiak P , Materna V , Matkowski R , et al. Relationship between cyclooxygenase 2 and P-glycoprotein expressions in invasive breast cancers and their prognostic significance . Breast Cancer Res . 2005 ; 7 : R862 - 70 .
26. Surowiak P , Pawełczyk K , Maciejczyk A , et al. Positive correlation between cyclooxygenase 2 and the expression of ABC transporters in non-small cell lung cancer . Anticancer Res . 2008 ; 28 : 2967 - 74 .
27. Arunasree KM , Roy KR , Anilkumar K , et al. Imatinib-resistant K562 cells are more sensitive to celecoxib, a selective COX-2 inhibitor: role of COX-2 and MDR-1. Leuk Res . 2008 ; 32 : 855 - 64 .
28. Arico S , Pattingre S , Bauvy C , et al. Celecoxib induces apoptosis by inhibiting 3-phosphoinositide-dependent protein kinese-1 activity in the human colon cancer HT-29 cell line . J Biol Chem . 2002 ; 277 : 27613 - 21 .
29. Zatelli MC , Luchin A , Tagliati F , et al. Cyclooxygenase-2 inhibitors prevent the development of chemoresistance phenotype in a breast cancer cell line by inhibiting glycoprotein p-170 expression . Endocr Relat Cancer . 2007 ; 14 : 1029 - 38 .
30. Kang HK , Lee E , Pyo H , Lim SJ. Cyclooxygenase-independent downregulation of multidrug resistance-associated protein-1 expression by celecoxib in human lung cancer cells . Mol Cancer Ther . 2005 ; 4 : 1358 - 63 .
31. Ko SH , Choi GJ , Lee JH , et al. Differential effects of selective cyclooxygenase-2 inhibitors in inhibiting proliferation and induction of apoptosis in oral squamous cell carcinoma . Oncol Rep . 2008 ; 19 : 425 - 33 .
32. Alahari SK , Reddig PJ , Juliano RL . Biological aspects of signal transduction by cell adhesion receptors . Int Rev Cytol . 2002 ; 220 : 145 - 84 .
33. Ozawa M , Baribault H , Kemler R. The cytoplasmic domain of the cell adhesion molecule uvomorulin associates with three independent proteins structurally related in different species . EMBO J . 1989 ; 8 : 1711 - 7 .
34. Kemler R. From cadherins to catenins: cytoplasmic protein interactions and regulation of cell adhesion . Trends Genet . 1993 ; 9 : 317 - 21 .
35. Fuchs SY , Ougolkov AV , Spiegelman VS , Minamoto T. Oncogenic beta-catenin signaling networks in colorectal cancer . Cell Cycle . 2005 ; 4 ( 11 ): 1522 - 39 .
36. Fouquet S , Lugo-Martínez VH , Faussat AM , Renaud F , Cardot P , Chambaz J , Pincon-Raymond M , Thenet S. Early loss of E-cadherin from cell-cell contacts is involved in the onset of anoikis in enterocytes . J Biol Chem . 2004 ; 41 : 43061 - 9 .
37. Bergin E , Levine JS , Koh JS , Lieberthal W. Mouse proximal tubular cell-cell adhesion inhibits apoptosis by a cadherin-dependent mechanism . Am J Physiol . 2000 ; 278 : 758 - 68 .
38. Carmeliet P , Lampugnani MG , Moons L , et al. Targeted deficiency or cytosolic truncation of the VE-cadherin gene in mice impairs VEGF-mediated endothelial survival and angiogenesis . Cell . 1999 ; 98 : 147 - 57 .
39. Tran NL , Adams DG , Vaillancourt RR , Heimark RL . Signal transduction from N-cadherin increases Bcl-2. Regulation of the phosphatidylinositol 3-kinase/Akt pathway by homophilic adhesion and actin cytoskeletal organization . J Biol Chem . 2002 ; 277 : 32905 - 14 .
40. Cook D , Fry MJ , Hughes K , Sumathipala R , Woodgett JR , Dale TC . Wingless inactivates glycogen synthase kinase-3 via an intracellular signaling pathway which involves a protein kinase C . EMBO J . 1996 ; 15 : 4526 - 36 .
41. Cadigan KM , Nusse R. Wnt signaling: a common theme in animal development . Genes Dev . 1997 ; 11 : 3286 - 305 .
42. Klein R , Nanduri V , Jing SA , et al. The trkB tyrosine protein kinase is a receptor for brain-derived neurotrophic factor and neurotrophin-3 . Cell . 1991 ; 66 : 395 - 403 .
43. Douma S , van Laar T , Zevenhoven J , Meuwissen R , van Garderen v , Peeper DS. Suppression of anoikis and induction of metastasis by the neurotrophic receptor TrkB . Nature . 2004 ; 430 : 1034 - 9 .