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Celecoxib inhibits STAT3 phosphorylation and suppresses cell migration and colony forming ability in rhabdomyosarcoma cells
Biochemical and Biophysical Research Communications 407 (2011) 450–455
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Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Celecoxib inhibits STAT3 phosphorylation and suppresses cell migration and colony forming ability in rhabdomyosarcoma cells Suzanne Reed a,b, Huameng Li c, Chenglong Li c, Jiayuh Lin a,⇑ a
Center for Childhood Cancer, The Research Institute at Nationwide Children’s Hospital, The Ohio State University, College of Medicine, Columbus, OH 43205, USA Division of Pediatric Hematology and Oncology, Nationwide Children’s Hospital, The Ohio State University, College of Medicine, Columbus, OH 43205, USA c Division of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, The Ohio State University, Columbus, OH 43205, USA b
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Article history: Received 2 March 2011 Available online 11 March 2011 Keywords: STAT3 Celecoxib Rhabdomyosarcoma
a b s t r a c t Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in the pediatric and adolescent population. Though treatments for localized disease have reasonable long-term success rates, if disease is diffuse at diagnosis, outcomes are far poorer. Additional and/or alternative therapies are critical for improved clinical outcomes. One potentially therapeutic target is the signal transducer and activator of transcription 3 (STAT3) pathway. STAT3 has been shown to have constitutive activation in human rhabdomyosarcoma cells; thus, inhibition of STAT3 signaling may be a mechanism to induce tumor cell death. Celecoxib has been shown, by computer modeling, to bind STAT3 at the SH2 domain and competitively inhibit native peptide binding necessary for phosphorylation and subsequent propagation of the STAT3 signaling cascade. We found that celecoxib inhibits IL-6-induced and persistent STAT3 phosphorylation and inhibits cell viability in human rhabdomyosarcoma cells. We found that genes downstream of STAT3 (BCL-2, survivin, cyclin D1) were downregulated with celecoxib. Celecoxib also inhibits colony formation and cell migration. Our results suggest that, though known more commonly as a cyclooxygenase-2 (COX-2) inhibitor, celecoxib could act through the STAT3 pathway as well. More importantly, its effect on cell migration and clonogenic colony forming ability make it a potentially useful therapeutic agent for rhabdomyosarcoma, especially in metastatic disease whose clinical outcome is marginal at best with current therapies. Ó 2011 Elsevier Inc. All rights reserved.
1. Introduction According to the National Cancer Institute, malignancy is the leading cause of death by disease in children between infancy and 15 years of age. Rhabdomyosarcoma, an often aggressive malignancy originating from skeletal muscle cells, is the most common soft tissue sarcoma in children and adolescents [1]. Prior to the use of multimodal therapy – including surgery, chemotherapy, and radiation therapy – disease-free survival was less than 30% [2]. Presently, with this multimodal therapy, greater than 70% of patients with localized rhabdomyosarcoma can be cured of their disease [3,4]. These improvements in outcome can be attributed to the use of intensive combination chemotherapy, more effective local therapy with surgery and radiation, and improved supportive care [5]. Though encouraging strides have been made for localized disease, metastatic rhabdomyosarcoma has not seen the same ⇑ Corresponding author. Address: Center for Childhood Cancer, The Research Institute at Nationwide Children’s Hospital, Department of Pediatrics, The Ohio State University, 700 Children’s Drive, Columbus, OH 43205, USA. E-mail address: [email protected] (J. Lin). 0006-291X/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2011.03.014
improvement in survival, and affected patients remain with dismal prognoses. The overall survival in metastatic rhabdomyosarcoma is approximately 30%, and this statistic has not significantly changed in more than 20 years [2–4]. Excluding patients younger than 10 years with embryonal RMS, which is a specific population with a better prognosis, patients with metastatic rhabdomyosarcoma have an estimated five-year failure-free survival of <20% [2–4]. Because of this, novel therapeutic agents are desperately needed to help target these particularly aggressive tumors. One such novel approach is through manipulation of signal transducers and activators of transcription (STATs) and, in particular, via the STAT3 signaling cascade. STAT3 is one of a group of transcription factors that was initially discovered as an acute phase reactant [6], but over time has been found to have many more roles. Its importance is multi-factorial; in addition to response to inflammation, STAT3 has been found to contribute to cell transformation, proliferation, and suppression of apoptosis [7,8]. It also has been well-defined in the literature as a mediator of angiogenesis and in malignant transformation of cell populations [6,9]. STAT3 has been demonstrated to be constitutively active in many tumor cell lines, most of which are considered adult malignancies. Constitutive STAT3 activation has been found in cancers of the breast,
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treatment, the cells were incubated for 72 h. Assay was completed by adding 25 lL 3-(4,5-dimethylthiazolyl)-2,5-diphenyltetrazolium bromide (MTT) according to manufacturer’s protocol (Roche Diagnostics, Mannheim, Germany). After incubation at 37 °C for 3 more hours, 100 lL of N,N-dimethylformamide solubilization solution was added to each well allowing absorbance to be measured by spectrophotometry. Half-maximal inhibitory concentrations (IC50) were determined using Calcusyn software (Biosoft, Cambridge, Great Britain). 2.4. Western blot analysis
Fig. 1. Computer modeling of celecoxib binding to STAT3 SH2 domain. Celecoxib is rendered in thick stick-ball model. The native pTyr-Leu706 phosphopeptide binding of the partnering SH2 in homo-dimerization is in thin stick-ball model. The docked binding modes show that celecoxib effectively competes with pTyr-Leu706 peptide for pTyr 705 binding site of STAT3 SH2.
lung, prostate, ovary, and pancreas, and also in melanoma and multiple myeloma [9–11]. More recently, STAT3 has also been found to be constitutively activated in human rhabdomyosarcoma cells and crucial for cell growth and survival of sarcoma cells [12,13]. Celecoxib is a selective COX-2 inhibitor, originally marketed for its anti-inflammatory properties, which has been gaining credit for its anti-tumor effects as well [14]. The mechanisms by which celecoxib exerts these anti-neoplastic effects are being investigated. One such mechanism, we suggest, is via the STAT3 pathway. In a computer binding model, celecoxib was shown to bind to STAT3 at the SH2 domain, blocking the native pTyr-Leu706 phosphopeptide and functionally preventing phosphorylation (Fig. 1). Using this computer modeling evidence, the objective of this study was to determine if celecoxib inhibits phosphorylation of STAT3 in human rhabdomyosarcoma cells in vitro and thus induces cell death. 2. Materials and methods 2.1. Cell lines Human rhabdomyosarcoma lines RH30 and RH28 were obtained from Dr. Peter Houghton’s lab and cell line RD2 was purchased from American Type Culture Collection (ATCC). SMS-CTR cell line was used for cytokine-induced STAT3 inhibition. All cell lines were maintained in Dulbecco’s Modified Eagle Medium, supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. The cell lines were stored in a humidified 37 °C incubator with 5% CO2. 2.2. Celecoxib Celecoxib powder (Catalog No. C-1502) was purchased from LC Laboratories. A 50 mmol/L solution was prepared by mixing powder dissolved in sterile dimethyl sulfoxide. This solution was stored at 20 °C. 2.3. MTT cell viability assay Rhabdomyosarcoma cells were seeded in 96-well plates. Five thousand cells were seeded per well. After seeding, the cells were incubated overnight. The following day, the cells were treated with escalating doses of celecoxib (serial dilutions prepared to achieve concentrations of: 10, 25, 50, 75, 100, and 125 lM). After
Rhabdomyosarcoma cells (RH30, RH28, and RD2) were seeded in sterile 10-cm plates in DMEM media with 10% FBS and 1% penicillin/streptomycin. Cells were incubated overnight at 37 °C. After incubation, one plate was treated with DMSO (as a control) and each of the other plates was treated with celecoxib. Escalating concentrations of 50, 75, and 100 lM celecoxib were prepared. The cells were treated for 18 h. Cells were then washed with cold purified buffered saline, collected, and lysed with cold, protease-containing RIPA buffer. Protein concentrations were determined using the BCA protein assay according to the manufacturer’s protocol (PIERCE Biotechnologies, Rockford, IL). Protein samples were separated by SDS gel and transferred to PVDF membranes. Membranes were then separately probed with antibodies (Cell Signaling Technology) for phospho-STAT3 (Tyrosine 705), STAT3, cleaved caspase-3, COX-2, and GAPDH. Specific protein was then identified by enhanced chemiluminescence. The above western blot method was used with SMS-CTR cells, with a few exceptions. These cells were seeded and grown in DMEM media with 10% FBS for 24 h, then media was removed and replaced with serum-free DMEM media. Cells were serum starved for 24 h, then treated with treated with DMSO (as a control) or celecoxib, at 50 and 75 lM, for 1 h. After 1 h, 25 ng/mL Interleukin-6 (IL-6) was added. One plate was treated with IL-6 only. After thirty more minutes, these treated cells were collected in the same fashion as the other cell lines, and subsequent western blotting technique was the same as indicated. 2.5. Reverse transcriptase-polymerase chain reaction (RT- PCR) Rhabdomyosarcoma cells were treated with celecoxib in different concentrations (50, 75, and 100 lM) or DMSO for 18 h. Cellular RNA was collected using RNeasy Kits (Qiagen, Valencia, CA). Reverse transcription was accomplished by Omniscript reverse transcription kit. Subsequent PCR amplification was performed under these specific conditions: 5 min at 94 °C followed by 20 cycles of 30 s at 94 °C, 30 s at 55 °C, and 30 s at 72 °C with a final extension of 5 min at 72 °C. Primer sequences used are as follows: Bcl-2: Forward: 50 -TCTTTGAGTTCGGTGGGGTC-30 Reverse: 50 -TGCATATTT GTTTGGGGCAGG-30 [15]. Cyclin D1: Forward: 50 -GCTGGAG CCCGTGAAAAAGA-30 Reverse: 50 -CTCCGCCTCTGGCATTTTG-30 [16]. Survivin: Forward: 50 -ACCAGGTGAGAAGTGAGGGA-30 Reverse: 50 -AACAGTAGAGGAGCCAGGGA-30 [17]. GAPDH: Forward: 50 -TGATGACATCAAGAAGGTGGTGAAG-30 Reverse: 50 -TCCTTGGA GGCCATGTGGGCAT-30 [18]. 2.6. Wound healing assay Rhabdomyosarcoma cells were seeded in 6-cm plates. When cells were 100% confluent, a wound was created using a pipette tip to make a linear scratch through the monolayer. Simultaneously, new medium containing celecoxib (50, 75, or 100 lM) or DMSO was added to the plates. The treatment-containing media was removed after 4 h and fresh media was added. After an additional 24 h, the degree of wound healing was evaluated using
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microscope imaging (Leica MZ 16FA microscope, Leica Microsystems, Bannockburn, IL).
2.7. Clonogenic colony formation assay Rhabdomyosarcoma cells were seeded on 10-cm plates at a density of 2000 cells per plate. Cells were then treated with DMSO or celecoxib (50, 75, or 100 lM) for 4 h. The cells were maintained at 37 °C and allowed to grow for 3 weeks. Colonies of cells were then fixed with cold methanol for 30 min and stained with 1% crystal violet.
3. Results 3.1. Computational modeling of celecoxib binding to STAT3 The druggable binding site of STAT3 SH2 domain (PDB code 1BG1) consists of three sub-pockets: pTyr705 binding site, Leu706 site, and a side sub-pocket. Molecular docking program MLSD based on AutoDock4 was used to dock celecoxib to the binding site on STAT3 SH2. Lamarckian Genetic Algorithms (LGA) was used as searching method in docking. Parameters and input files of docking simulations were prepared as previously reported [19,20]. Docking simulations were performed for 75 runs, with 3.5 million energy evaluations in each run. Computational docking resulted in a major cluster with 88% conformers, with a lowest binding energy of 7.2 kcal/mole. As shown in Fig. 1, celecoxib docked to the major pTyr705 binding site and side sub-pocket. Binding mode analysis also found that sulfonamide moiety of celecoxib formed two hydrogen bonds with residues Glu612 and Arg609 of the receptor, respectively. Computer modeling shows that celecoxib could effectively compete with pTyr-Leu706 peptide for pTyr 705 binding site of STAT3 SH2.
3.2. Cell viability and proliferation is decreased by celecoxib Cell viability assays were used to identify inhibitory effect of celecoxib on rhabdomyosarcoma cells. A dose-dependent inhibition in cell viability was demonstrated after treatment. IC50 values for each cell line treated with celecoxib were determined by Calcusyn software. IC50 values are as follows: RH30 (61.05 lM); RH28 (63.3 lM); RD2 (43.5 lM). 3.3. STAT3 phosphorylation is inhibited by celecoxib To investigate inhibition of STAT3 phosphorylation, western blot analysis was used to detect presence of phosphorylated STAT3 (PSTAT3) after treatment with celecoxib. Cells from each RMS cell line (RH30, RH28, and RD2) were plated and treated with escalating doses of celecoxib: 50, 75, or 100 lM. DMSO was used as the control group. As shown in Fig. 2A, we found a decrease in the amount of PSTAT3 with increasing doses of celecoxib. Membranes were stripped then probed for total STAT3, which demonstrated consistent expression with escalating celecoxib doses. This suggests that celecoxib could inhibit STAT3 phosphorylation, and the decrease in PSTAT3 would not be explained by a constitutional decrease in total STAT3. In addition, we separately probed membranes for COX-2 and found consistent COX-2 expression with different celecoxib doses. This invariable expression of COX-2 further suggests the mechanism of inhibition of cell viability is independent of the COX-2 pathway. GAPDH, used as a control, showed consistent expression with escalating doses of celecoxib. As additional evidence of inhibition of STAT3 phosphorylation, we used RT-PCR to amplify genes known to be expressed downstream of STAT3. As illustrated in Fig. 3, downstream genes bcl-2, survivin, cyclin D showed decreased expression with increasing doses of celecoxib in RH30, RH28, and RD2 RMS cells. This yields additional evidence that celecoxib inhibits STAT3 phosphorylation. GAPDH, used as a control, showed consistent expression with escalating celecoxib doses.
Fig. 2. (A) Western blot analysis of RMS cells treated with celecoxib, demonstrating inhibition of STAT3 phosphorylation in RMS cells. RH30, RH28, and RD2 RMS cell lines show a decrease in expression of phosphorylated STAT3 in response to increasing doses of celecoxib. Total STAT3 expression and COX-2 expression remain stable. (B) Western blot analysis of RMS cells with minimal endogenous phosphorylated STAT3 expression (SMS-CTR) demonstrating increased expression of PSTAT3 with IL-6 alone, then subsequent inhibition with celecoxib. Total STAT3 expression remains stable.
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Fig. 3. RT-PCR analysis of RMS cells treated with celecoxib, demonstrating decreased expression of genes downstream of STAT3. RH30, RH28, and RD2 RMS cell lines show a decrease in expression of downstream genes: BCL-2, cyclin D, survivin. Degree of expression decrease is correlated with increased celecoxib doses.
Fig. 4. (A) Wound healing assay demonstrating impaired wound healing in RMS cells after treatment with celecoxib. RMS cell lines RH30, RH28, and RD2 show more impaired wound healing, and ultimately no wound healing (in 100 lM treatment group), with increasing doses of celecoxib. (B) Clonogenic colony forming assay demonstrating that celecoxib inhibits colony formation in RMS cell lines, RH30 and RD2. Again, degree of inhibition is correlated with increasing celecoxib doses.
3.4. Celecoxib inhibits IL-6-induced STAT3 phosphorylation in RMS cells To investigate if celecoxib could inhibit IL-6 induced STAT3 phosphorylation, we chose a RMS cell line with low endogenous PSTAT3 expression, SMS-CTR. We used IL-6 to induce STAT3
expression while treating with celecoxib. We again used western blot analysis to detect PSTAT3. As seen in Fig. 2B, PSTAT3 had increased expression with IL-6 alone, then demonstrated subsequent decreased expression with treatment with celecoxib. Consistent expression of total STAT3 again suggests that this is specific inhibition of phosphorylation of STAT3 rather than inhibition of STAT3
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globally. GAPDH was used as a control and also showed consistent expression. 3.5. Celecoxib impairs cell migration and colony forming ability It has been established that STAT3 is important in cell migration. We used a wound healing assay to examine effect of STAT3 inhibition on cell migration. Our results demonstrated that with increasing doses of celecoxib, cell migration was increasingly inhibited. This is represented by the progressive inability of RH30, RH28 and RD2 cells to heal wounds with higher doses of celecoxib. Treatment of cells with DMSO was used as a control. This is shown in Fig. 4. A clonogenic colony formation assay was performed to investigate the recovery from Celecoxib treatment. Our results demonstrated that a progressive decrease in colony formation with increasing celecoxib doses (Fig. 4). 4. Discussion STAT3 has been widely described to be influential in cell growth and proliferation, and specifically in the pathological, unregulated cell growth and proliferation that is associated with neoplasms [21]. As evidence of this, many different types of malignancies demonstrate constitutive, unregulated activation of STAT3 when compared with normal cells. This constitutive activation offers an accessible and potentially clinically relevant therapeutic target, especially in malignancies which have been traditionally difficult to treat. Here we were able to show that celecoxib interrupts the phosphorylation of STAT3 in RMS cells, a crucial step in perpetuating downstream signaling; subsequently, downstream genes are underexpressed, ultimately resulting in cell death. Celecoxib also was shown to inhibit formation of clonogenic colonies and wound healing in RMS cells, properties which could also be beneficial in the treatment of malignancies. Though several mechanisms for celecoxib’s anti-tumor effect have been proposed [22–25], its effect on the STAT3 signaling cascade in RMS cells has not yet been described. Further, several studies indicate that the anti-tumor effect of celecoxib is, at least in part, via a COX-2-independent mechanism [26–28]. A COX-2 independent mechanism for celecoxib activity is also suggested by our results. In addition, there is evidence that novel celecoxib analogs, without COX-2 inhibition activity, cause cell death in cancer cells [29]. This further suggests that celecoxib could work through a COX-2 independent mechanism. Our results indicate that the STAT3 pathway is one of these COX-2 independent mechanisms. IL-6, a proinflammatory cytokine, has been implicated as a factor in tumor growth, as has its relationship to STAT3 [30,31]. It has been suggested that IL-6 is a key mediator in an autocrine pathway which allows for continuous and unregulated cell signaling and subsequent tumorigenesis. Our results indicating that celecoxib is able to inhibit IL-6-induced STAT3 phosphorylation may suggest that it could be useful for targeted therapy, especially in malignancies that are known to have aberrant IL-6 expression. Exploration of IL-6 expression in human rhabdomyosarcoma cells in vitro and in vivo may be an area that warrants more investigation. Though sometimes the mechanism by which a particular drug exerts its therapeutic effect is unclear, understanding mechanisms can be particularly beneficial in chemotherapy. Multimodal therapy is critical in the treatment of many cancers, including rhabdomyosarcoma. Drug synergy and compatibility with other chemotherapy and treatment modalities can mean the difference between disease treatment and disease cure. Celecoxib has been previously reported to have synergistic effects with radiation therapy in the treatment of other cancers [32–34]. Radiation therapy is
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Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Celecoxib inhibits STAT3 phosphorylation and suppresses cell migration and colony forming ability in rhabdomyosarcoma cells Suzanne Reed a,b, Huameng Li c, Chenglong Li c, Jiayuh Lin a,⇑ a
Center for Childhood Cancer, The Research Institute at Nationwide Children’s Hospital, The Ohio State University, College of Medicine, Columbus, OH 43205, USA Division of Pediatric Hematology and Oncology, Nationwide Children’s Hospital, The Ohio State University, College of Medicine, Columbus, OH 43205, USA c Division of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, The Ohio State University, Columbus, OH 43205, USA b
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Article history: Received 2 March 2011 Available online 11 March 2011 Keywords: STAT3 Celecoxib Rhabdomyosarcoma
a b s t r a c t Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in the pediatric and adolescent population. Though treatments for localized disease have reasonable long-term success rates, if disease is diffuse at diagnosis, outcomes are far poorer. Additional and/or alternative therapies are critical for improved clinical outcomes. One potentially therapeutic target is the signal transducer and activator of transcription 3 (STAT3) pathway. STAT3 has been shown to have constitutive activation in human rhabdomyosarcoma cells; thus, inhibition of STAT3 signaling may be a mechanism to induce tumor cell death. Celecoxib has been shown, by computer modeling, to bind STAT3 at the SH2 domain and competitively inhibit native peptide binding necessary for phosphorylation and subsequent propagation of the STAT3 signaling cascade. We found that celecoxib inhibits IL-6-induced and persistent STAT3 phosphorylation and inhibits cell viability in human rhabdomyosarcoma cells. We found that genes downstream of STAT3 (BCL-2, survivin, cyclin D1) were downregulated with celecoxib. Celecoxib also inhibits colony formation and cell migration. Our results suggest that, though known more commonly as a cyclooxygenase-2 (COX-2) inhibitor, celecoxib could act through the STAT3 pathway as well. More importantly, its effect on cell migration and clonogenic colony forming ability make it a potentially useful therapeutic agent for rhabdomyosarcoma, especially in metastatic disease whose clinical outcome is marginal at best with current therapies. Ó 2011 Elsevier Inc. All rights reserved.
1. Introduction According to the National Cancer Institute, malignancy is the leading cause of death by disease in children between infancy and 15 years of age. Rhabdomyosarcoma, an often aggressive malignancy originating from skeletal muscle cells, is the most common soft tissue sarcoma in children and adolescents [1]. Prior to the use of multimodal therapy – including surgery, chemotherapy, and radiation therapy – disease-free survival was less than 30% [2]. Presently, with this multimodal therapy, greater than 70% of patients with localized rhabdomyosarcoma can be cured of their disease [3,4]. These improvements in outcome can be attributed to the use of intensive combination chemotherapy, more effective local therapy with surgery and radiation, and improved supportive care [5]. Though encouraging strides have been made for localized disease, metastatic rhabdomyosarcoma has not seen the same ⇑ Corresponding author. Address: Center for Childhood Cancer, The Research Institute at Nationwide Children’s Hospital, Department of Pediatrics, The Ohio State University, 700 Children’s Drive, Columbus, OH 43205, USA. E-mail address: [email protected] (J. Lin). 0006-291X/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2011.03.014
improvement in survival, and affected patients remain with dismal prognoses. The overall survival in metastatic rhabdomyosarcoma is approximately 30%, and this statistic has not significantly changed in more than 20 years [2–4]. Excluding patients younger than 10 years with embryonal RMS, which is a specific population with a better prognosis, patients with metastatic rhabdomyosarcoma have an estimated five-year failure-free survival of <20% [2–4]. Because of this, novel therapeutic agents are desperately needed to help target these particularly aggressive tumors. One such novel approach is through manipulation of signal transducers and activators of transcription (STATs) and, in particular, via the STAT3 signaling cascade. STAT3 is one of a group of transcription factors that was initially discovered as an acute phase reactant [6], but over time has been found to have many more roles. Its importance is multi-factorial; in addition to response to inflammation, STAT3 has been found to contribute to cell transformation, proliferation, and suppression of apoptosis [7,8]. It also has been well-defined in the literature as a mediator of angiogenesis and in malignant transformation of cell populations [6,9]. STAT3 has been demonstrated to be constitutively active in many tumor cell lines, most of which are considered adult malignancies. Constitutive STAT3 activation has been found in cancers of the breast,
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treatment, the cells were incubated for 72 h. Assay was completed by adding 25 lL 3-(4,5-dimethylthiazolyl)-2,5-diphenyltetrazolium bromide (MTT) according to manufacturer’s protocol (Roche Diagnostics, Mannheim, Germany). After incubation at 37 °C for 3 more hours, 100 lL of N,N-dimethylformamide solubilization solution was added to each well allowing absorbance to be measured by spectrophotometry. Half-maximal inhibitory concentrations (IC50) were determined using Calcusyn software (Biosoft, Cambridge, Great Britain). 2.4. Western blot analysis
Fig. 1. Computer modeling of celecoxib binding to STAT3 SH2 domain. Celecoxib is rendered in thick stick-ball model. The native pTyr-Leu706 phosphopeptide binding of the partnering SH2 in homo-dimerization is in thin stick-ball model. The docked binding modes show that celecoxib effectively competes with pTyr-Leu706 peptide for pTyr 705 binding site of STAT3 SH2.
lung, prostate, ovary, and pancreas, and also in melanoma and multiple myeloma [9–11]. More recently, STAT3 has also been found to be constitutively activated in human rhabdomyosarcoma cells and crucial for cell growth and survival of sarcoma cells [12,13]. Celecoxib is a selective COX-2 inhibitor, originally marketed for its anti-inflammatory properties, which has been gaining credit for its anti-tumor effects as well [14]. The mechanisms by which celecoxib exerts these anti-neoplastic effects are being investigated. One such mechanism, we suggest, is via the STAT3 pathway. In a computer binding model, celecoxib was shown to bind to STAT3 at the SH2 domain, blocking the native pTyr-Leu706 phosphopeptide and functionally preventing phosphorylation (Fig. 1). Using this computer modeling evidence, the objective of this study was to determine if celecoxib inhibits phosphorylation of STAT3 in human rhabdomyosarcoma cells in vitro and thus induces cell death. 2. Materials and methods 2.1. Cell lines Human rhabdomyosarcoma lines RH30 and RH28 were obtained from Dr. Peter Houghton’s lab and cell line RD2 was purchased from American Type Culture Collection (ATCC). SMS-CTR cell line was used for cytokine-induced STAT3 inhibition. All cell lines were maintained in Dulbecco’s Modified Eagle Medium, supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. The cell lines were stored in a humidified 37 °C incubator with 5% CO2. 2.2. Celecoxib Celecoxib powder (Catalog No. C-1502) was purchased from LC Laboratories. A 50 mmol/L solution was prepared by mixing powder dissolved in sterile dimethyl sulfoxide. This solution was stored at 20 °C. 2.3. MTT cell viability assay Rhabdomyosarcoma cells were seeded in 96-well plates. Five thousand cells were seeded per well. After seeding, the cells were incubated overnight. The following day, the cells were treated with escalating doses of celecoxib (serial dilutions prepared to achieve concentrations of: 10, 25, 50, 75, 100, and 125 lM). After
Rhabdomyosarcoma cells (RH30, RH28, and RD2) were seeded in sterile 10-cm plates in DMEM media with 10% FBS and 1% penicillin/streptomycin. Cells were incubated overnight at 37 °C. After incubation, one plate was treated with DMSO (as a control) and each of the other plates was treated with celecoxib. Escalating concentrations of 50, 75, and 100 lM celecoxib were prepared. The cells were treated for 18 h. Cells were then washed with cold purified buffered saline, collected, and lysed with cold, protease-containing RIPA buffer. Protein concentrations were determined using the BCA protein assay according to the manufacturer’s protocol (PIERCE Biotechnologies, Rockford, IL). Protein samples were separated by SDS gel and transferred to PVDF membranes. Membranes were then separately probed with antibodies (Cell Signaling Technology) for phospho-STAT3 (Tyrosine 705), STAT3, cleaved caspase-3, COX-2, and GAPDH. Specific protein was then identified by enhanced chemiluminescence. The above western blot method was used with SMS-CTR cells, with a few exceptions. These cells were seeded and grown in DMEM media with 10% FBS for 24 h, then media was removed and replaced with serum-free DMEM media. Cells were serum starved for 24 h, then treated with treated with DMSO (as a control) or celecoxib, at 50 and 75 lM, for 1 h. After 1 h, 25 ng/mL Interleukin-6 (IL-6) was added. One plate was treated with IL-6 only. After thirty more minutes, these treated cells were collected in the same fashion as the other cell lines, and subsequent western blotting technique was the same as indicated. 2.5. Reverse transcriptase-polymerase chain reaction (RT- PCR) Rhabdomyosarcoma cells were treated with celecoxib in different concentrations (50, 75, and 100 lM) or DMSO for 18 h. Cellular RNA was collected using RNeasy Kits (Qiagen, Valencia, CA). Reverse transcription was accomplished by Omniscript reverse transcription kit. Subsequent PCR amplification was performed under these specific conditions: 5 min at 94 °C followed by 20 cycles of 30 s at 94 °C, 30 s at 55 °C, and 30 s at 72 °C with a final extension of 5 min at 72 °C. Primer sequences used are as follows: Bcl-2: Forward: 50 -TCTTTGAGTTCGGTGGGGTC-30 Reverse: 50 -TGCATATTT GTTTGGGGCAGG-30 [15]. Cyclin D1: Forward: 50 -GCTGGAG CCCGTGAAAAAGA-30 Reverse: 50 -CTCCGCCTCTGGCATTTTG-30 [16]. Survivin: Forward: 50 -ACCAGGTGAGAAGTGAGGGA-30 Reverse: 50 -AACAGTAGAGGAGCCAGGGA-30 [17]. GAPDH: Forward: 50 -TGATGACATCAAGAAGGTGGTGAAG-30 Reverse: 50 -TCCTTGGA GGCCATGTGGGCAT-30 [18]. 2.6. Wound healing assay Rhabdomyosarcoma cells were seeded in 6-cm plates. When cells were 100% confluent, a wound was created using a pipette tip to make a linear scratch through the monolayer. Simultaneously, new medium containing celecoxib (50, 75, or 100 lM) or DMSO was added to the plates. The treatment-containing media was removed after 4 h and fresh media was added. After an additional 24 h, the degree of wound healing was evaluated using
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microscope imaging (Leica MZ 16FA microscope, Leica Microsystems, Bannockburn, IL).
2.7. Clonogenic colony formation assay Rhabdomyosarcoma cells were seeded on 10-cm plates at a density of 2000 cells per plate. Cells were then treated with DMSO or celecoxib (50, 75, or 100 lM) for 4 h. The cells were maintained at 37 °C and allowed to grow for 3 weeks. Colonies of cells were then fixed with cold methanol for 30 min and stained with 1% crystal violet.
3. Results 3.1. Computational modeling of celecoxib binding to STAT3 The druggable binding site of STAT3 SH2 domain (PDB code 1BG1) consists of three sub-pockets: pTyr705 binding site, Leu706 site, and a side sub-pocket. Molecular docking program MLSD based on AutoDock4 was used to dock celecoxib to the binding site on STAT3 SH2. Lamarckian Genetic Algorithms (LGA) was used as searching method in docking. Parameters and input files of docking simulations were prepared as previously reported [19,20]. Docking simulations were performed for 75 runs, with 3.5 million energy evaluations in each run. Computational docking resulted in a major cluster with 88% conformers, with a lowest binding energy of 7.2 kcal/mole. As shown in Fig. 1, celecoxib docked to the major pTyr705 binding site and side sub-pocket. Binding mode analysis also found that sulfonamide moiety of celecoxib formed two hydrogen bonds with residues Glu612 and Arg609 of the receptor, respectively. Computer modeling shows that celecoxib could effectively compete with pTyr-Leu706 peptide for pTyr 705 binding site of STAT3 SH2.
3.2. Cell viability and proliferation is decreased by celecoxib Cell viability assays were used to identify inhibitory effect of celecoxib on rhabdomyosarcoma cells. A dose-dependent inhibition in cell viability was demonstrated after treatment. IC50 values for each cell line treated with celecoxib were determined by Calcusyn software. IC50 values are as follows: RH30 (61.05 lM); RH28 (63.3 lM); RD2 (43.5 lM). 3.3. STAT3 phosphorylation is inhibited by celecoxib To investigate inhibition of STAT3 phosphorylation, western blot analysis was used to detect presence of phosphorylated STAT3 (PSTAT3) after treatment with celecoxib. Cells from each RMS cell line (RH30, RH28, and RD2) were plated and treated with escalating doses of celecoxib: 50, 75, or 100 lM. DMSO was used as the control group. As shown in Fig. 2A, we found a decrease in the amount of PSTAT3 with increasing doses of celecoxib. Membranes were stripped then probed for total STAT3, which demonstrated consistent expression with escalating celecoxib doses. This suggests that celecoxib could inhibit STAT3 phosphorylation, and the decrease in PSTAT3 would not be explained by a constitutional decrease in total STAT3. In addition, we separately probed membranes for COX-2 and found consistent COX-2 expression with different celecoxib doses. This invariable expression of COX-2 further suggests the mechanism of inhibition of cell viability is independent of the COX-2 pathway. GAPDH, used as a control, showed consistent expression with escalating doses of celecoxib. As additional evidence of inhibition of STAT3 phosphorylation, we used RT-PCR to amplify genes known to be expressed downstream of STAT3. As illustrated in Fig. 3, downstream genes bcl-2, survivin, cyclin D showed decreased expression with increasing doses of celecoxib in RH30, RH28, and RD2 RMS cells. This yields additional evidence that celecoxib inhibits STAT3 phosphorylation. GAPDH, used as a control, showed consistent expression with escalating celecoxib doses.
Fig. 2. (A) Western blot analysis of RMS cells treated with celecoxib, demonstrating inhibition of STAT3 phosphorylation in RMS cells. RH30, RH28, and RD2 RMS cell lines show a decrease in expression of phosphorylated STAT3 in response to increasing doses of celecoxib. Total STAT3 expression and COX-2 expression remain stable. (B) Western blot analysis of RMS cells with minimal endogenous phosphorylated STAT3 expression (SMS-CTR) demonstrating increased expression of PSTAT3 with IL-6 alone, then subsequent inhibition with celecoxib. Total STAT3 expression remains stable.
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Fig. 3. RT-PCR analysis of RMS cells treated with celecoxib, demonstrating decreased expression of genes downstream of STAT3. RH30, RH28, and RD2 RMS cell lines show a decrease in expression of downstream genes: BCL-2, cyclin D, survivin. Degree of expression decrease is correlated with increased celecoxib doses.
Fig. 4. (A) Wound healing assay demonstrating impaired wound healing in RMS cells after treatment with celecoxib. RMS cell lines RH30, RH28, and RD2 show more impaired wound healing, and ultimately no wound healing (in 100 lM treatment group), with increasing doses of celecoxib. (B) Clonogenic colony forming assay demonstrating that celecoxib inhibits colony formation in RMS cell lines, RH30 and RD2. Again, degree of inhibition is correlated with increasing celecoxib doses.
3.4. Celecoxib inhibits IL-6-induced STAT3 phosphorylation in RMS cells To investigate if celecoxib could inhibit IL-6 induced STAT3 phosphorylation, we chose a RMS cell line with low endogenous PSTAT3 expression, SMS-CTR. We used IL-6 to induce STAT3
expression while treating with celecoxib. We again used western blot analysis to detect PSTAT3. As seen in Fig. 2B, PSTAT3 had increased expression with IL-6 alone, then demonstrated subsequent decreased expression with treatment with celecoxib. Consistent expression of total STAT3 again suggests that this is specific inhibition of phosphorylation of STAT3 rather than inhibition of STAT3
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globally. GAPDH was used as a control and also showed consistent expression. 3.5. Celecoxib impairs cell migration and colony forming ability It has been established that STAT3 is important in cell migration. We used a wound healing assay to examine effect of STAT3 inhibition on cell migration. Our results demonstrated that with increasing doses of celecoxib, cell migration was increasingly inhibited. This is represented by the progressive inability of RH30, RH28 and RD2 cells to heal wounds with higher doses of celecoxib. Treatment of cells with DMSO was used as a control. This is shown in Fig. 4. A clonogenic colony formation assay was performed to investigate the recovery from Celecoxib treatment. Our results demonstrated that a progressive decrease in colony formation with increasing celecoxib doses (Fig. 4). 4. Discussion STAT3 has been widely described to be influential in cell growth and proliferation, and specifically in the pathological, unregulated cell growth and proliferation that is associated with neoplasms [21]. As evidence of this, many different types of malignancies demonstrate constitutive, unregulated activation of STAT3 when compared with normal cells. This constitutive activation offers an accessible and potentially clinically relevant therapeutic target, especially in malignancies which have been traditionally difficult to treat. Here we were able to show that celecoxib interrupts the phosphorylation of STAT3 in RMS cells, a crucial step in perpetuating downstream signaling; subsequently, downstream genes are underexpressed, ultimately resulting in cell death. Celecoxib also was shown to inhibit formation of clonogenic colonies and wound healing in RMS cells, properties which could also be beneficial in the treatment of malignancies. Though several mechanisms for celecoxib’s anti-tumor effect have been proposed [22–25], its effect on the STAT3 signaling cascade in RMS cells has not yet been described. Further, several studies indicate that the anti-tumor effect of celecoxib is, at least in part, via a COX-2-independent mechanism [26–28]. A COX-2 independent mechanism for celecoxib activity is also suggested by our results. In addition, there is evidence that novel celecoxib analogs, without COX-2 inhibition activity, cause cell death in cancer cells [29]. This further suggests that celecoxib could work through a COX-2 independent mechanism. Our results indicate that the STAT3 pathway is one of these COX-2 independent mechanisms. IL-6, a proinflammatory cytokine, has been implicated as a factor in tumor growth, as has its relationship to STAT3 [30,31]. It has been suggested that IL-6 is a key mediator in an autocrine pathway which allows for continuous and unregulated cell signaling and subsequent tumorigenesis. Our results indicating that celecoxib is able to inhibit IL-6-induced STAT3 phosphorylation may suggest that it could be useful for targeted therapy, especially in malignancies that are known to have aberrant IL-6 expression. Exploration of IL-6 expression in human rhabdomyosarcoma cells in vitro and in vivo may be an area that warrants more investigation. Though sometimes the mechanism by which a particular drug exerts its therapeutic effect is unclear, understanding mechanisms can be particularly beneficial in chemotherapy. Multimodal therapy is critical in the treatment of many cancers, including rhabdomyosarcoma. Drug synergy and compatibility with other chemotherapy and treatment modalities can mean the difference between disease treatment and disease cure. Celecoxib has been previously reported to have synergistic effects with radiation therapy in the treatment of other cancers [32–34]. Radiation therapy is
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