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Celecoxib enhanced the cytotoxic effect of cisplatin in drug-resistant human gastric cancer cells by inhibition of cyclooxygenase-2
Author’s Accepted Manuscript Celecoxib enhanced the cytotoxic effect of cisplatin in drug-resistant Human gastric Cancer cells by inhibition of cyclooxygenase-2 Hong-Bin Xu, Fu-Ming Shen, Qian-Zhou Lv www.elsevier.com
PII: DOI: Reference:
S0014-2999(15)30258-2 http://dx.doi.org/10.1016/j.ejphar.2015.09.025 EJP70232
To appear in: European Journal of Pharmacology Received date: 6 July 2015 Revised date: 15 September 2015 Accepted date: 15 September 2015 Cite this article as: Hong-Bin Xu, Fu-Ming Shen and Qian-Zhou Lv, Celecoxib enhanced the cytotoxic effect of cisplatin in drug-resistant Human gastric Cancer cells by inhibition of cyclooxygenase-2, European Journal of Pharmacology, http://dx.doi.org/10.1016/j.ejphar.2015.09.025 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Celecoxib enhanced the cytotoxic effect of cisplatin in drug-resistant human gastric cancer cells by inhibition of cyclooxygenase-2 Hong-Bin Xu1, 2, Fu-Ming Shen2, Qian-Zhou Lv1* 1
Department of Pharmacy, Zhongshan Hospital, Fudan University, Shanghai
200032, China 2
Department of Pharmacy, Shanghai Tenth People’s Hospital, Tongji University
School of Medicine, Shanghai 200072, China. *
Corresponding to Qian-Zhou Lv: Tel: +86-21-34160880; Fax: +86-21-34160880;
E-mail: [email protected].
Abstract Recently studies indicated that cyclooxygenase-2 might induce P-glycoprotein expression, and was involved in the development of drug resistance phenotype in human gastric cancer cells. The present study was to explore the correlation of celecoxib, a cyclooxygenase-2 specific inhibitor, and P-glycoprotein in drug-resistant gastric cancer cells. The results showed the over-expression of cyclooxygenase-2 and P-glycoprotein in cisplatin-resistant gastric cancer SGC-7901 cells (SGC-7901/DDP), suggesting the possible involvement of cyclooxygenase-2 in the development of P-glycoprotein-mediated drug resistance. Celecoxib was more effective in SGC-7901/DDP cells with a lower inhibitory concentration compared to that in SGC-7901 cells, supporting such a cyclooxygenase-2-dependent drug resistance in SGC-7901/DDP cells. Further studies revealed down-regulation of cyclooxygenase-2 1
and P-glycoprotein expression by celecoxib, and a decline in prostaglandin E2 release and protein kinase A level. Celecoxib-induced apoptosis of SGC-7901/DDP cells led to increased p53 expression, decreased Bcl-2/Bax ratio and up-regulated caspase-3 level. Also, celecoxib induced apoptosis in SGC-7901/DDP cells synergistically with cisplatin. Our study suggested that celecoxib might enhance the cytotoxic effect of chemotherapeutic agents in drug-resistant human gastric cancer cells through a cyclooxygenase-2-dependent manner. Keyword: celecoxib; cyclooxygenase-2; P-glycoprotein; gastric cancer 1. Introduction Gastric cancer is the fourth most common malignancy, and the second leading cause of cancer-related death worldwide (Felay et al., 2010). Chemotherapy plays an important role in the management of patients with gastric cancer. However, the treatment response rate is low and cases of complete remission are rare due to the development of multidrug resistance (MDR). The MDR phenotype is characterized by the over-expression of P-glycoprotein in plasma membrane that works as a pump to extrude anticancer drugs from cells (Gottesman and Pastan 1993). Recently studies reported a close association between cyclooxygenase-2 (COX-2) and P-glycoprotein in human gastric cancer cells (Chen et al., 2013; Gu and Chen, 2012). One study revealed that COX-2 inhibitor could reduce chemotherapeutic agent-induced COX-2 and P-glycoprotein expression in human gastric cancer cells. It suggested that COX-2 might induce P-glycoprotein expression, and was involved in the development of drug resistance phenotype in human gastric cancer cells (Gu and 2
Chen, 2012). The fact that COX-2 inhibitors blocking the COX-2-mediated increase in P-glycoprotein expression and activity in other drug-resistant cancer cells supports such a possibility (Arunasree et al., 2008; Ziemann et al., 2006). However, another study showed that COX-2 inhibitor could decrease the accumulation of chemotherapeutic agent in human gastric cancer cells, and antagonized chemotherapeutic agent-induced cytotoxicity and apoptosis through a COX-2-independent manner (Chen et al., 2013). Therefore, it is not clear whether COX-2 inhibitors could be used as synergic agents to enhance cytotoxicity of chemotherapeutic agents in drug-resistant human gastric cancer. Cisplatin is one of the most effective chemotherapeutic agents for the treatment of gastric cancer (Pasini et al., 2011).
In the present study, cisplatin-resistant human
gastric cancer SGC-7901 cells (SGC-7901/DDP) were developed by continuous exposure of cells to cisplatin. We studied the cytotoxic and pro-apoptotic effects of celecoxib, a COX-2 selective inhibitor, in combination with cisplatin on SGC-7901/DDP cells, to elucidate the possible value and underlying mechanisms of COX-2 selective inhibitor for treatment with gastric cancer. We demonstrated that COX-2 and P-glycoprotein were over-expressed in SGC-7901/DDP cells compared to parental SGC-7901 cells, and celecoxib could inhibit P-glycoprotein expression in a COX-2-dependent way. Celecoxib enhanced cisplatin-induced apoptosis in SGC-7901/DDP cells through prostaglandin E2 (PGE2)-protein kinase A (PKA)-mediated pathway. These results indicated COX-2 inhibitors and chemotherapeutic agents used in combination might produce a synergistic anticancer 3
effect for the treatment of gastric cancer. 2. Materials and Methods 2.1. Chemicals and Drugs Celecoxib, cisplatin, PGE2, 3-(4,5-dimethylthiazol-2-yl)- 2,5- diphenyl tetrazolium bromide (MTT), RIPA lysis buffer and other reagent grade chemicals were purchased from Sigma Chemical Co. (St Louis, MO, USA). The purity of celecoxib was determined to be ≧ 98% by high-performance liquid chromatography. FITC-annexin V and propidium iodide (PI) were obtained from BD Bioscienses (Franklin Lakes, NJ, USA). Antibodies against P-glycoprotein, PKA and prostaglandin E receptor 2 (EP2) were obtained from Abcam (Cambridge, UK). Antibodies for COX-2 and other primary antibodies were purchased from Cell Signaling Technology (Beverly, MA). Cell culture media and supplements were products of GIBCO-BRL (Rockville, MD, USA). 2.2. Cell lines and culture conditions The SGC-7901/DDP cells was developed from the parental SGC-7901 cells by stepwise selection for resistance with increasing concentration of cisplatin and maintained in the presence of 0.5 μg/ml cisplatin. Cells were grown in RPMI 1640 containing 10% fetal calf serum and 300 mg/l glutamine at 37 °C in 5% CO2. 2.3. Cell proliferation and apoptosis assay The cell proliferation was determined by MTT assay (Hansen et al., 1989). Cells (5×104 per well) were seeded to 96-well culture plate and cultured with pharmacological agents at 37 °C for 24 h. After treatment, MTT was added at 37 °C 4
for 2 h, and cell proliferation was analyzed by an ELISA reader (Bio-tek Instruments, VT, USA) at 570 nm. Apoptosis was assessed by labeling cells with annexin V-FITC and PI (Gong et al., 1994). Briefly, cells (5 × 105 per well) were seeded into 6-well plates and then treated with pharmacological agents at 37 °C for 24 h. After treatment, annexin V-FITC and PI double-staining were performed according to manufacturer’s instruction, and cell apoptosis was analyzed by flow cytometry (FACSCalibur, BD Bioscience). 2.4. Western blot analysis After treatment with pharmacological agents, cells were lysed in RIPA buffer. After shook for 30 min and centrifuged (10,000 × g) for 10 min at 37 ℃, the supernatants were collected. The protein content was determined according to the Bradford method (Bradford, 1976). For western blot analysis, equal amount of total cell lysate (100 µg) was resolved on 8-12% SDS-PAGE gels along with protein molecular weight standards, and then transferred onto nitrocellulose membranes. The membranes were blocked for 1 h at room temperature, then incubated with appropriate primary antibodies at 4 ℃ for 8-12 h. The blots were incubated with the secondary antibodies. The bands were visualized with ECL plus detection system (Amersham Pharmacia Biotech., Piscataway, NJ). 2.5. PGE2 estimation After treatment with pharmacological agents, the levels of PGE2 in cells were estimated as per manufacturer’s instructions (Uscn, USA). 2.6. Statistical Analysis 5
Data were expressed as mean ± S.D., and the significance of the difference between groups was determined by ANOVA followed by the Bonferroni post hoc test. P-values below 0.05 were regarded as statistically significant. 3. Results 3.1. Celecoxib inhibited COX-2 and P-glycoprotein over-expression in SGC-7901/DDP cells As shown in Fig. 1, SGC-7901/DDP cells showed over-expression of both COX-2 and P-glycoprotein as compared with SGC-7901 cells. Meanwhile, the results indicated the down-regulation in the protein levels of COX-2 and P-glycoprotein by celecoxib in SGC-7901/DDP cells (Fig. 2), suggesting a possible role for COX-2 in the development of P-glycoprotein-mediated drug resistance in SGC-7901/DDP cells. 3.2. Celecoxib induced apoptosis in SGC-7901/DDP cells and acted synergistically with cisplatin The MTT results showed that a dose-dependent decrease in the growth of cells was observed with increasing concentrations of cisplatin and celecoxib (Fig. 3A-B). Meanwhile, celecoxib enhanced the inhibitory effect of cisplatin on the growth of SGC/7901/DDP cells (Fig. 3D). In the presence of 3 µM celecoxib, the IC50 (concentration resulting in 50% inhibition of cell growth) of cisplatin for SGC-7901/DDP cells was decreased from 19.98 to 14.57 µM (Table 1). In addition, celecoxib showed more potent inhibition in the growth of SGC-7901/DDP cells (IC50 = 35.45 µM) than in SGC-7901 cells (IC50 = 115.08 µM) (Table 1). Therefore, SGC-7901/DDP cells are more sensitive to celecoxib than SGC-7901 cells, either 6
alone or in combination with cisplatin. When PGE2 was applied alone, there was no significant change in cells viabilities (Fig. 3C). However, PGE2 reduced the inhibitory effect of cisplatin on the growth of SGC-7901/DDP cells (Fig. 3D). In the presence of 3 µM PGE2, the IC50 of cisplatin for SGC-7901/DDP cells was increased from 19.98 to 22.17 µM (Table 1). Apoptosis was quantified by using flow cytometer to explore the mechanism of celecoxib-induced cytotoxicity in SGC-7901/DDP cells. After treatment with cisplatin (10 µM) or celecoxib (10 µM) alone, it showed 13.90% and 11.87% cells undergoing apoptosis, respectively (Fig. 4). Further, after treatment with both cisplatin (10 µM) and celecoxib (10 µM), there was a significant increase (26.50%) in the percent apoptosis. However, when cells was treated with cisplatin (10 µM) in combination with PGE2 (10 µM), there was a decrease (9.33%) in the percent apoptosis. These findings suggested that celecoxib induced apoptosis in SGC-7901/DDP cells and had synergy with cisplatin. Celecoxib might enhance the sensitivity of SGC-7901/DDP cells to cisplatin through inhibiting COX-2-mediated p-glycoprotein expression 3.3. Celecoxib down-regulated P-glycoprotein expression through PGE2-PKA-mediated pathway Previous studies reported that PGE2, a predominant metabolic product of COX-2, up-regulated P-glycoprotein expression in cancer cells through binding to the prostaglandin E receptor and activating the PKA-related pathway (Bai et al., 2010; Ziemann et al., 2006). Therefore, the levels of EP2, PKA, cAMP-response element binding protein (CREB) and P-glycoprotein was monitored after cells treatment with 7
PGE2 and celecoxib. The results showed PGE2 (10 µM) induced P-glycoprotein expression in SGC-7901/DDP cells through binding to EP2 and activating PKA and CREB. In contrast, celecoxib (10 µM) inhibited the expression of P-glycoprotein through blocking EP2, and inactivating PKA and CREB (Fig. 5). The levels of PGE2 were also determined to further examine the involvement of COX-2 in the development of drug resistance phenotype. As shown in Fig. 6, there was a significant increase in the PGE2 levels in SGC-7901/DDP cells as compared to SGC-7901 cells. After SGC-7901/DDP cells treatment with celecoxib, it showed a significant decline in the levels of PGE2. These results suggested that COX-2 might play a role in the regulation of P-glycoprotein expression in SGC-7901/DDP cells via PGE2-PKA-mediated pathway. 3.4. Involvement of p53, Bcl-2, caspase-3 in celecoxib-induced apoptosis The activation of p53 leads to either apoptosis or cell cycle arrest and thereby suppresses tumor formation. Therefore, targeting p53 is an important strategy in cancer treatment (Smith et al., 1998; Soussi 2007). We measured the level of p53 in celecoxib-treated SGC-7901/DDP cells. The up-regulation of p53 protein expression in SGC-7901/DDP cells suggested that p53 might be important for the induction of apoptosis mediated by celecoxib (Fig. 7). The Bcl-2 family proteins also play important roles in regulation of apoptosis (Chao and Korsmeyer, 1998). To gain further insights into the mechanism of apoptosis induction in our model, we determined the effects of celecoxib treatment on levels of Bcl-2 family proteins. As shown in Fig. 7, celecoxib (10 µM) treatment caused a marked decrease in the level of 8
Bcl-2 protein. Meanwhile, the levels of Bax and Bcl-xl proteins had no change after treatment with celecoxib. These results suggested that celecoxib-induced cell death might be partly regulated by Bcl-2 family proteins. Caspases play critical roles in execution of apoptosis program (Ghavami et al., 2009). Next, we explored the possibility of whether celecoxib induced cell death was mediated by caspases. The analysis demonstrated increased level of caspase-3 at 24 h (Fig. 7), thus indicating that celecoxib-induced apoptosis may be associated with caspases activation. The levels of p53, Bcl-2 family and caspase-3 had almost no changed after treatment with PGE2 (10 µM) 4. Discussion COX-2 induces the expression of P-glycoprotein in cancer cells, which causes drug resistance, suggesting that COX-2 inhibition might reduce the chemo-resistance phenotype (Arunasree et al., 2008; Ratnasinghe et al., 2001). The presented study also indicates an over-expression of COX-2 and P-glycoprotein in cisplatin-resistant gastric cancer cells, and thereby increasing the survival of these cells despite cisplatin treatment at high concentrations. Celecoxib, a COX-2-specific inhibitor, down-regulates the expression of P-glycoprotein through a COX-2-dependent manner, thus sensitizing drug-resistant cells to chemotherapeutic agent. The results are in line with the previous study, where COX-2-mediated induction of P-glycoprotein expression in drug-resistant human cancer cells is inhibited by celecoxib, and the cytotoxic effects of chemotherapeutic agents to drug-resistant human cancer cells were increased (Kallea et al., 2010). 9
Previous researches reported the significant association between PGE2 and P-glycoprotein expression in drug resistant human cancer cells (Ratnasinghe et al., 2001; Ziemann et al., 2006). PGE2 exerts its effects by binding to and activating prostaglandin E receptor, which cooperated in P-glycoprotein expression regulation, and linked to the activation of cAMP-dependent PKA (Bai et al., 2010; Ziemann et al., 2006). Prostaglandin E receptor/PKA pathway might be important in the regulation of P-glycoprotein expression in drug-resistant cancer cells. EP2 is the predominant receptor in mediating PGE2-induced P-glycoprotein expression (Bai et al., 2010). In the present study also EP2, PKA along with P-glycoprotein was up-regulated in SGC-7901/DDP cells treated with PGE2, suggesting the possible role of COX-2 inhibitor in regulating PGE2-mediated P-glycoprotein expression in SGC-7901/DDP cells. In fact, western blot analysis in the presence of celecoxib in SGC-7901/DDP cells demonstrated down-regulation of P-glycoprotein as well as EP2, PKA expression in SGC-7901/DDP cells. Furthermore, the present study has demonstrated that the production of PGE2 was higher in SGC-7901/DDP cells than in SGC-7901 cells, and increased in SGC-7901/DDP cells following treatment with cisplatin. Celecoxib suppressed cisplatin-induced PGE2 production in SGC-7901/DDP cells. These findings suggested that celecoxib might down-regulated P-glycoprotein expression through PGE2-EP2/PKA-mediated pathway. CREB is a transcription factor that regulates a wide variety of genes by binding to cAMP response element (Conkright et al., 2005; Ghosh et al., 2007). Activation of 10
CREB in cancer cells induces COX-2 transcription, leading to tumor growth through inhibition of apoptosis and increased angiogenesis. CREB-mediated activation of COX-2 as a potential signaling pathway in cancer cells can be targeted for cancer management. Our results indicated the expression of CREB in SGC-7901/DDP cells, suggesting the involvement of CREB in COX-2 mediated cisplatin resistance. Also the up-regulation of CREB is observed in SGC-7901/DDP cells treated with PGE2. However, celecoxib inhibited the CREB activity in SGC-7901/DDP cells, and thereby might restore the sensitivity of SGC-7901/DDP cells to apoptotic stimuli through down-regulation of COX-2. Chemo-resistance represents a major obstacle to cisplatin containing-regimens in the effective treatment of gastric cancer (Pasini et al., 2011). The previous studies have demonstrated that resistance to cisplatin treatment might be due to not only the over-expression of ATP-binding cassette transporters but also the lack of p53 gene (Tang et al., 2013; Zhou et al., 2012). P53-dependent cell cycle arrest is an important component of the cellular response to stress (Luu et al., 2002). When cells receive anticancer drugs such as cisplatin, p53 protein is accumulated (Li et al., 1998; Li et al., 2000; Smith et al., 1995). The increased p53 can trans-activate its downstream target genes to induce cell apoptosis. In addition, accumulating studies found significant correlations between p53 and P-glycoprotein. P53 could sensitize the drug-resistant cancer cells to chemicals through down-regulation of P-glycoprotein expression (Kong et al., 2012). In the present study, we demonstrated the expression of p53 was up-regulated in SGC-7901/DDP cells treated with celecoxib. It suggested that 11
celecoxib might enhance cisplatin-induced p53 expression in SGC-7901/DDP cells. Further research is underway in our lab to investigate whether celcecoxib increases the accumulation of cisplatin in SGC-7901/DDP cells. P53-dependent apoptosis is activated by the Bax/mitochondrial/caspase-9 pathway. Bcl-2 and Bax expression are regulated by p53 both in vitro and in vivo (Miyashita et al., 1994). Therefore, we sought to gain insight into the mechanism of apoptosis induction by celecoxib. Expression of the Bcl-2 family members was examined in celecoxib-treated SGC-7901/DDP cells. We observed down-regulation of anti-apoptotic member Bcl-2 expression; there was no change in the expression of Bax and Bcl-xl. Decreased in the ratio of Bcl-2/Bax could cause changes in the membrane potential of mitochondria, consequently, cytochrome C and other polypeptides were released from the inter-membrane space of mitochondria into cytoplasm. Once released, cytochrome C could activate caspases (Li et al., 1997; Stennicke et al., 1999). The observation of caspase-3 activation further confirmed that the promotion of apoptosis by celecoxib involved a caspase-dependent pathway. In conclusion, our findings suggested that COX-2 and P-glycoprotein over-expression might be responsible for the development of resistance to cisplatin in SGC-7901/DDP cells. Celecoxib, a selective COX-2 inhibitor, might enhance the cytotoxic effect of cisplatin in drug-resistant human gastric cancer SGC-7901/DDP cells through a cyclooxygenase-2-dependent manner. Acknowledgements This work was partly supported by the Scientific Research Foundation of Shanghai 12
Pharmaceutical Society (No. 2014-YY-02-13) and Natural Science Foundation of Shanghai (No. 14ZR1432500). Conflicts of interest statement None declared.
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Table 1 Effect of celecoxib or prostaglandin E2 on cisplatin cytotoxicity in SGC-7901 and SGC-7901/DDP cells
a
Compound
SGC-7901 (IC50 μM)
SGC-7901/DDP (IC50 μM)
Cisplatin
0.452
19.98
Celecoxib
115.08
35.45
Cisplatin + celecoxib (3 μM)
0.410
14.57
Cisplatin + prostaglandin E2 (3 μM)a
0.481
22.17
MTT assay showed that prostaglandin E2 (1-100μM) had no damage on the viability
of SGC-7901 and SGC-7901/DDP cells.
18
Fig. 1. Cyclooxygenase-2 and P-glycoprotein were over-expression in SGC-7901/DDP cells. Representative western blots of proteins (A), quantitated data from immunoblots for cyclooxygenase-2 and P-glycoprotein (B) are shown.
Lane 1:
SGC-7901 cells; Lane 2: SGC-7901/DDP cells. Data were expressed as mean ± S.D. (n = 3). Fig. 2. Celecoxib inhibited cyclooxygenase-2 and P-glycoprotein expression in SGC-7901/DDP cells. Representative western blots of proteins (A), quantitated data from immunoblots for cyclooxygenase-2 and P-glycoprotein (B) are shown. SGC-7901/DDP cells were treated with celecoxib (10 µM), as indicated, for 24 h. Total cell lysate was harvested and subjected to western blot analysis with antibodies against indicated proteins. Lane 1: control (SGC-7901/DDP cells without treatment); Lane 2: SGC-7901/DDP cells treated with 10 µM celecoxib. Data were expressed as mean ± S.D. (n = 3). Fig. 3. Celecoxib inhibited proliferation in SGC-7901/DDP cells and acted synergistically with cisplatin. (A) Cells were cultured for 24 h with or without cisplatin and cell viability was analyzed by MTT assay; (B) cells were cultured for 24 h with or without celecoxib and cell viability was analyzed by MTT assay; (C) cells were cultured for 24 h with or without prostaglandin E2 and cell viability was analyzed by MTT assay; (D) cells were cultured for 24 h with cisplatin in presence or absence of celecoxib (3 µM) or prostaglandin E2 (3 µM) and cell viability was analyzed by MTT assay. Data were expressed as mean ± S.D. (n = 4). Fig. 4. Celecoxib induced apoptosis in SGC-7901/DDP cells and acted synergistically 19
with cisplatin. (A) SGC-7901/DDP cells were cultured for 24 h with cisplatin (10 µM), celecoxib (10 µM) and prostaglandin E2 (10 µM) alone or in combination and apoptotic cells were analyzed by flow cytometry; (B) the percentages of apoptotic cells from flow cytometer analysis. Data were expressed as mean ± S.D. (n = 3). Fig. 5. Celecoxib down-regulated the expression of P-glycoprotein expression through EP2/PKA-mediated pathway. Representative western blots of proteins (A), quantitated data from immunoblots for EP2, PKA, CREB, p-CREB and P-glycoprotein (B) are shown. SGC-7901/DDP cells were treated with prostaglandin E2 (10 µM) and celecoxib (10 µM), as indicated, for 24 h. Total cell lysate was harvested and subjected to western blot analysis with antibodies against indicated proteins. Lane 1: control (SGC-7901/DDP cells without treatment); Lane 2: SGC-7901/DDP cells treated with 10 µM prostaglandin E2; Lane 3: SGC-7901/DDP cells treated with 10 µM celecoxib. Data were expressed as mean ± S.D. (n = 3). * P < 0.05 compared with SGC-7901/DDP cells without treatment. Fig. 6. Celecoxib decreased the release of prostaglandin E2 in SGC-7901/DDP cells. After cells were incubated with celecoxib (0.3, 1 and 3 µM) for 24 h, prostaglandin E2 levels were evaluated by ELISA method. Data were expressed as mean ± S.D. (n = 3). *
P < 0.05 compared with SGC-7901/DDP cells in the presence of 1 µM cisplatin.
Fig. 7. Celecoxib induced SGC-7901/DDP cells apoptosis through increasing p53 expression, decreasing Bcl-2/Bax ratio and up-regulating caspase-3 level. Representative western blots of proteins (A), quantitated data from immunoblots for p53, Bcl-2, Bax, Bcl-xl, and caspase-3 (B) are shown. SGC-7901/DDP cells were 20
treated with prostaglandin E2 (10 µM) and celecoxib (10 µM), as indicated, for 24 h. Total cell lysate was harvested and subjected to western blot analysis with antibodies against indicated proteins. Lane 1: control (SGC-7901/DDP cells without treatment); Lane 2: SGC-7901/DDP cells treated with 10 µM prostaglandin E2; Lane 3: SGC-7901/DDP cells treated with 10 µM celecoxib. Data were expressed as mean ± S.D. (n = 3). * P < 0.05 compared with SGC-7901/DDP cells without treatment.
21
Fig. 1 A 1
2
Cyclooxygenase-2 β-actin
P-glycoprotein β-actin B 15 SGC-7901 SGC-7901/DDP
Fold
10
5
0 Cyclooxygenase-2
P-glycoprotein
Fig. 2 A
1
2
Cyclooxygenase-2 β-actin
P-glycoprotein β-actin
B
120 Control
Protein level (% of control)
Celecoxib (10uM)
60
0 Cyclooxygenase-2
P-glycoprotein
Fig. 3 A
Proliferation inhibition rate (% of control)
100 SGC-7901/DDP SGC-7901
50
0 40
20
10
5
2.5
1.25
0.625
0.3125 0.15625
Cisplatin (uM)
B 100 SGC-7901/DDP Proliferation inhibition rate (% of control)
SGC-7901
50
0 200
100
50
25 Celecoxib (uM)
12.5
6.25
3.125
C 100
Proliferation inhibintion rate (% of control)
SGC-7901/DDP SGC-7901
50
0 100
50
25 12.5 Prostaglandin E2 (uM)
6.25
3.125
D Cisplatin Cisplatin + Celecoxib 3uM Cisplatin + Prostaglandin E2 3uM 100
SGC-7901/DDP
Proliferation inhibition rate (% of control)
SGC-7901
50
0 10
5
2.5
1.25
0.625 0.3125 0.1563
40
Cisplatin (uM )
20
10
5
2.5
1.25
0.625
Fig. 4 A Control
Cisplatin (10µM)
Celecoxib (10µM)
Cisplatin (10µM) + celecoxib (10µM)
Prostaglandin E2 (10µM)
Cisplatin (10µM) + Prostaglandin E2 (10µM)
B 40
% Apoptosis
30
20
10
0 Control
Cisplatin(10µM)
Celecoxib (10µM) Cisplatin (10µM) + Celecoxib (10µM)
Prostaglandin E2
Cisplatin (10µM) + Prostagladin E2 (10µM)
Fig. 5 A
1
2
3
EP2 PKA CREB P-CREB P-glycoprotein β-actin B 160
Protein level (% of control)
140
Control
*
Prostaglandin E2 (10uM) Celecoxib (10uM)
*
*
*
120
*
100
*
*
*
80 60 40
*
20 0 EP2
PKA
CREB
p-CREB
P-glycoprotein
Fig. 6
Prostaglandin E2 (pg/mL)
150
SGC-7901
SGC-7901/DDP
100 *
50
0 Control
Cisplatin 1 µM
Cisplatin 1 µM + Cisplatin 1 µM + Cisplatin 1 µM + Celecoxib 0.3 µM Celecoxib 1 µM Celecoxib 3 µM
Control
Cisplatin 1 µM
Cisplatin 1 µM + Cisplatin 1 µM + Cisplatin 1 µM + Celecoxib 0.3 µM Celecoxib 1 µM Celecoxib 3 µM
Fig. 7 A
1
2
3
P53 Bcl-2 Bax Bcl-xl Caspase-3 β-actin B 180
Control
*
Prostaglandin E2 (10uM)
Protein level (% of control)
Celecoxib (10uM)
* 120
60
*
0 p53
Bcl-2
Bax
Bcl-xl
Caspase-3
PII: DOI: Reference:
S0014-2999(15)30258-2 http://dx.doi.org/10.1016/j.ejphar.2015.09.025 EJP70232
To appear in: European Journal of Pharmacology Received date: 6 July 2015 Revised date: 15 September 2015 Accepted date: 15 September 2015 Cite this article as: Hong-Bin Xu, Fu-Ming Shen and Qian-Zhou Lv, Celecoxib enhanced the cytotoxic effect of cisplatin in drug-resistant Human gastric Cancer cells by inhibition of cyclooxygenase-2, European Journal of Pharmacology, http://dx.doi.org/10.1016/j.ejphar.2015.09.025 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Celecoxib enhanced the cytotoxic effect of cisplatin in drug-resistant human gastric cancer cells by inhibition of cyclooxygenase-2 Hong-Bin Xu1, 2, Fu-Ming Shen2, Qian-Zhou Lv1* 1
Department of Pharmacy, Zhongshan Hospital, Fudan University, Shanghai
200032, China 2
Department of Pharmacy, Shanghai Tenth People’s Hospital, Tongji University
School of Medicine, Shanghai 200072, China. *
Corresponding to Qian-Zhou Lv: Tel: +86-21-34160880; Fax: +86-21-34160880;
E-mail: [email protected].
Abstract Recently studies indicated that cyclooxygenase-2 might induce P-glycoprotein expression, and was involved in the development of drug resistance phenotype in human gastric cancer cells. The present study was to explore the correlation of celecoxib, a cyclooxygenase-2 specific inhibitor, and P-glycoprotein in drug-resistant gastric cancer cells. The results showed the over-expression of cyclooxygenase-2 and P-glycoprotein in cisplatin-resistant gastric cancer SGC-7901 cells (SGC-7901/DDP), suggesting the possible involvement of cyclooxygenase-2 in the development of P-glycoprotein-mediated drug resistance. Celecoxib was more effective in SGC-7901/DDP cells with a lower inhibitory concentration compared to that in SGC-7901 cells, supporting such a cyclooxygenase-2-dependent drug resistance in SGC-7901/DDP cells. Further studies revealed down-regulation of cyclooxygenase-2 1
and P-glycoprotein expression by celecoxib, and a decline in prostaglandin E2 release and protein kinase A level. Celecoxib-induced apoptosis of SGC-7901/DDP cells led to increased p53 expression, decreased Bcl-2/Bax ratio and up-regulated caspase-3 level. Also, celecoxib induced apoptosis in SGC-7901/DDP cells synergistically with cisplatin. Our study suggested that celecoxib might enhance the cytotoxic effect of chemotherapeutic agents in drug-resistant human gastric cancer cells through a cyclooxygenase-2-dependent manner. Keyword: celecoxib; cyclooxygenase-2; P-glycoprotein; gastric cancer 1. Introduction Gastric cancer is the fourth most common malignancy, and the second leading cause of cancer-related death worldwide (Felay et al., 2010). Chemotherapy plays an important role in the management of patients with gastric cancer. However, the treatment response rate is low and cases of complete remission are rare due to the development of multidrug resistance (MDR). The MDR phenotype is characterized by the over-expression of P-glycoprotein in plasma membrane that works as a pump to extrude anticancer drugs from cells (Gottesman and Pastan 1993). Recently studies reported a close association between cyclooxygenase-2 (COX-2) and P-glycoprotein in human gastric cancer cells (Chen et al., 2013; Gu and Chen, 2012). One study revealed that COX-2 inhibitor could reduce chemotherapeutic agent-induced COX-2 and P-glycoprotein expression in human gastric cancer cells. It suggested that COX-2 might induce P-glycoprotein expression, and was involved in the development of drug resistance phenotype in human gastric cancer cells (Gu and 2
Chen, 2012). The fact that COX-2 inhibitors blocking the COX-2-mediated increase in P-glycoprotein expression and activity in other drug-resistant cancer cells supports such a possibility (Arunasree et al., 2008; Ziemann et al., 2006). However, another study showed that COX-2 inhibitor could decrease the accumulation of chemotherapeutic agent in human gastric cancer cells, and antagonized chemotherapeutic agent-induced cytotoxicity and apoptosis through a COX-2-independent manner (Chen et al., 2013). Therefore, it is not clear whether COX-2 inhibitors could be used as synergic agents to enhance cytotoxicity of chemotherapeutic agents in drug-resistant human gastric cancer. Cisplatin is one of the most effective chemotherapeutic agents for the treatment of gastric cancer (Pasini et al., 2011).
In the present study, cisplatin-resistant human
gastric cancer SGC-7901 cells (SGC-7901/DDP) were developed by continuous exposure of cells to cisplatin. We studied the cytotoxic and pro-apoptotic effects of celecoxib, a COX-2 selective inhibitor, in combination with cisplatin on SGC-7901/DDP cells, to elucidate the possible value and underlying mechanisms of COX-2 selective inhibitor for treatment with gastric cancer. We demonstrated that COX-2 and P-glycoprotein were over-expressed in SGC-7901/DDP cells compared to parental SGC-7901 cells, and celecoxib could inhibit P-glycoprotein expression in a COX-2-dependent way. Celecoxib enhanced cisplatin-induced apoptosis in SGC-7901/DDP cells through prostaglandin E2 (PGE2)-protein kinase A (PKA)-mediated pathway. These results indicated COX-2 inhibitors and chemotherapeutic agents used in combination might produce a synergistic anticancer 3
effect for the treatment of gastric cancer. 2. Materials and Methods 2.1. Chemicals and Drugs Celecoxib, cisplatin, PGE2, 3-(4,5-dimethylthiazol-2-yl)- 2,5- diphenyl tetrazolium bromide (MTT), RIPA lysis buffer and other reagent grade chemicals were purchased from Sigma Chemical Co. (St Louis, MO, USA). The purity of celecoxib was determined to be ≧ 98% by high-performance liquid chromatography. FITC-annexin V and propidium iodide (PI) were obtained from BD Bioscienses (Franklin Lakes, NJ, USA). Antibodies against P-glycoprotein, PKA and prostaglandin E receptor 2 (EP2) were obtained from Abcam (Cambridge, UK). Antibodies for COX-2 and other primary antibodies were purchased from Cell Signaling Technology (Beverly, MA). Cell culture media and supplements were products of GIBCO-BRL (Rockville, MD, USA). 2.2. Cell lines and culture conditions The SGC-7901/DDP cells was developed from the parental SGC-7901 cells by stepwise selection for resistance with increasing concentration of cisplatin and maintained in the presence of 0.5 μg/ml cisplatin. Cells were grown in RPMI 1640 containing 10% fetal calf serum and 300 mg/l glutamine at 37 °C in 5% CO2. 2.3. Cell proliferation and apoptosis assay The cell proliferation was determined by MTT assay (Hansen et al., 1989). Cells (5×104 per well) were seeded to 96-well culture plate and cultured with pharmacological agents at 37 °C for 24 h. After treatment, MTT was added at 37 °C 4
for 2 h, and cell proliferation was analyzed by an ELISA reader (Bio-tek Instruments, VT, USA) at 570 nm. Apoptosis was assessed by labeling cells with annexin V-FITC and PI (Gong et al., 1994). Briefly, cells (5 × 105 per well) were seeded into 6-well plates and then treated with pharmacological agents at 37 °C for 24 h. After treatment, annexin V-FITC and PI double-staining were performed according to manufacturer’s instruction, and cell apoptosis was analyzed by flow cytometry (FACSCalibur, BD Bioscience). 2.4. Western blot analysis After treatment with pharmacological agents, cells were lysed in RIPA buffer. After shook for 30 min and centrifuged (10,000 × g) for 10 min at 37 ℃, the supernatants were collected. The protein content was determined according to the Bradford method (Bradford, 1976). For western blot analysis, equal amount of total cell lysate (100 µg) was resolved on 8-12% SDS-PAGE gels along with protein molecular weight standards, and then transferred onto nitrocellulose membranes. The membranes were blocked for 1 h at room temperature, then incubated with appropriate primary antibodies at 4 ℃ for 8-12 h. The blots were incubated with the secondary antibodies. The bands were visualized with ECL plus detection system (Amersham Pharmacia Biotech., Piscataway, NJ). 2.5. PGE2 estimation After treatment with pharmacological agents, the levels of PGE2 in cells were estimated as per manufacturer’s instructions (Uscn, USA). 2.6. Statistical Analysis 5
Data were expressed as mean ± S.D., and the significance of the difference between groups was determined by ANOVA followed by the Bonferroni post hoc test. P-values below 0.05 were regarded as statistically significant. 3. Results 3.1. Celecoxib inhibited COX-2 and P-glycoprotein over-expression in SGC-7901/DDP cells As shown in Fig. 1, SGC-7901/DDP cells showed over-expression of both COX-2 and P-glycoprotein as compared with SGC-7901 cells. Meanwhile, the results indicated the down-regulation in the protein levels of COX-2 and P-glycoprotein by celecoxib in SGC-7901/DDP cells (Fig. 2), suggesting a possible role for COX-2 in the development of P-glycoprotein-mediated drug resistance in SGC-7901/DDP cells. 3.2. Celecoxib induced apoptosis in SGC-7901/DDP cells and acted synergistically with cisplatin The MTT results showed that a dose-dependent decrease in the growth of cells was observed with increasing concentrations of cisplatin and celecoxib (Fig. 3A-B). Meanwhile, celecoxib enhanced the inhibitory effect of cisplatin on the growth of SGC/7901/DDP cells (Fig. 3D). In the presence of 3 µM celecoxib, the IC50 (concentration resulting in 50% inhibition of cell growth) of cisplatin for SGC-7901/DDP cells was decreased from 19.98 to 14.57 µM (Table 1). In addition, celecoxib showed more potent inhibition in the growth of SGC-7901/DDP cells (IC50 = 35.45 µM) than in SGC-7901 cells (IC50 = 115.08 µM) (Table 1). Therefore, SGC-7901/DDP cells are more sensitive to celecoxib than SGC-7901 cells, either 6
alone or in combination with cisplatin. When PGE2 was applied alone, there was no significant change in cells viabilities (Fig. 3C). However, PGE2 reduced the inhibitory effect of cisplatin on the growth of SGC-7901/DDP cells (Fig. 3D). In the presence of 3 µM PGE2, the IC50 of cisplatin for SGC-7901/DDP cells was increased from 19.98 to 22.17 µM (Table 1). Apoptosis was quantified by using flow cytometer to explore the mechanism of celecoxib-induced cytotoxicity in SGC-7901/DDP cells. After treatment with cisplatin (10 µM) or celecoxib (10 µM) alone, it showed 13.90% and 11.87% cells undergoing apoptosis, respectively (Fig. 4). Further, after treatment with both cisplatin (10 µM) and celecoxib (10 µM), there was a significant increase (26.50%) in the percent apoptosis. However, when cells was treated with cisplatin (10 µM) in combination with PGE2 (10 µM), there was a decrease (9.33%) in the percent apoptosis. These findings suggested that celecoxib induced apoptosis in SGC-7901/DDP cells and had synergy with cisplatin. Celecoxib might enhance the sensitivity of SGC-7901/DDP cells to cisplatin through inhibiting COX-2-mediated p-glycoprotein expression 3.3. Celecoxib down-regulated P-glycoprotein expression through PGE2-PKA-mediated pathway Previous studies reported that PGE2, a predominant metabolic product of COX-2, up-regulated P-glycoprotein expression in cancer cells through binding to the prostaglandin E receptor and activating the PKA-related pathway (Bai et al., 2010; Ziemann et al., 2006). Therefore, the levels of EP2, PKA, cAMP-response element binding protein (CREB) and P-glycoprotein was monitored after cells treatment with 7
PGE2 and celecoxib. The results showed PGE2 (10 µM) induced P-glycoprotein expression in SGC-7901/DDP cells through binding to EP2 and activating PKA and CREB. In contrast, celecoxib (10 µM) inhibited the expression of P-glycoprotein through blocking EP2, and inactivating PKA and CREB (Fig. 5). The levels of PGE2 were also determined to further examine the involvement of COX-2 in the development of drug resistance phenotype. As shown in Fig. 6, there was a significant increase in the PGE2 levels in SGC-7901/DDP cells as compared to SGC-7901 cells. After SGC-7901/DDP cells treatment with celecoxib, it showed a significant decline in the levels of PGE2. These results suggested that COX-2 might play a role in the regulation of P-glycoprotein expression in SGC-7901/DDP cells via PGE2-PKA-mediated pathway. 3.4. Involvement of p53, Bcl-2, caspase-3 in celecoxib-induced apoptosis The activation of p53 leads to either apoptosis or cell cycle arrest and thereby suppresses tumor formation. Therefore, targeting p53 is an important strategy in cancer treatment (Smith et al., 1998; Soussi 2007). We measured the level of p53 in celecoxib-treated SGC-7901/DDP cells. The up-regulation of p53 protein expression in SGC-7901/DDP cells suggested that p53 might be important for the induction of apoptosis mediated by celecoxib (Fig. 7). The Bcl-2 family proteins also play important roles in regulation of apoptosis (Chao and Korsmeyer, 1998). To gain further insights into the mechanism of apoptosis induction in our model, we determined the effects of celecoxib treatment on levels of Bcl-2 family proteins. As shown in Fig. 7, celecoxib (10 µM) treatment caused a marked decrease in the level of 8
Bcl-2 protein. Meanwhile, the levels of Bax and Bcl-xl proteins had no change after treatment with celecoxib. These results suggested that celecoxib-induced cell death might be partly regulated by Bcl-2 family proteins. Caspases play critical roles in execution of apoptosis program (Ghavami et al., 2009). Next, we explored the possibility of whether celecoxib induced cell death was mediated by caspases. The analysis demonstrated increased level of caspase-3 at 24 h (Fig. 7), thus indicating that celecoxib-induced apoptosis may be associated with caspases activation. The levels of p53, Bcl-2 family and caspase-3 had almost no changed after treatment with PGE2 (10 µM) 4. Discussion COX-2 induces the expression of P-glycoprotein in cancer cells, which causes drug resistance, suggesting that COX-2 inhibition might reduce the chemo-resistance phenotype (Arunasree et al., 2008; Ratnasinghe et al., 2001). The presented study also indicates an over-expression of COX-2 and P-glycoprotein in cisplatin-resistant gastric cancer cells, and thereby increasing the survival of these cells despite cisplatin treatment at high concentrations. Celecoxib, a COX-2-specific inhibitor, down-regulates the expression of P-glycoprotein through a COX-2-dependent manner, thus sensitizing drug-resistant cells to chemotherapeutic agent. The results are in line with the previous study, where COX-2-mediated induction of P-glycoprotein expression in drug-resistant human cancer cells is inhibited by celecoxib, and the cytotoxic effects of chemotherapeutic agents to drug-resistant human cancer cells were increased (Kallea et al., 2010). 9
Previous researches reported the significant association between PGE2 and P-glycoprotein expression in drug resistant human cancer cells (Ratnasinghe et al., 2001; Ziemann et al., 2006). PGE2 exerts its effects by binding to and activating prostaglandin E receptor, which cooperated in P-glycoprotein expression regulation, and linked to the activation of cAMP-dependent PKA (Bai et al., 2010; Ziemann et al., 2006). Prostaglandin E receptor/PKA pathway might be important in the regulation of P-glycoprotein expression in drug-resistant cancer cells. EP2 is the predominant receptor in mediating PGE2-induced P-glycoprotein expression (Bai et al., 2010). In the present study also EP2, PKA along with P-glycoprotein was up-regulated in SGC-7901/DDP cells treated with PGE2, suggesting the possible role of COX-2 inhibitor in regulating PGE2-mediated P-glycoprotein expression in SGC-7901/DDP cells. In fact, western blot analysis in the presence of celecoxib in SGC-7901/DDP cells demonstrated down-regulation of P-glycoprotein as well as EP2, PKA expression in SGC-7901/DDP cells. Furthermore, the present study has demonstrated that the production of PGE2 was higher in SGC-7901/DDP cells than in SGC-7901 cells, and increased in SGC-7901/DDP cells following treatment with cisplatin. Celecoxib suppressed cisplatin-induced PGE2 production in SGC-7901/DDP cells. These findings suggested that celecoxib might down-regulated P-glycoprotein expression through PGE2-EP2/PKA-mediated pathway. CREB is a transcription factor that regulates a wide variety of genes by binding to cAMP response element (Conkright et al., 2005; Ghosh et al., 2007). Activation of 10
CREB in cancer cells induces COX-2 transcription, leading to tumor growth through inhibition of apoptosis and increased angiogenesis. CREB-mediated activation of COX-2 as a potential signaling pathway in cancer cells can be targeted for cancer management. Our results indicated the expression of CREB in SGC-7901/DDP cells, suggesting the involvement of CREB in COX-2 mediated cisplatin resistance. Also the up-regulation of CREB is observed in SGC-7901/DDP cells treated with PGE2. However, celecoxib inhibited the CREB activity in SGC-7901/DDP cells, and thereby might restore the sensitivity of SGC-7901/DDP cells to apoptotic stimuli through down-regulation of COX-2. Chemo-resistance represents a major obstacle to cisplatin containing-regimens in the effective treatment of gastric cancer (Pasini et al., 2011). The previous studies have demonstrated that resistance to cisplatin treatment might be due to not only the over-expression of ATP-binding cassette transporters but also the lack of p53 gene (Tang et al., 2013; Zhou et al., 2012). P53-dependent cell cycle arrest is an important component of the cellular response to stress (Luu et al., 2002). When cells receive anticancer drugs such as cisplatin, p53 protein is accumulated (Li et al., 1998; Li et al., 2000; Smith et al., 1995). The increased p53 can trans-activate its downstream target genes to induce cell apoptosis. In addition, accumulating studies found significant correlations between p53 and P-glycoprotein. P53 could sensitize the drug-resistant cancer cells to chemicals through down-regulation of P-glycoprotein expression (Kong et al., 2012). In the present study, we demonstrated the expression of p53 was up-regulated in SGC-7901/DDP cells treated with celecoxib. It suggested that 11
celecoxib might enhance cisplatin-induced p53 expression in SGC-7901/DDP cells. Further research is underway in our lab to investigate whether celcecoxib increases the accumulation of cisplatin in SGC-7901/DDP cells. P53-dependent apoptosis is activated by the Bax/mitochondrial/caspase-9 pathway. Bcl-2 and Bax expression are regulated by p53 both in vitro and in vivo (Miyashita et al., 1994). Therefore, we sought to gain insight into the mechanism of apoptosis induction by celecoxib. Expression of the Bcl-2 family members was examined in celecoxib-treated SGC-7901/DDP cells. We observed down-regulation of anti-apoptotic member Bcl-2 expression; there was no change in the expression of Bax and Bcl-xl. Decreased in the ratio of Bcl-2/Bax could cause changes in the membrane potential of mitochondria, consequently, cytochrome C and other polypeptides were released from the inter-membrane space of mitochondria into cytoplasm. Once released, cytochrome C could activate caspases (Li et al., 1997; Stennicke et al., 1999). The observation of caspase-3 activation further confirmed that the promotion of apoptosis by celecoxib involved a caspase-dependent pathway. In conclusion, our findings suggested that COX-2 and P-glycoprotein over-expression might be responsible for the development of resistance to cisplatin in SGC-7901/DDP cells. Celecoxib, a selective COX-2 inhibitor, might enhance the cytotoxic effect of cisplatin in drug-resistant human gastric cancer SGC-7901/DDP cells through a cyclooxygenase-2-dependent manner. Acknowledgements This work was partly supported by the Scientific Research Foundation of Shanghai 12
Pharmaceutical Society (No. 2014-YY-02-13) and Natural Science Foundation of Shanghai (No. 14ZR1432500). Conflicts of interest statement None declared.
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Table 1 Effect of celecoxib or prostaglandin E2 on cisplatin cytotoxicity in SGC-7901 and SGC-7901/DDP cells
a
Compound
SGC-7901 (IC50 μM)
SGC-7901/DDP (IC50 μM)
Cisplatin
0.452
19.98
Celecoxib
115.08
35.45
Cisplatin + celecoxib (3 μM)
0.410
14.57
Cisplatin + prostaglandin E2 (3 μM)a
0.481
22.17
MTT assay showed that prostaglandin E2 (1-100μM) had no damage on the viability
of SGC-7901 and SGC-7901/DDP cells.
18
Fig. 1. Cyclooxygenase-2 and P-glycoprotein were over-expression in SGC-7901/DDP cells. Representative western blots of proteins (A), quantitated data from immunoblots for cyclooxygenase-2 and P-glycoprotein (B) are shown.
Lane 1:
SGC-7901 cells; Lane 2: SGC-7901/DDP cells. Data were expressed as mean ± S.D. (n = 3). Fig. 2. Celecoxib inhibited cyclooxygenase-2 and P-glycoprotein expression in SGC-7901/DDP cells. Representative western blots of proteins (A), quantitated data from immunoblots for cyclooxygenase-2 and P-glycoprotein (B) are shown. SGC-7901/DDP cells were treated with celecoxib (10 µM), as indicated, for 24 h. Total cell lysate was harvested and subjected to western blot analysis with antibodies against indicated proteins. Lane 1: control (SGC-7901/DDP cells without treatment); Lane 2: SGC-7901/DDP cells treated with 10 µM celecoxib. Data were expressed as mean ± S.D. (n = 3). Fig. 3. Celecoxib inhibited proliferation in SGC-7901/DDP cells and acted synergistically with cisplatin. (A) Cells were cultured for 24 h with or without cisplatin and cell viability was analyzed by MTT assay; (B) cells were cultured for 24 h with or without celecoxib and cell viability was analyzed by MTT assay; (C) cells were cultured for 24 h with or without prostaglandin E2 and cell viability was analyzed by MTT assay; (D) cells were cultured for 24 h with cisplatin in presence or absence of celecoxib (3 µM) or prostaglandin E2 (3 µM) and cell viability was analyzed by MTT assay. Data were expressed as mean ± S.D. (n = 4). Fig. 4. Celecoxib induced apoptosis in SGC-7901/DDP cells and acted synergistically 19
with cisplatin. (A) SGC-7901/DDP cells were cultured for 24 h with cisplatin (10 µM), celecoxib (10 µM) and prostaglandin E2 (10 µM) alone or in combination and apoptotic cells were analyzed by flow cytometry; (B) the percentages of apoptotic cells from flow cytometer analysis. Data were expressed as mean ± S.D. (n = 3). Fig. 5. Celecoxib down-regulated the expression of P-glycoprotein expression through EP2/PKA-mediated pathway. Representative western blots of proteins (A), quantitated data from immunoblots for EP2, PKA, CREB, p-CREB and P-glycoprotein (B) are shown. SGC-7901/DDP cells were treated with prostaglandin E2 (10 µM) and celecoxib (10 µM), as indicated, for 24 h. Total cell lysate was harvested and subjected to western blot analysis with antibodies against indicated proteins. Lane 1: control (SGC-7901/DDP cells without treatment); Lane 2: SGC-7901/DDP cells treated with 10 µM prostaglandin E2; Lane 3: SGC-7901/DDP cells treated with 10 µM celecoxib. Data were expressed as mean ± S.D. (n = 3). * P < 0.05 compared with SGC-7901/DDP cells without treatment. Fig. 6. Celecoxib decreased the release of prostaglandin E2 in SGC-7901/DDP cells. After cells were incubated with celecoxib (0.3, 1 and 3 µM) for 24 h, prostaglandin E2 levels were evaluated by ELISA method. Data were expressed as mean ± S.D. (n = 3). *
P < 0.05 compared with SGC-7901/DDP cells in the presence of 1 µM cisplatin.
Fig. 7. Celecoxib induced SGC-7901/DDP cells apoptosis through increasing p53 expression, decreasing Bcl-2/Bax ratio and up-regulating caspase-3 level. Representative western blots of proteins (A), quantitated data from immunoblots for p53, Bcl-2, Bax, Bcl-xl, and caspase-3 (B) are shown. SGC-7901/DDP cells were 20
treated with prostaglandin E2 (10 µM) and celecoxib (10 µM), as indicated, for 24 h. Total cell lysate was harvested and subjected to western blot analysis with antibodies against indicated proteins. Lane 1: control (SGC-7901/DDP cells without treatment); Lane 2: SGC-7901/DDP cells treated with 10 µM prostaglandin E2; Lane 3: SGC-7901/DDP cells treated with 10 µM celecoxib. Data were expressed as mean ± S.D. (n = 3). * P < 0.05 compared with SGC-7901/DDP cells without treatment.
21
Fig. 1 A 1
2
Cyclooxygenase-2 β-actin
P-glycoprotein β-actin B 15 SGC-7901 SGC-7901/DDP
Fold
10
5
0 Cyclooxygenase-2
P-glycoprotein
Fig. 2 A
1
2
Cyclooxygenase-2 β-actin
P-glycoprotein β-actin
B
120 Control
Protein level (% of control)
Celecoxib (10uM)
60
0 Cyclooxygenase-2
P-glycoprotein
Fig. 3 A
Proliferation inhibition rate (% of control)
100 SGC-7901/DDP SGC-7901
50
0 40
20
10
5
2.5
1.25
0.625
0.3125 0.15625
Cisplatin (uM)
B 100 SGC-7901/DDP Proliferation inhibition rate (% of control)
SGC-7901
50
0 200
100
50
25 Celecoxib (uM)
12.5
6.25
3.125
C 100
Proliferation inhibintion rate (% of control)
SGC-7901/DDP SGC-7901
50
0 100
50
25 12.5 Prostaglandin E2 (uM)
6.25
3.125
D Cisplatin Cisplatin + Celecoxib 3uM Cisplatin + Prostaglandin E2 3uM 100
SGC-7901/DDP
Proliferation inhibition rate (% of control)
SGC-7901
50
0 10
5
2.5
1.25
0.625 0.3125 0.1563
40
Cisplatin (uM )
20
10
5
2.5
1.25
0.625
Fig. 4 A Control
Cisplatin (10µM)
Celecoxib (10µM)
Cisplatin (10µM) + celecoxib (10µM)
Prostaglandin E2 (10µM)
Cisplatin (10µM) + Prostaglandin E2 (10µM)
B 40
% Apoptosis
30
20
10
0 Control
Cisplatin(10µM)
Celecoxib (10µM) Cisplatin (10µM) + Celecoxib (10µM)
Prostaglandin E2
Cisplatin (10µM) + Prostagladin E2 (10µM)
Fig. 5 A
1
2
3
EP2 PKA CREB P-CREB P-glycoprotein β-actin B 160
Protein level (% of control)
140
Control
*
Prostaglandin E2 (10uM) Celecoxib (10uM)
*
*
*
120
*
100
*
*
*
80 60 40
*
20 0 EP2
PKA
CREB
p-CREB
P-glycoprotein
Fig. 6
Prostaglandin E2 (pg/mL)
150
SGC-7901
SGC-7901/DDP
100 *
50
0 Control
Cisplatin 1 µM
Cisplatin 1 µM + Cisplatin 1 µM + Cisplatin 1 µM + Celecoxib 0.3 µM Celecoxib 1 µM Celecoxib 3 µM
Control
Cisplatin 1 µM
Cisplatin 1 µM + Cisplatin 1 µM + Cisplatin 1 µM + Celecoxib 0.3 µM Celecoxib 1 µM Celecoxib 3 µM
Fig. 7 A
1
2
3
P53 Bcl-2 Bax Bcl-xl Caspase-3 β-actin B 180
Control
*
Prostaglandin E2 (10uM)
Protein level (% of control)
Celecoxib (10uM)
* 120
60
*
0 p53
Bcl-2
Bax
Bcl-xl
Caspase-3