Arch Gynecol Obstet DOI 10.1007/s00404-014-3555-3
GYNECOLOGIC ONCOLOGY
Celecoxib induces epithelial-mesenchymal transition in epithelial ovarian cancer cells via regulating ZEB1 expression Ruihan Liu • Jingfang Zheng • Chao Li • Yingxin Pang • Qiaomei Zheng • Xiaoxuan Xu Peishu Liu
•
Received: 10 October 2014 / Accepted: 13 November 2014 Ó Springer-Verlag Berlin Heidelberg 2014
Abstract Purpose The purpose of our study was to investigate the therapeutic potential of Celecoxib for epithelial ovarian cancer, especially on cellular morphological changes, proliferation invasion and epithelial-mesenchymal transition (EMT). Method The MTT and transwell assays were performed to evaluate the effect of Celecoxib on proliferation and invasion ability of ovarian cancer cell lines, respectively. Western blot was carried out to detect the expression of epithelial phenotypes, E-cadherin and Keratin, and mesenchymal phenotypes, N-cadherin and Vimentin, as well as p-AKT, p-ERK and ZEB1. ZEB1 small-interfering RNA (siRNA) was used to downregulate the expression of ZEB1 to further inquiring into the downstream of Celecoxibinduced EMT. Results Cellular morphological assessment revealed that both A2780 and SKOV3 cells gradually appeared in the morphology of mesenchymal cells after Celecoxib treatment. The MTT assay demonstrated that celecoxib had no effect on cell proliferation. Transwell assay showed that Celecoxib significantly increased the cell invasion ability. Western blot data proved that the expression of E-cadherin
R. Liu and J. Zheng have contributed equally. R. Liu J. Zheng Y. Pang Q. Zheng X. Xu P. Liu (&) Department of Obstetrics and Gynecology, Qilu Hospital of Shangdong University, No. 107 Wen Hua Xi Road, Jinan 250012, Shandong, China e-mail: [email protected] C. Li Department of Neurosurgery, Qilu Hospital of Shangdong University, No. 107 Wen Hua Xi Road, Jinan 250012, Shandong, China
and keratin was elevated, whereas the expression of N-cadherin and Vimentin was decreased in a dose-dependent manner compared with the untreated cells, the expression of p-AKT, p-ERK and ZEB1 was also obviously elevated. However, ZEB1 siRNA reversed Celecoxib-induced E-cadherin expression and N-cadherin expression, as well as cellular invasiveness. Conclusion Our results indicated that Celecoxib might induce EMT and increase cellular invasiveness in ovarian cancer cells in vitro, which also implied that it needed a comprehensive evaluation in preclinical researches before introducing Celecoxib into the clinical regimen. Keywords
Celecoxib Ovarian cancer EMT ZEB1
Introduction Ovarian cancer has the highest mortality rate among all gynecological malignancies. Epithelial ovarian cancer (EOC) accounts for 90 % of all ovarian cancers [1]. Despite advances in surgery and chemotherapy, the 5-year survival rate has remained approximately only 30 %, which is due to diagnosis at the advanced stage [2]. It has been considered that the leading cause of relapse and death from patients with ovarian cancer is metastasis. Metastasisrelevant events, such as the omentum, peritoneum, diaphragm, and small bowel mesentery, have been confirmed that they are poor prognostic factors for EOC patients [3]. Hence, it is crucial for clarifying the relevant mechanism which prompts ovarian cancer metastasis. Compelling evidence suggested that inflammation has been implicated in cancer initiation and progression [4]. Cyclooxygenase-2 (Cox-2), generated in inflammatory processes, is an inducible enzyme necessary for converting
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arachidonic acid into prostaglandin [5]. Studies indicated that Cox-2 plays crucial roles in invasion, metastasis and epithelial-mesenchymal transition (EMT) in human epithelial tumors [6–8]. These findings supported the appealing strategy of applying Celecoxib, a selective inhibitor of Cox-2, as an attractive compound for anticancer treatment. Several studies suggested that Celecoxib could evoke cell cycle arrest, apoptosis, and anti-angiogenesis [9]. Therefore, a number of clinical trials have been performed to inspect the effect of Celecoxib on the adjuvant therapy of cancers such as non-small cell lung cancer (NSCLC), colorectal cancer (CRC), and oral premalignant lesions, as well as in the treatment of EOC [10–13]. However, these trials demonstrated that using Celecoxib as adjuvant therapy failed to improve the prognosis of patients. Although a study on 45 recurrent patients with ovarian cancer treated with Celecoxib and carboplatin showed promising activity, another clinical trial on 151 patients with ovarian cancer who were treated with Celecoxib and docetaxel plus carboplatin showed no improvement in progression-free survival and overall survival [13, 14]. The mechanisms of these controversial therapeutic effects of Celecoxib in ovarian cancer remain to be investigated. Herein, we tried to investigate the effect of Celecoxib on ovarian cancer cells in vitro. In this study, we found that Celecoxib induced EMT and upregulated an EMT-related transcription factor, ZEB1. Downregulation of ZEB1 reversed Celecoxib-induced EMT and cellular invasiveness. Our experiments suggested that Celecoxib could act in part through a mechanism involving ZEB1 upregulation in ovarian cancer cells. This might provide a possible reason to explain the failure of Celecoxib in improving the prognosis of patients with ovarian cancer.
Aldrich, St Louis, MO, USA) at 50 mM and stored in small aliquots at -20 °C. Throughout this research, all vehicle control cells were treated with DMSO and the DMSO’s final concentration did not exceed 0.1 % (v/v). The medium with or without Celecoxib was replaced every 24 h. Morphological assessment and image capture Cells were seeded into 6-well plates and then treated with 10 mM Celecoxib or equal volume of DMSO as control. After treatment for 48 h, cells were washed to remove dead cells. Morphological changes were monitored using an Olympus IX51 inverted microscope (Olympus Optical, Melville, NY). Pictures were taken using 209 magnification. Cells which presented spindle-like morphology and cellular protrusions were considered as having undergone EMT. Cell viability assay For the cell viability experiment, both cell lines were seeded at a concentration of 3 9 103 cells per well into 96-wells plates. After incubation with 10 mM Celecoxib for 48 h, 10 lL MTT (Sigma–Aldrich, St Louis, MO, USA) solution (5 mg/mL) was added to the culture medium and incubated for another 4 h at 37 °C. The mixture was discarded and the insoluble formazan was dissolved by adding 100 lL DMSO to each well. Cell growth was determined by measuring the absorbance at 570 and 620 nm (background) using a plate reader (BioRad, Hercules, CA). Results are representative of three independent experiments in triplicates. Transwell invasion assay
Materials and methods Cell culture and reagents Human ovarian epithelial cancer cell lines, A2780 and SKOV3 were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The A2780 cells were maintained in RPMI 1640 medium (Gibco, Auckland, New Zealand) supplemented with 2.0 g/L NaHCO3, 10 % fetal bovine serum (TBD, Tianjin, China), and antibiotics (Invitrogen Life Technologies, Carlsbad, CA, USA) at 37 °C in a humidified 5 % CO2 atmosphere. SKOV3 cells were cultured in McCoy’s 5A medium (Sigma–Aldrich, St Louis, MO, USA) with 2.2 g/L NaHCO3, 10 % FBS and antibiotics. Cells were seeded in 6-well plates until reaching a confluence of 70–80 %. Cells were treated with Celecoxib (Sigma–Aldrich, St Louis, MO, USA) as indicated. Celecoxib was dissolved in dimethyl sulfoxide (DMSO, Sigma–
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Invasion assays were conducted using a 24-well transwell chambers (8 lm pore size; Corning Costar, Cambridge, MA) coated with 50 mL of Matrigel (1:4 dilution in serumfree medium; BD Biosciences, San Jose, CA). Upon reaching confluence of 75–80 % with Celecoxib treatment for 48 h, 5 9 104 A2780 cells or 3 9 104 SKOV3 cells were suspended in 100 lL serum-free RPMI-1640 and seeded into the upper chamber. Medium with 10 % FBS as a chemoattractant was added into the bottom compartment of the chamber. After 24 h incubation, non-invaded cells on the upper surface of the filter were removed. Those that invaded to the underside of the filter were fixed with methanol, stained with crystal violet solution, photographed using Olympus IX51 inverted microscope, and cells were counted in five random fields (1009) of each chamber. The average cell numbers of three duplicate assays for each experimental condition were used for statistical analysis.
Arch Gynecol Obstet Fig. 1 Celecoxib downregulates Cox-2 expression and induces cell morphological changes. A2780 and SKOV3 ovarian cancer cell lines were treated with various concentrations of Celecoxib as indicated for 48 h. a, b Downregulation of Cox-2 was evaluated by western blot analysis in both cell lines. c Representative phase-contrast images of both cell lines treated with 10 lM Celecoxib for 48 h. Original magnification, 920
Fig. 2 Celecoxib promotes cell invasion and has no effect on cell proliferation. Invasive assessment in both A2780 and SKOV3 cell lines was detected using transwell assay. a Representative images of A2780 and SKOV3 cells that invaded through the filters (9100 magnification). b Quantification of invasive cells. The bars indicate mean ± SD. The significance of the difference was assessed using student’s t test. ***P B 0.001. c Effect of Celecoxib on the proliferation of A2780 and SKOV3 cells. Both cell lines were incubated with Celecoxib for 48 h and cell numbers was determined by MTT assay
Protein extraction and western blot After transfection and/or treatment with Celecoxib as indicated, the entire cellular protein was extracted using the RIPA (radio immunoprecipitation assay) buffer (50 mM Tris-pH 7.4, 150 mM NaCl, 1 % Triton X-100, and 1 %
sodium deoxycholate, 0.1 % SDS) supplemented with protease inhibitor mix (Thermo Fisher Scientific Inc., Waltham, MA) and transferred to microcentrifuge tubes. Western blotting was performed according to the manufacturer’s instruction. Antibodies included anti-Cox-2, antiZEB1, anti-E-cadherin, anti-N-cadherin, anti-Vimentin,
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Arch Gynecol Obstet Fig. 3 Celecoxib promotes ovarian cancer cells to undergo EMT. EMT hallmarks were examined using western blot analysis. a, b EMT hallmarks in A2780 and SKOV3 after treated with Celecoxib for 48 h. c, d Semiquantification of western blot bands shown in a. Densitometric analysis of E-cadherin and N-cadherin bands were performed and normalized with that of GAPDH. The bars indicate mean ± SD. The significance of the difference was assessed using student’s t test. *P B 0.05, ***P B 0.001
anti-keratin, anti-Phospho-Akt (Ser473), anti-PhosphoERK (Thr202/Tyr204) (Cell Signaling Technology, Danvers, MA, USA) and anti-glyceraldehyde3-phosphate dehydrogenase (GAPDH, Santa Cruz Biotechnology, Dallas, TX, USA). The working concentration of primary and secondary antibodies about western blotting was 1:1,000–2,000 and 1:5,000, respectively. The antigen– antibody immunocomplexes were detected using enhanced chemiluminescent substrate (Thermo Fisher Scientific Inc., Waltham, MA). The band of GAPDH served as a loading control. Densitometric quantification was conducted by ImageJ software (NIH, Bethesda, MD, USA). siRNA transfection Small-interfering RNA (siRNA) specifically targeting human ZEB1 and scrambled negative siRNA control were purchased from Invitrogen (Invitrogen Life Technologies, Carlsbad, CA, USA). The ZEB1 siRNA sequences were as follows: 50 -GCUGAGAAGCCUGAGUCCUCUGUUU-30 . Cells were seeded into 6-well plates overnight to reach 30–50 % confluence. Then they were transfected with 20 nM of siRNA or negative control using the Lipofectamine 2000 (Invitrogen Life Technologies, Carlsbad, CA, USA) in OPTI-MEM media (Gibco, Auckland, New Zealand). Six hours later, the transfected cells were maintained
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in complete medium supplemented with 10 nM Celecoxib. After 48 h, the cells were harvested and used for tumor cell invasion assay and western blot analysis of gene expression. Statistical analysis All of the experiments were repeated three times in triplicate. Data were showed as a mean ± standard deviation. Comparison between groups was assessed using two-tailed Student’s t test and P values of less than 0.05 were considered to be statistically significant (*). All the statistical analyses were performed using GraphPad Prism Version5.01 (GraphPad Software, San Diego, CA, USA).
Results Celecoxib induces cell morphological changes and promotes cell invasion The effect of Celecoxib on Cox-2 expression in A2780 and SKOV3 ovarian cancer cells lines was detected following treatment with various concentrations of Celecoxib as indicated. Celecoxib treatment effectively decreased Cox-2 expression in a dose-dependent manner (Fig. 1a, b). Both
Arch Gynecol Obstet Fig. 4 Celecoxib activates PI3K/AKT and MEK/ERK signaling pathways and upregulates ZEB1 expression. The expression of p-AKT, p-ERK, ZEB1, Snail and Slug were detected by western blot analysis. a, b The effect of Celecoxib on signal pathways after 1 h treatment in both cell lines. c, d Semiquantification of western blot bands shown in a. Densitometric analysis of p-AKT and p-ERK bands were performed and normalized with that of GAPDH. The bars indicate mean ± SD. The significance of the difference was assessed using student’s t test. *P B 0.05, **P B 0.01, ***P B 0.001. e, f The effect of Celecoxib on EMT-related transcription factors after 6 h treatment in A2780 and SKOV3 cells, respectively
A2780 and SKOV3 cells gradually appeared in the morphology of mesenchymal cells compared with the untreated cells (Fig. 1c). Next, the effect of Celecoxib on cell invasion ability was measured using matrigel invasion assay. As shown in Fig. 2a, b, the invasion abilities were significantly increased compared with the control group (P B 0.001) in both cell lines. However, Celecoxib had no effect on cell proliferation (Fig. 2c, P = 0.131 and 0.088). These findings suggested that Celecoxib was able to change the cellular morphology and increase the invasion of ovarian cancer cells. The above changes are the characteristics of EMT. Celecoxib alters EMT phenotypes Then, we detected the alterations of EMT hallmarks following treatment with different concentrations of Celecoxib. After 48 h of treatment, we examined the expression
of epithelial phenotypes, E-cadherin and keratin, and mesenchymal phenotypes, N-cadherin and Vimentin. The results showed that the expression of E-cadherin and keratin was elevated, whereas the expression of N-cadherin and Vimentin was decreased in a dose-dependent manner in both A2780 and SKOV3 cells (Fig. 3a, b). These data suggested that Celecoxib indeed induced EMT in ovarian caner cells. Celecoxib modulates ZEB1 expression through the PI3K/AKT and MEK/ERK signaling pathways To elucidate the mechanism by which Celecoxib induced EMT, two common EMT-related signaling pathways PI3K/ AKT and MEK/ERK were detected in both cell lines. As shown in Fig. 4a, b, after treatment with Celecoxib 1 h, the expression of p-AKT and p-ERK was increased in a dosedependent manner. The EMT-related transcription factors
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Arch Gynecol Obstet Fig. 5 Upregulation of ZEB1 mainly accounts for Celecoxibinduced EMT in ovarian cancer cells. a, b Disruption of ZEB1 using siRNA reversed Celecoxib-induced EMT. c, d Semiquantification of western blot bands shown in a. Densitometric analysis of E-cadherin and N-cadherin bands were performed and normalized with that of GAPDH. The bars indicate mean ± SD. The significance of the difference was assessed using student’s t test. *P B 0.05, **P B 0.01, ***P B 0.001
including ZEB1, Snail and Slug were also determined. It is apparent that ZEB1 was substantially upregulated by Celecoxib. However, the expression of Snail and Slug was barely affected (Fig. 4e, f). Taken together, these results suggested that Celecoxib-induced EMT might by increasing ZEB1 expression via PI3K/AKT and MEK/ERK signaling pathways. Downregulation of ZEB1 impedes Celecoxibstimulated cell invasion and EMT To further determine the effect of ZEB1 in EMT and invasiveness which was stimulated by Celecoxib, siRNA strategy was used in both cell lines. Compared with the Celecoxib treatment group, the expression of ZEB1 was effectively reduced in the group which accepted interference treatment preferentially (Fig. 5a, b). The changes of the protein levels of E-cadherin and N-cadherin in the Celecoxib treatment cells were consistent with previous findings (Fig. 5a, b). Downregulation of ZEB1 expression reversed Celecoxib-induced E-cadherin expression and N-cadherin expression (Fig. 5a, b). The invasive capabilities of cells after Celecoxib treatment with or without ZEB1 siRNA pretreatment were determined. As shown in Fig. 6, downregulation of ZEB1 expression significantly decreased the number of invasion cells. These results
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showed the key role that ZEB1 played in Celecoxibinduced EMT.
Discussion Ovarian cancer is a highly metastatic disease and more than 70 % of the patients are diagnosed at advanced stage with widespread intraperitoneal invasion and metastasis [15]. It has been considered that invasion and metastasis largely contribute to the high mortality in ovarian cancer patients. EMT is a biological process characterized by losing of epithelial properties and gaining of mesenchymal traits during embryogenesis [16]. In recent decades, many studies demonstrated that EMT also aberrantly activated during tumor progression. This process endows malignant cells with invasiveness and metastasis features and is associated with inferior prognosis of cancer patients [17]. To increase migratory and invasiveness capacity, the cells modify their morphology, they become polarized and extend protrusions [18]. In our study, after treatment with selective Cox-2 inhibitor Celecoxib, both A2780 and SKOV3 cells gradually appeared in the morphology of mesenchymal cells and the invasion ability was significantly increased. Celecoxib-induced downregulation of E-cadherin expression accompanied by an
Arch Gynecol Obstet Fig. 6 Downregulation of ZEB1 impedes Celecoxibstimulated cell invasion in ovarian cancer cells. a Representative images of cells that invaded through the filters (9100 magnification). Cells were pretreated with ZEB1 siRNA or negative control and then treated with Celecoxib. After 48 h, cells were seeded into the upper chamber of the transwell system and incubated for another 24 h. b, c Quantification of invasive cells as shown in a. The bars indicate mean ± SD. The significance of the difference was assessed using student’s t test.*P B 0.05, ***P B 0.001
upregulation of N-cadherin expression, further verified the happening of Celecoxib-induced EMT. This result is consistent with a previous study which confirmed that Celecoxib could induce the change of cellular shape and invasiveness via EMT in lung cancer in vitro [19]. The above findings suggested that Celecoxib could trigger the occurrence of EMT, which might provide some explanations to interpret the failure of Celecoxib in improving the prognosis of cancer patients. The detection of the signaling pathways of Celecoxibinduced EMT could elucidate the mechanism behind and provide some enlightenment to overcome it. EMT is regulated by a series of signaling pathways including MEK/ ERK, PI3K/Akt, Smads, and b-catenin, as well as cell surface protein such as integrins [20]. Activation of PI3K/ AKT and MEK/ERK pathways was essential for inducing EMT in ovarian cancer [21]. By analyzing these two common EMT-related signaling pathways, we found that
PI3K/AKT and MEK/ERK simultaneously activated in both cell lines after treatment with Celecoxib. Despite of the different activated upstream molecules, the pathways manipulating EMT appeared to converge on a panel of transcription factors including Snail, Slug and ZEB1, which directly bind to and repress the activity of the E-cadherin promoter [22]. The expression of ZEB1 was substantially elevated by Celecoxib treatment in this study. A recent report demonstrated that ZEB1 was an important EMT regulator and activated the NF-jB complex in breast cancer, which is in consistent with our findings [23]. Another study also showed that ZEB1 implicated in EMT in ovarian cancer cells under a control of microRNA-200 [24]. To show the significance of ZEB1 in the regulation of EMT and cell invasive ability, ZEB1 expression was transiently downregulated in ovarian cancer cells. Downregulation of ZEB1 reversed Celecoxib-induced E-cadherin expression and N-cadherin expression, as well as cellular
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invasiveness regardless of Celecoxib treatment or not. It was also noticed that ZEB1 could enhance the invasive potential of ovarian cancer cells by regulating MMP3 [25]. Likewise, Yang et al. [26] have indicated that upregulation of ZEB1 sensitized lung cancer cells to metastasis. In concert with our findings, those reports supported the crucial role of ZEB1 in regulating EMT and invasiveness. However, these findings seem in conflict with the previous studies which confirmed that higher Cox-2 expression was associated with inferior prognosis in ovarian cancer patients and Cox-2 inhibitors had antitumor effect [27]. Whereas, Wang et al. [19] have demonstrated that Celecoxib could induce EMT in Cox-2 non-expression cancer cells. In other words, Celecoxib-induced EMT is independent of its Cox-2 inhibitory activity. It was also worth noting that in previous studies, Celecoxib at the concentrations of up to 50–100 lM exhibited anticarcinogenic effects [9]. The concentrations of experiments in vitro were much higher than the maximum blood concentrations in human, which was only approximately 5.6 lM after administration of a single oral dose of Celecoxib 800 mg [28]. In this study, we applied Celecoxib in a range of 5–15 lM, which is believed to be more close to physiologically relevant conditions. Celecoxib did not apparently affect the proliferation of ovarian cancer cell under those concentrations. The above could give some explanations for the controversial therapeutic effects of Celecoxib in vitro and in vivo. In summary, our studies provide insight into the possible mechanisms of the failure of selective Cox-2 inhibitor Celecoxib treatment in clinical trials. We have shown that Celecoxib treatment could induce EMT and increase the cellular invasiveness in EOC cells via activating PI3K/ AKT and MEK/ERK signaling pathways and ZEB1 was an important regulator. These findings reminded us that introducing Celecoxib into the clinical regimen must follow the antecedence of comprehensive evaluation in preclinical researches. Acknowledgments This work was supported by the National Natural Science Foundation of China (81370696 and 81101984), the Science and Technology Development Planning of Shandong (2013GGE27031), Promotive research fund for excellent young and middle-aged scientists of Shandong Province (BS2011YY019). Conflict of interest
We declare that we have no conflict of interest.
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25. Sun N, Zhang Q, Xu C, Zhao Q, Ma Y (2014) Molecular regulation of ovarian cancer cell invasion. Tumour Biol. 15 Aug [Epub ahead of print] 26. Yang Y, Ahn YH, Chen Y, Tan X, Guo L (2014) ZEB1 sensitizes lung adenocarcinoma to metastasis suppression by PI3K antagonism. J Clin Invest 124(6):2696–2708 27. Lee JY, Myung SK, Song YS (2013) Prognostic role of cyclooxygenase-2 in epithelial ovarian cancer: a meta-analysis of observational studies. Gynecol Oncol 129(3):613–619 28. Niederberger E, Tegeder I, Vetter G, Schmidtko A, Schmidt H (2001) Celecoxib loses its anti-inflammatory efficacy at high doses through activation of NF-kappaB. FASEB J 15(9):1622–1624
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Celecoxib induces epithelial-mesenchymal transition in epithelial ovarian cancer cells via regulating ZEB1 expression Ruihan Liu • Jingfang Zheng • Chao Li • Yingxin Pang • Qiaomei Zheng • Xiaoxuan Xu Peishu Liu
•
Received: 10 October 2014 / Accepted: 13 November 2014 Ó Springer-Verlag Berlin Heidelberg 2014
Abstract Purpose The purpose of our study was to investigate the therapeutic potential of Celecoxib for epithelial ovarian cancer, especially on cellular morphological changes, proliferation invasion and epithelial-mesenchymal transition (EMT). Method The MTT and transwell assays were performed to evaluate the effect of Celecoxib on proliferation and invasion ability of ovarian cancer cell lines, respectively. Western blot was carried out to detect the expression of epithelial phenotypes, E-cadherin and Keratin, and mesenchymal phenotypes, N-cadherin and Vimentin, as well as p-AKT, p-ERK and ZEB1. ZEB1 small-interfering RNA (siRNA) was used to downregulate the expression of ZEB1 to further inquiring into the downstream of Celecoxibinduced EMT. Results Cellular morphological assessment revealed that both A2780 and SKOV3 cells gradually appeared in the morphology of mesenchymal cells after Celecoxib treatment. The MTT assay demonstrated that celecoxib had no effect on cell proliferation. Transwell assay showed that Celecoxib significantly increased the cell invasion ability. Western blot data proved that the expression of E-cadherin
R. Liu and J. Zheng have contributed equally. R. Liu J. Zheng Y. Pang Q. Zheng X. Xu P. Liu (&) Department of Obstetrics and Gynecology, Qilu Hospital of Shangdong University, No. 107 Wen Hua Xi Road, Jinan 250012, Shandong, China e-mail: [email protected] C. Li Department of Neurosurgery, Qilu Hospital of Shangdong University, No. 107 Wen Hua Xi Road, Jinan 250012, Shandong, China
and keratin was elevated, whereas the expression of N-cadherin and Vimentin was decreased in a dose-dependent manner compared with the untreated cells, the expression of p-AKT, p-ERK and ZEB1 was also obviously elevated. However, ZEB1 siRNA reversed Celecoxib-induced E-cadherin expression and N-cadherin expression, as well as cellular invasiveness. Conclusion Our results indicated that Celecoxib might induce EMT and increase cellular invasiveness in ovarian cancer cells in vitro, which also implied that it needed a comprehensive evaluation in preclinical researches before introducing Celecoxib into the clinical regimen. Keywords
Celecoxib Ovarian cancer EMT ZEB1
Introduction Ovarian cancer has the highest mortality rate among all gynecological malignancies. Epithelial ovarian cancer (EOC) accounts for 90 % of all ovarian cancers [1]. Despite advances in surgery and chemotherapy, the 5-year survival rate has remained approximately only 30 %, which is due to diagnosis at the advanced stage [2]. It has been considered that the leading cause of relapse and death from patients with ovarian cancer is metastasis. Metastasisrelevant events, such as the omentum, peritoneum, diaphragm, and small bowel mesentery, have been confirmed that they are poor prognostic factors for EOC patients [3]. Hence, it is crucial for clarifying the relevant mechanism which prompts ovarian cancer metastasis. Compelling evidence suggested that inflammation has been implicated in cancer initiation and progression [4]. Cyclooxygenase-2 (Cox-2), generated in inflammatory processes, is an inducible enzyme necessary for converting
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arachidonic acid into prostaglandin [5]. Studies indicated that Cox-2 plays crucial roles in invasion, metastasis and epithelial-mesenchymal transition (EMT) in human epithelial tumors [6–8]. These findings supported the appealing strategy of applying Celecoxib, a selective inhibitor of Cox-2, as an attractive compound for anticancer treatment. Several studies suggested that Celecoxib could evoke cell cycle arrest, apoptosis, and anti-angiogenesis [9]. Therefore, a number of clinical trials have been performed to inspect the effect of Celecoxib on the adjuvant therapy of cancers such as non-small cell lung cancer (NSCLC), colorectal cancer (CRC), and oral premalignant lesions, as well as in the treatment of EOC [10–13]. However, these trials demonstrated that using Celecoxib as adjuvant therapy failed to improve the prognosis of patients. Although a study on 45 recurrent patients with ovarian cancer treated with Celecoxib and carboplatin showed promising activity, another clinical trial on 151 patients with ovarian cancer who were treated with Celecoxib and docetaxel plus carboplatin showed no improvement in progression-free survival and overall survival [13, 14]. The mechanisms of these controversial therapeutic effects of Celecoxib in ovarian cancer remain to be investigated. Herein, we tried to investigate the effect of Celecoxib on ovarian cancer cells in vitro. In this study, we found that Celecoxib induced EMT and upregulated an EMT-related transcription factor, ZEB1. Downregulation of ZEB1 reversed Celecoxib-induced EMT and cellular invasiveness. Our experiments suggested that Celecoxib could act in part through a mechanism involving ZEB1 upregulation in ovarian cancer cells. This might provide a possible reason to explain the failure of Celecoxib in improving the prognosis of patients with ovarian cancer.
Aldrich, St Louis, MO, USA) at 50 mM and stored in small aliquots at -20 °C. Throughout this research, all vehicle control cells were treated with DMSO and the DMSO’s final concentration did not exceed 0.1 % (v/v). The medium with or without Celecoxib was replaced every 24 h. Morphological assessment and image capture Cells were seeded into 6-well plates and then treated with 10 mM Celecoxib or equal volume of DMSO as control. After treatment for 48 h, cells were washed to remove dead cells. Morphological changes were monitored using an Olympus IX51 inverted microscope (Olympus Optical, Melville, NY). Pictures were taken using 209 magnification. Cells which presented spindle-like morphology and cellular protrusions were considered as having undergone EMT. Cell viability assay For the cell viability experiment, both cell lines were seeded at a concentration of 3 9 103 cells per well into 96-wells plates. After incubation with 10 mM Celecoxib for 48 h, 10 lL MTT (Sigma–Aldrich, St Louis, MO, USA) solution (5 mg/mL) was added to the culture medium and incubated for another 4 h at 37 °C. The mixture was discarded and the insoluble formazan was dissolved by adding 100 lL DMSO to each well. Cell growth was determined by measuring the absorbance at 570 and 620 nm (background) using a plate reader (BioRad, Hercules, CA). Results are representative of three independent experiments in triplicates. Transwell invasion assay
Materials and methods Cell culture and reagents Human ovarian epithelial cancer cell lines, A2780 and SKOV3 were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The A2780 cells were maintained in RPMI 1640 medium (Gibco, Auckland, New Zealand) supplemented with 2.0 g/L NaHCO3, 10 % fetal bovine serum (TBD, Tianjin, China), and antibiotics (Invitrogen Life Technologies, Carlsbad, CA, USA) at 37 °C in a humidified 5 % CO2 atmosphere. SKOV3 cells were cultured in McCoy’s 5A medium (Sigma–Aldrich, St Louis, MO, USA) with 2.2 g/L NaHCO3, 10 % FBS and antibiotics. Cells were seeded in 6-well plates until reaching a confluence of 70–80 %. Cells were treated with Celecoxib (Sigma–Aldrich, St Louis, MO, USA) as indicated. Celecoxib was dissolved in dimethyl sulfoxide (DMSO, Sigma–
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Invasion assays were conducted using a 24-well transwell chambers (8 lm pore size; Corning Costar, Cambridge, MA) coated with 50 mL of Matrigel (1:4 dilution in serumfree medium; BD Biosciences, San Jose, CA). Upon reaching confluence of 75–80 % with Celecoxib treatment for 48 h, 5 9 104 A2780 cells or 3 9 104 SKOV3 cells were suspended in 100 lL serum-free RPMI-1640 and seeded into the upper chamber. Medium with 10 % FBS as a chemoattractant was added into the bottom compartment of the chamber. After 24 h incubation, non-invaded cells on the upper surface of the filter were removed. Those that invaded to the underside of the filter were fixed with methanol, stained with crystal violet solution, photographed using Olympus IX51 inverted microscope, and cells were counted in five random fields (1009) of each chamber. The average cell numbers of three duplicate assays for each experimental condition were used for statistical analysis.
Arch Gynecol Obstet Fig. 1 Celecoxib downregulates Cox-2 expression and induces cell morphological changes. A2780 and SKOV3 ovarian cancer cell lines were treated with various concentrations of Celecoxib as indicated for 48 h. a, b Downregulation of Cox-2 was evaluated by western blot analysis in both cell lines. c Representative phase-contrast images of both cell lines treated with 10 lM Celecoxib for 48 h. Original magnification, 920
Fig. 2 Celecoxib promotes cell invasion and has no effect on cell proliferation. Invasive assessment in both A2780 and SKOV3 cell lines was detected using transwell assay. a Representative images of A2780 and SKOV3 cells that invaded through the filters (9100 magnification). b Quantification of invasive cells. The bars indicate mean ± SD. The significance of the difference was assessed using student’s t test. ***P B 0.001. c Effect of Celecoxib on the proliferation of A2780 and SKOV3 cells. Both cell lines were incubated with Celecoxib for 48 h and cell numbers was determined by MTT assay
Protein extraction and western blot After transfection and/or treatment with Celecoxib as indicated, the entire cellular protein was extracted using the RIPA (radio immunoprecipitation assay) buffer (50 mM Tris-pH 7.4, 150 mM NaCl, 1 % Triton X-100, and 1 %
sodium deoxycholate, 0.1 % SDS) supplemented with protease inhibitor mix (Thermo Fisher Scientific Inc., Waltham, MA) and transferred to microcentrifuge tubes. Western blotting was performed according to the manufacturer’s instruction. Antibodies included anti-Cox-2, antiZEB1, anti-E-cadherin, anti-N-cadherin, anti-Vimentin,
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Arch Gynecol Obstet Fig. 3 Celecoxib promotes ovarian cancer cells to undergo EMT. EMT hallmarks were examined using western blot analysis. a, b EMT hallmarks in A2780 and SKOV3 after treated with Celecoxib for 48 h. c, d Semiquantification of western blot bands shown in a. Densitometric analysis of E-cadherin and N-cadherin bands were performed and normalized with that of GAPDH. The bars indicate mean ± SD. The significance of the difference was assessed using student’s t test. *P B 0.05, ***P B 0.001
anti-keratin, anti-Phospho-Akt (Ser473), anti-PhosphoERK (Thr202/Tyr204) (Cell Signaling Technology, Danvers, MA, USA) and anti-glyceraldehyde3-phosphate dehydrogenase (GAPDH, Santa Cruz Biotechnology, Dallas, TX, USA). The working concentration of primary and secondary antibodies about western blotting was 1:1,000–2,000 and 1:5,000, respectively. The antigen– antibody immunocomplexes were detected using enhanced chemiluminescent substrate (Thermo Fisher Scientific Inc., Waltham, MA). The band of GAPDH served as a loading control. Densitometric quantification was conducted by ImageJ software (NIH, Bethesda, MD, USA). siRNA transfection Small-interfering RNA (siRNA) specifically targeting human ZEB1 and scrambled negative siRNA control were purchased from Invitrogen (Invitrogen Life Technologies, Carlsbad, CA, USA). The ZEB1 siRNA sequences were as follows: 50 -GCUGAGAAGCCUGAGUCCUCUGUUU-30 . Cells were seeded into 6-well plates overnight to reach 30–50 % confluence. Then they were transfected with 20 nM of siRNA or negative control using the Lipofectamine 2000 (Invitrogen Life Technologies, Carlsbad, CA, USA) in OPTI-MEM media (Gibco, Auckland, New Zealand). Six hours later, the transfected cells were maintained
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in complete medium supplemented with 10 nM Celecoxib. After 48 h, the cells were harvested and used for tumor cell invasion assay and western blot analysis of gene expression. Statistical analysis All of the experiments were repeated three times in triplicate. Data were showed as a mean ± standard deviation. Comparison between groups was assessed using two-tailed Student’s t test and P values of less than 0.05 were considered to be statistically significant (*). All the statistical analyses were performed using GraphPad Prism Version5.01 (GraphPad Software, San Diego, CA, USA).
Results Celecoxib induces cell morphological changes and promotes cell invasion The effect of Celecoxib on Cox-2 expression in A2780 and SKOV3 ovarian cancer cells lines was detected following treatment with various concentrations of Celecoxib as indicated. Celecoxib treatment effectively decreased Cox-2 expression in a dose-dependent manner (Fig. 1a, b). Both
Arch Gynecol Obstet Fig. 4 Celecoxib activates PI3K/AKT and MEK/ERK signaling pathways and upregulates ZEB1 expression. The expression of p-AKT, p-ERK, ZEB1, Snail and Slug were detected by western blot analysis. a, b The effect of Celecoxib on signal pathways after 1 h treatment in both cell lines. c, d Semiquantification of western blot bands shown in a. Densitometric analysis of p-AKT and p-ERK bands were performed and normalized with that of GAPDH. The bars indicate mean ± SD. The significance of the difference was assessed using student’s t test. *P B 0.05, **P B 0.01, ***P B 0.001. e, f The effect of Celecoxib on EMT-related transcription factors after 6 h treatment in A2780 and SKOV3 cells, respectively
A2780 and SKOV3 cells gradually appeared in the morphology of mesenchymal cells compared with the untreated cells (Fig. 1c). Next, the effect of Celecoxib on cell invasion ability was measured using matrigel invasion assay. As shown in Fig. 2a, b, the invasion abilities were significantly increased compared with the control group (P B 0.001) in both cell lines. However, Celecoxib had no effect on cell proliferation (Fig. 2c, P = 0.131 and 0.088). These findings suggested that Celecoxib was able to change the cellular morphology and increase the invasion of ovarian cancer cells. The above changes are the characteristics of EMT. Celecoxib alters EMT phenotypes Then, we detected the alterations of EMT hallmarks following treatment with different concentrations of Celecoxib. After 48 h of treatment, we examined the expression
of epithelial phenotypes, E-cadherin and keratin, and mesenchymal phenotypes, N-cadherin and Vimentin. The results showed that the expression of E-cadherin and keratin was elevated, whereas the expression of N-cadherin and Vimentin was decreased in a dose-dependent manner in both A2780 and SKOV3 cells (Fig. 3a, b). These data suggested that Celecoxib indeed induced EMT in ovarian caner cells. Celecoxib modulates ZEB1 expression through the PI3K/AKT and MEK/ERK signaling pathways To elucidate the mechanism by which Celecoxib induced EMT, two common EMT-related signaling pathways PI3K/ AKT and MEK/ERK were detected in both cell lines. As shown in Fig. 4a, b, after treatment with Celecoxib 1 h, the expression of p-AKT and p-ERK was increased in a dosedependent manner. The EMT-related transcription factors
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Arch Gynecol Obstet Fig. 5 Upregulation of ZEB1 mainly accounts for Celecoxibinduced EMT in ovarian cancer cells. a, b Disruption of ZEB1 using siRNA reversed Celecoxib-induced EMT. c, d Semiquantification of western blot bands shown in a. Densitometric analysis of E-cadherin and N-cadherin bands were performed and normalized with that of GAPDH. The bars indicate mean ± SD. The significance of the difference was assessed using student’s t test. *P B 0.05, **P B 0.01, ***P B 0.001
including ZEB1, Snail and Slug were also determined. It is apparent that ZEB1 was substantially upregulated by Celecoxib. However, the expression of Snail and Slug was barely affected (Fig. 4e, f). Taken together, these results suggested that Celecoxib-induced EMT might by increasing ZEB1 expression via PI3K/AKT and MEK/ERK signaling pathways. Downregulation of ZEB1 impedes Celecoxibstimulated cell invasion and EMT To further determine the effect of ZEB1 in EMT and invasiveness which was stimulated by Celecoxib, siRNA strategy was used in both cell lines. Compared with the Celecoxib treatment group, the expression of ZEB1 was effectively reduced in the group which accepted interference treatment preferentially (Fig. 5a, b). The changes of the protein levels of E-cadherin and N-cadherin in the Celecoxib treatment cells were consistent with previous findings (Fig. 5a, b). Downregulation of ZEB1 expression reversed Celecoxib-induced E-cadherin expression and N-cadherin expression (Fig. 5a, b). The invasive capabilities of cells after Celecoxib treatment with or without ZEB1 siRNA pretreatment were determined. As shown in Fig. 6, downregulation of ZEB1 expression significantly decreased the number of invasion cells. These results
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showed the key role that ZEB1 played in Celecoxibinduced EMT.
Discussion Ovarian cancer is a highly metastatic disease and more than 70 % of the patients are diagnosed at advanced stage with widespread intraperitoneal invasion and metastasis [15]. It has been considered that invasion and metastasis largely contribute to the high mortality in ovarian cancer patients. EMT is a biological process characterized by losing of epithelial properties and gaining of mesenchymal traits during embryogenesis [16]. In recent decades, many studies demonstrated that EMT also aberrantly activated during tumor progression. This process endows malignant cells with invasiveness and metastasis features and is associated with inferior prognosis of cancer patients [17]. To increase migratory and invasiveness capacity, the cells modify their morphology, they become polarized and extend protrusions [18]. In our study, after treatment with selective Cox-2 inhibitor Celecoxib, both A2780 and SKOV3 cells gradually appeared in the morphology of mesenchymal cells and the invasion ability was significantly increased. Celecoxib-induced downregulation of E-cadherin expression accompanied by an
Arch Gynecol Obstet Fig. 6 Downregulation of ZEB1 impedes Celecoxibstimulated cell invasion in ovarian cancer cells. a Representative images of cells that invaded through the filters (9100 magnification). Cells were pretreated with ZEB1 siRNA or negative control and then treated with Celecoxib. After 48 h, cells were seeded into the upper chamber of the transwell system and incubated for another 24 h. b, c Quantification of invasive cells as shown in a. The bars indicate mean ± SD. The significance of the difference was assessed using student’s t test.*P B 0.05, ***P B 0.001
upregulation of N-cadherin expression, further verified the happening of Celecoxib-induced EMT. This result is consistent with a previous study which confirmed that Celecoxib could induce the change of cellular shape and invasiveness via EMT in lung cancer in vitro [19]. The above findings suggested that Celecoxib could trigger the occurrence of EMT, which might provide some explanations to interpret the failure of Celecoxib in improving the prognosis of cancer patients. The detection of the signaling pathways of Celecoxibinduced EMT could elucidate the mechanism behind and provide some enlightenment to overcome it. EMT is regulated by a series of signaling pathways including MEK/ ERK, PI3K/Akt, Smads, and b-catenin, as well as cell surface protein such as integrins [20]. Activation of PI3K/ AKT and MEK/ERK pathways was essential for inducing EMT in ovarian cancer [21]. By analyzing these two common EMT-related signaling pathways, we found that
PI3K/AKT and MEK/ERK simultaneously activated in both cell lines after treatment with Celecoxib. Despite of the different activated upstream molecules, the pathways manipulating EMT appeared to converge on a panel of transcription factors including Snail, Slug and ZEB1, which directly bind to and repress the activity of the E-cadherin promoter [22]. The expression of ZEB1 was substantially elevated by Celecoxib treatment in this study. A recent report demonstrated that ZEB1 was an important EMT regulator and activated the NF-jB complex in breast cancer, which is in consistent with our findings [23]. Another study also showed that ZEB1 implicated in EMT in ovarian cancer cells under a control of microRNA-200 [24]. To show the significance of ZEB1 in the regulation of EMT and cell invasive ability, ZEB1 expression was transiently downregulated in ovarian cancer cells. Downregulation of ZEB1 reversed Celecoxib-induced E-cadherin expression and N-cadherin expression, as well as cellular
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invasiveness regardless of Celecoxib treatment or not. It was also noticed that ZEB1 could enhance the invasive potential of ovarian cancer cells by regulating MMP3 [25]. Likewise, Yang et al. [26] have indicated that upregulation of ZEB1 sensitized lung cancer cells to metastasis. In concert with our findings, those reports supported the crucial role of ZEB1 in regulating EMT and invasiveness. However, these findings seem in conflict with the previous studies which confirmed that higher Cox-2 expression was associated with inferior prognosis in ovarian cancer patients and Cox-2 inhibitors had antitumor effect [27]. Whereas, Wang et al. [19] have demonstrated that Celecoxib could induce EMT in Cox-2 non-expression cancer cells. In other words, Celecoxib-induced EMT is independent of its Cox-2 inhibitory activity. It was also worth noting that in previous studies, Celecoxib at the concentrations of up to 50–100 lM exhibited anticarcinogenic effects [9]. The concentrations of experiments in vitro were much higher than the maximum blood concentrations in human, which was only approximately 5.6 lM after administration of a single oral dose of Celecoxib 800 mg [28]. In this study, we applied Celecoxib in a range of 5–15 lM, which is believed to be more close to physiologically relevant conditions. Celecoxib did not apparently affect the proliferation of ovarian cancer cell under those concentrations. The above could give some explanations for the controversial therapeutic effects of Celecoxib in vitro and in vivo. In summary, our studies provide insight into the possible mechanisms of the failure of selective Cox-2 inhibitor Celecoxib treatment in clinical trials. We have shown that Celecoxib treatment could induce EMT and increase the cellular invasiveness in EOC cells via activating PI3K/ AKT and MEK/ERK signaling pathways and ZEB1 was an important regulator. These findings reminded us that introducing Celecoxib into the clinical regimen must follow the antecedence of comprehensive evaluation in preclinical researches. Acknowledgments This work was supported by the National Natural Science Foundation of China (81370696 and 81101984), the Science and Technology Development Planning of Shandong (2013GGE27031), Promotive research fund for excellent young and middle-aged scientists of Shandong Province (BS2011YY019). Conflict of interest
We declare that we have no conflict of interest.
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