- Email: [email protected]
NF-kB and L1CAM activities
Accepted Manuscript Celecoxib suppresses proliferation and metastasis of pancreatic cancer cells by down-regulating STAT3 / NF-kB and L1CAM activities Chaohui Zuo, Yuan Hong, Xiaoxin Qiu, Darong Yang, Nianli Liu, Xinyi Sheng, Kunyan Zhou, Bo Tang, Shuhan Xiong, Min Ma, Zhuo Liu PII:
S1424-3903(18)30030-9
DOI:
10.1016/j.pan.2018.02.006
Reference:
PAN 839
To appear in:
Pancreatology
Received Date: 16 August 2017 Revised Date:
31 January 2018
Accepted Date: 12 February 2018
Please cite this article as: Zuo C, Hong Y, Qiu X, Yang D, Liu N, Sheng X, Zhou K, Tang B, Xiong S, Ma M, Liu Z, Celecoxib suppresses proliferation and metastasis of pancreatic cancer cells by downregulating STAT3 / NF-kB and L1CAM activities, Pancreatology (2018), doi: 10.1016/j.pan.2018.02.006. 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 proof before it is published in its final 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.
ACCEPTED MANUSCRIPT Celecoxib suppresses proliferation and metastasis of pancreatic cancer cells by down-regulating STAT3 / NF-kB and L1CAM activities
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Running title: Celecoxib inhibit pancreatic cancer via L1CAM/NFkB
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Bo Tang2, Shuhan Xiong4, Min Ma1, Zhuo Liu1
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Chaohui Zuo1*#, Yuan Hong2*, Xiaoxin Qiu2, Darong Yang3, Nianli Liu3, Xinyi Sheng2, Kunyan Zhou1,
Department of Gastroduodenal and Pancreatic Surgery, Hunan Cancer Hospital and The Affiliated
Cancer Hospital of Xiangya School of Medicine, Central South University. No 283 Tongzipo Road, Changsha 410013, China.
Graduates School, University of South China, 28 West Changsheng Road, Hengyang 421001, China.
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Department of Molecular Medicine, College of Biology, State Key Laboratory of Chemo / Biosensing
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and Chemometrics, Hunan University. No 2 Lushan South road, Changsha 410082, China
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School of Public Health, Jilin University. No 2699 Qianjin Street, Changchun, 130021, China
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* Chaohui Zuo and Yuan Hong contribute equally in this study.
#
Correspondence to
Chaohui Zuo Department of Gastroduodenal and Pancreatic Surgery, Hunan Cancer Hospital and The Affiliated Cancer Hospital of Xiangya School of Medicine, Central South University. No 283 Tongzipo Road,
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ACCEPTED MANUSCRIPT Changsha 410013, China. Tel: +86-13875911328 Fax: +86-731-89762142
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Email: [email protected]
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ACCEPTED MANUSCRIPT Abstract Objective: To explore the molecular mechanisms of celecoxib-induced pancreatic cancer suppression in vivo and in vitro. Methods: The anti-pancreatic cancer activities of celecoxib (0, 20, 60 and 100
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µmol/L) were investigated by cell viability and migration of Panc-1 and Bxpc-3 cells in vitro. The expression of L1CAM in pancreatic cancer and adjacent tissues was compared using immunohistochemistry. The expressions of L1CAM, STAT3, p-STAT3, NF-κB, p-NF-κB were
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determined by western blotting, and cell invasive ability was determined by wound healing assay in
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L1CAM-silenced and over-expressed Panc-1and Bxpc-3 cells. Results: The expression of L1CAM in pancreatic carcinoma was stronger than that in the adjacent tissues and L1CAM could increase the growth and invasion of pancreatic cancer cells. Over-expression of L1CAM activated the STAT3/NF-κB signaling pathway in Panc-1 and Bxpc-3 pancreatic cancer cells and celecoxib inhibited
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their viability and the expressions of STAT3, p-STAT3, NF-κB, p-NF-κB as well as full length L1CAM in a concentration dependent manner. Conclusions: L1CAM was highly expressed in pancreatic cancer tissue and positively correlated with age, TNM staging and tumor differentiation. L1CAM activated the
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STAT/NF-κB signaling pathway and celecoxib could inhibit the activity of L1CAM, STAT3 and the
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NF-κB signaling pathway resulting in decreased growth and invasion of pancreatic cancer cells.
Keywords: pancreatic cancer; celecoxib; L1CAM; STAT3/NF-kB
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ACCEPTED MANUSCRIPT Introduction Pancreatic cancer is a most aggressive gastrointestinal malignancy and the 4th leading cause of carcinoma-related mortality in the US. In recent years, the incidence of pancreatic cancer has increased
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significantly. There were 48,960 new cases estimated in 2015, and about 40,560 cases die of this disease in America. Regardless of decades of endeavor, pancreatic cancer is still has a poor prognosis with the mortality being comparable to the morbidity 1. The incidence and mortality of pancreatic
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cancer is also increasing in China. In 2015, the incidence of pancreatic cancer in China was
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90.1/100,000, and the mortality was 79.4/100,000 people ranking it as the 6th leading cause of cancer-related death 2. Even though only resection can provide the possibility of cure, most pancreatic cancer patients miss the chance due to no early specific and sensitive clinical diagnostic technique, which results in just 15% of pancreatic cancer patients being diagnosed at an early stage so the surgery
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can be performed 3. Consequently, systemic chemotherapy has grown to be a most essential therapeutic intervention for advanced pancreatic cancer patients. It has been reported that COX-2 is highly expressed and closely related to the development of pancreatic cancers
4
and thus COX-2 inhibitors
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such as celecoxib have the potential to suppress their invasion and metastasis.
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STAT3 is a transcriptional factor, which is intimately involved in cell proliferation, differentiation and apoptosis, and abnormal activation is associated with poor prognosis of solid tumors 5. NF-κB is up-regulated in over 70% of pancreatic cancer cell lines and tumor tissues 6 and has been proven to be associated with angiogenesis, invasion, and metastasis of pancreatic cancer 7. Both NF-κB and STAT3 have been reported to participate in the invasion, metastasis, angiogenesis and immune escape of cancer cells, and STAT3/NF-κB signaling is a major regulator of tumor angiogenesis and invasiveness 8. In addition, it has been reported that COX-2 activates the STAT3/NF-κB signaling pathway to promote
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ACCEPTED MANUSCRIPT invasive growth and metastasis of pancreatic cancer 9. L1 cell adhesion molecule (L1CAM) is a glycoprotein with a molecular weight of 200-220 KDa, and belongs to the immune protein superfamily. It is expressed in neurons to play a role in neuronal migration, axonal growth, bundles and elongation, 10
.
A 200 kDa soluble
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whereas in tumors it has been associated with cell invasion and motility
ectodomain of L1CAM can be cleaved proximal to the plasma membrane by specific metalloproteinases
11, 12
. In pancreatic cancer, it has been reported that L1CAM is up-regulated
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and
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closely related with invasive growth, metastasis and prognosis of pancreatic cancer 10, 14 and is believed
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to be a good target for treatment 15. In addition, expression of COX-2 and L1CAM in pancreatic cancer tissues were both enhanced, suggesting that COX-2 and L1CAM could be targets for pancreatic cancer therapy
16
. However, the involvement of L1CAM in celecoxib-induced anti-cancer efficacy in
pancreatic cancer remains unclear.
Compounds and reagents
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Materials and Methods
Antibodies against STAT3 and NF-κB were purchased from Wuhan Boster Biological Technology, Ltd.
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(Wuhan, China); antibodies against p-STAT3 and p-NF-κB were obtained from Cell Signaling
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Technology (Danvers, USA); the antibody against L1CAM was bought from Santa-Cruz (Santa-Cruz, USA); the MTT kit came from Sigma-Aldrich (St. Louis, USA); celecoxib was purchased from Pfizer Inc. (New York, USA); L1CAM plasmid was purchased from Beijing Yi Qiao Shenzhou Biotechnology Co., Ltd.(Beijing, China); L1CAM siRNA was purchased from GenePharma Co., Ltd (Shanghai, China). The used transfection reagent was HiPerFect (Qiagen, Hilden, Germany). Cell lines and culture The human pancreatic cancer cells, Panc-1 and Bxpc-3, were obtained from the cell bank of the cancer research institute in the central south university. Both cell lines were cultured in DMEM medium 5
ACCEPTED MANUSCRIPT containing 10% FBS, 20 mmol/L NaHCO3, 100 U/mL penicillin and 100 µg/mL streptomycin, at 37°C in a 5% CO2 atmosphere incubator. Panc-1 and Bxpc-3 cells were treated with various concentrations of celecoxib.
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Cancer and adjacent tissue of pancreatic cancer patients A total of 56 pancreatic cancer tissue specimens (TNM stages II-III), and the matched adjacent tissue (5 cm from the cancerous tissue) were collected by stomach and duodenum surgical resection from
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patients in the Hunan Provincial Tumor Hospital from 2010 to 2015, and stored at -80°C. All of the
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patients did not accept preoperative chemotherapy or radiotherapy. Western blot
Pancreatic cancer cells in 6-well plates pretreated with PBS or different concentration of celecoxib were washed twice with cold PBS, and then re-suspended in RIPA cell lysis buffer (50mM Tris-HCl,
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PH7.4, 150mMNaCl, 1% NP40, 0.5% Sodium deoxycholate, 0.1% SDS) with proteinase inhibitors (100:1) and incubated on ice for 15 min followed by scratch. The supernatant of the cell lysate was collected by centrifugation at 13,000 g and 4°C for 5 min. The supernatant was transferred into new 1.5
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mL EP tubes, and the protein concentrations were determined, using the bicinchoninic acid (BCA)
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method according to the manufacturer’s protocol. A total of 25 µg of proteins mixed with the same volume of 2 × loading buffer were run on 10% SDS-PAGE gels and subsequently transferred to polyvinylidene fluoride (PVDF) membranes (120 V × 90 min). Membranes were blocked with 5% nonfat milk for 1 h at room temperature, and then incubated overnight at 4°C or 1 h at room temperature with primary antibodies as indicated. The membranes were washed with TBST 3 times × 5 min and further incubated with an appropriate second antibody for 1 h at room temperature. Signals were developed using an enhanced chemiluminescent (ECL) detection kit (Pierce, Rockford, USA) at
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ACCEPTED MANUSCRIPT room temperature for 5 min after further washing with TBST 3 times × 5 min. The bands of the target proteins were obtained and the gray value of each band was calculated using Image J software. Cell viability assay
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Cell viability was measured using a MTT assay. Briefly, the logarithmic phase pancreatic cancer cells were seeded at a density of 5,000 cells/100 µL/well, in 96-well plates; the marginal wells were filled with sterile PBS. The cells were incubated at 37°C in a 5% CO2 incubator overnight until the cells were
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attached to the bottom (i.e. a monolayer of cells covered the bottom of the wells), and treated with 0, 20, 60 or 100 µM of celecoxib with 5 duplicates of each concentration for 24 h. Then, 20 µL of MTT (5
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mg/mL) was added to each well and cultured for 3-4 h. The wells with the same volume of serum free medium, MTT and DMSO as the test wells were designated as the blank control group. The wells with the same number of pancreatic cancer cells and the same volume of serum free medium, MTT and
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DMSO as in the test wells were the control group. After termination of the reaction, the optical density (O.D.) value of each well was measured with a microplate reader at a wavelength of 570 nm. The cell viability and inhibition rate were calculated according to the following equations, respectively: cell
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viability = (OD value of test drug group - OD value of blank group) / (OD value of control group - OD
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value of blank group) × 100%; inhibition rate = (1- OD value of test drug group) / OD value of control group.
Wound healing assay
The migration abilities of wild type pancreatic cancer cells, L1CAM silenced and over-expressed pancreatic cancer cells were determined using the wound healing assay. Briefly, logarithmic phase pancreatic cancer cells were seeded at a density of 8 × 105 cells/mL/well, in 6-well plates, in triplicate. The cells were pretreated as described above for the indicated groups. Five straight lines at intervals of
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ACCEPTED MANUSCRIPT 1 cm were marked using a pen on the back of the well with pretreated cells. A straight line, perpendicular to the 5 lines on the back of each well at the center, was gently scraped using the tip of a disposable pipette, when the pancreatic cancer cells were grown to 95% confluence. The wells were
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then rinsed with sterile PBS 3 times and filled with serum-free DMEM medium. The wound healing was observed and photographed at 0, 6 and 24 h. Fluorescence microscope observations
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The coverslips were soaked in 75% alcohol and placed in the wells of 6 well-plates with 1 mL
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PBS/well. Cells were seeded onto the coverslips at a concentration of 5 × 105 cells/mL/well after washing the coverslips twice with 2 mL of PBS. The medium was discarded and plates washed once with 2 mL of PBS. A total of 2 mL of acetone was added into each well, which were then placed in a freezer at -20°C for 8 min, and then the acetone was discarded. The coverslip was moved to the
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microscope slide with cells upward and washed 3 times × 5min with PBS, followed by the addition of 250 µL sheep serum to block for 30 min. Immediately, 250 µL of primary antibody was added and cells incubated at room temperature for 60 min after discarding the sheep serum, and then washed 3 times ×
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5min with PBS. Then, 250 µL of the second antibody was added and the cells incubated at room
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temperature for 60 min in the dark, and then washed 3 times × 5 min with PBS. The air-dried coverslips with treated cells were placed carefully, face down on a new microscope slide with 10 µL of DAPI so that DAPI could be expanded slowly without bubbles. Specimens were observed and photographed under a fluorescence microscope at a 360~400 nm excitation wavelength. Cell transfection with L1CAM expressing plasmid and L1CAM siRNA Pancreatic cancer cells were seeded in 6-well plates at a concentration of 6 × 105/well and incubated overnight at 37°C in a 5% CO2 incubator. The target plasmid and siRNA were diluted with Opti-MEM 8
ACCEPTED MANUSCRIPT serum-free medium and transfected with HiPerFect reagent before transfection, gently mixed, and allowed to stand at room temperature for 20 min. The solution was added to the cell culture dish and shaken back and forth to mix it with the cell culture medium. Cells transfected with scrambled siRNA
indicated time points according to experimental requirements. Immunohistochemistry
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or empty plasmid were used as the corresponding controls. Gene expression was measured at the
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Tumor tissue was fixed in 10% neutral buffered paraformaldehyde at 4°C for 24 h. Sections (5 µm thick) were deparaffinized, rehydrated with PBS (pH 7.4), treated with aqueous 3% H2O2 for 10 min,
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and the antigen retrieved in 0.1% trypsin (M/V) at 37°C for 10 min. Sections were blocked with 5% normal goat serum at room temperature for 30 min, then 1:100 anti-L1CAM antibody was applied and sections incubated at 4°C overnight. Next, after being washed 2 times × 5 min with PBS sections were
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incubated with a second antibody for 1 h at 37°C. Then they were incubated with diaminobenzidine (DAB) for 5 min and counterstained with hematoxylin after being washed in running water. After gradient ethanol dehydration and xylene for 3 min, the sections were then mounted with neutral gum
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after being made transparent with xylene.
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Observation and evaluation criteria (using the quartile method) Images were acquired with an optical microscope (Olympus IX70, Japan). PBS was used as the negative control instead of primary antibody. The expression level of the target protein in the tissues was determined according to the positive cell numbers in the visual field 17. First five areas with the highest number of L1CAM -positive cells were selected. Then, these cells were counted (per mm2) at higher (×400) magnification. Finally, L1CAM positive cells were scored into four categories: 1 (< 25%); 2 (25% to 50%); 3 (50% to 75%); and 4 (> 75%) and the staining intensity of positive cells was
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ACCEPTED MANUSCRIPT scored as weak, moderate and strong. The final score was the product of the intensity and the percentages of L1CAM expressing cells. Ethics statement
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The protocol of this study was approved by the ethics committee of the Hunan Cancer Hospital and The Affiliated Cancer Hospital of Xiangya School of Medicine, Central South University and all participants provided written informed consent. Statistical analysis
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All statistical analyses were performed with SSPS for Windows (version 18.0. Chicago: SPSS Inc). The measurement data were analyzed using a paired t-test. The count data was expressed as a percentage
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and compared using a χ2 test. Comparisons between multiple groups were performed using variance analysis of repeated measurement data. P < 0.05 was taken to indicate a statistically significant difference. Results
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Celecoxib inhibited cell viability of pancreatic cancer cells Panc-1 and Bxpc-3 To investigate the inhibitory effect of celecoxib on pancreatic cancer cell viability, the Panc-1 and Bxpc-3 cells were challenged with celecoxib for 24, 36 and 48 h at final concentrations of 0, 20, 60 or
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100 µM, and the relative viable cell numbers were analyzed by MTT assay. As shown in Figure 1A
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and 1B, celecoxib significantly inhibited the viability of both Panc-1 (Figure 1A) and Bxpc-3 (Figure 1B) cells in a dose- and time-dependent manner, and the difference between each celecoxib treatment group and the blank control group was statistically significant (P < 0.05). The inhibition rate of celecoxib at final concentrations of 0, 20, 60 or 100 µM on the Panc-1 cells at 24 h treatment was 0%, 12%, 28% and 61% respectively; after 36 h treatment the inhibition rate was 0%, 19%, 35% and 78% respectively, and after 48 h 0%, 23%, 41% and 86%. The inhibition rate of celecoxib at the final concentrations of 0, 20, 60 or 100 µM on the Bxpc-3 cells after 24 h treatment was 0%, 8%, 14% and
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ACCEPTED MANUSCRIPT 36% respectively; after 36 h 0%, 4%, 26% and 58%; and after 48 h treatment 0%, 19%, 37% and 76%. These data reveal the anticancer activity of celecoxib in pancreatic cancer cells, with Panc-1 cells being more sensitive to celecoxib than Bxpc-3 cells.
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Up-regulation of L1CAM in human pancreatic cancer cells A total of 56 pancreatic cancer tissue specimens and matched adjacent tissues were collected from patients in the Hunan Provincial Tumor Hospital from 2008 to 2014. The 56 pancreatic cancer patients
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included 36 males and 20 females; the median age was 59 years (39 to 78 years) at pathological
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diagnosis. There were 48 cases of ductal adenocarcinoma and 8 cases of mucinous cystadenocarcinoma; 3 cases of stage I, 8 cases of stage II, 18 cases of stage III and 27 cases of stage IV, according to the TNM staging
18
. All these patients received radical surgery (radical pancreaticoduodenectomy or
pancreatic tail resection), without radiotherapy and chemotherapy before the surgery.
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The expression of L1CAM in 56 pancreatic cancer tissue specimens and matched adjacent tissues was detected by immunohistochemistry. Our results showed that L1CAM was strongly positively expressed in 21 pancreatic cancer tissue specimens, positively expressed in 10, and weakly positively expressed
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in 25 versus the matched adjacent tissues, which showed negative expression of L1CAM (Figure 2).
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The expression of L1CAM was negatively correlated with age, the TNM stage and tumor differentiation status (Table 1). Down-regulation of L1CAM, STAT3 and NF-κB in Panc-1 and Bxpc-3 cells by celecoxib To demonstrate whether the anti-cancer effect of celecoxib was associated with L1CAM and related mechanisms in pancreatic cancer cells, the total protein of Panc-1 and Bxpc-3 cells, treated with different concentrations of celecoxib (0, 20, 60, 100 µM) for 24 h, was subjected to western blot analysis to determine the expression levels of L1CAM, STAT3, p-STAT3, NF-κB and p-NF-κB. The
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ACCEPTED MANUSCRIPT results showed that the expression of STAT3, p-STAT3, NF-κB and p-NF-κB decreased with an increase in the concentration of celecoxib in Panc-1 and Bxpc-3 cells (Figure 3 A); the appearance of full length L1CAM (230 kDa) was also reduced by celecoxib in a concentration-dependent manner,
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which was most obvious in Bxpc-3 cells at a concentration of 100 µM (Figure 3B). Effects of over-expression of L1CAM and L1CAM silencing on migration and invasion of pancreatic cancer cells.
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To demonstrate further that L1CAM promotes the migration of pancreatic cancer cells, the L1CAM
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gene was over-expressed or silenced in Panc-1 and Bxpc-3 cells (Figure 4A) and the migration abilities of the L1CAM over-expressing Panc-1 (Panc-1-Ov) cells, L1CAM over-expressed Bxpc-3 (Bxpc-3-Ov)cells, L1CAM silenced Panc-1 cells (Panc-1-Si) and L1CAM silenced Bxpc-3 (Bxpc-3-Si) cells were measured using wound healing assays and compared with the corresponding control cells
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(Panc-1-Co or Bxpc-3-Co), which were the cells transfected with scrambled siRNA. Figure 4B shows the sizes of the gaps in wound healing assays at the indicated time points. Our results showed that compared with the corresponding control cells, the migration abilities of Panc-1-Si and
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Bxpc-3-Si were decreased, while the migration abilities of Panc-1-Ov and Bxpc-3-Ov cells were
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increased, which was reflected in more closed gaps in the wound healing assays. These results further confirmed the importance of L1CAM in promoting pancreatic cancer metastasis. L1CAM promotes phosphorylated STAT3 and NF-κB To confirm further that the effect of L1CAM in promoting pancreatic cancer viability and metastasis is related to STAT3/NF-κB, the levels of p-STAT3 and p-NF-κB in Panc-1-Ov and Bxpc-3-Ov cells were detected using an immunofluorescence assay. The results revealed that the expression of p-STAT3 and p-NF-κB were increased in Panc-1-Ov and Bxpc-3-Ov cells, which indicated that L1CAM could
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ACCEPTED MANUSCRIPT stimulate the phosphorylation level of STAT3 and NF-κB (Figure 5). This finding was consistent with the results of our western blot assays. The results confirmed that L1CAM could activate the STAT3/NF-κB signaling pathway by increasing the expression of p-STAT3 and p-NF-κB. Discussion
cancer cells, which is in agreement with the literature
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In the present study we found that L1CAM, NF-κB and STAT3 were enhanced expressed in pancreatic . In addition, we found that
down-regulation of L1CAM led to reduced migration of Bxpc-3 and Panc-1 cells, which is in line with
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previous reports that L1CAM triggers cell migration and invasion 20. Moreover, celecoxib could inhibit the expressions of STAT3, p-STAT3, NF-κB and p-NF-κB in Panc-1 and Bxpc-3 cells in a
constitutive NF-κB activity
22, 23
21
and leads to
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concentration-dependent manner. L1CAM has been reported to activate STAT3
, while interactions between STAT3 and NF-κB promote the
development and progression cancers 24. In addition, previous research revealed that COX-2 inhibitors downregulated STAT3 and NF-κB activities
9, 25
thereby exhibiting anti-cancer properties. However,
though a previous study reported common over-expression of L1CAM, COX-2 and epidermal growth factor receptor (EGFR) in anaplastic pancreatic cancer, a link between their activation has not been 16
. L1CAM can be cleaved by the metalloproteinases ADAM10 and ADAM17
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further described
leading to the release of a 200 kDa soluble ectodomain with the 32 kDa transmembrane stump retaining in the plasma membrane 11. The results of our western blot analysis suggested that celecoxib
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might induce enhanced ectodomain shedding, but further studies are necessary, since there are no information in the literature about transcriptional regulations of L1CAM.
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Taken together, our data showed that LICAM activates the STAT3/NF-kB signaling pathway and enhances the proliferation and invasion of pancreatic cancer cells, which is inhibited by silencing of L1CAM. Celecoxib reduced the expression of L1CAM, STAT3 and NF-kB in pancreatic cancer cells.
Acknowledgments This study was supported by a grant from the Development and reform commission of Hunan Province Industry Research Project (2015-99), and the Hunan Province Tumor Hospital Science and Technology 13
ACCEPTED MANUSCRIPT Project (K2013).
Conflict of interests
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The authors declare no conflict of interests for this research.
Authors’ contribution
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CZ was responsible for the conception and design of the study. XQ, MM, BT, YH and SX were
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responsible for acquisition of data and analyzed the data; furthermore, KZ was in charge of statistical analysis. DY ZL and NL drafted the manuscript; XS and CZ revised and commented the draft. All
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authors approved the final version of the manuscript.
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ACCEPTED MANUSCRIPT Tables Table 1. Correlation of L1CAM expression in human pancreatic cancer tissues and the clinical characteristics of patients
Characteristics
Case number Low (-/+)
High (++/+++)
Age
0.005
15
> 55
34
10
UICC stage I+II
11
III+IV
45
3
17
28
15
11
4
41
14
27
0.038
0.001
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Poor
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ACCEPTED MANUSCRIPT Figure legends Figure 1. A). Inhibition rate of celecoxib on the viability of pancreatic cancer cells of Panc-1. B). Inhibition rate of celecoxib on the viability of pancreatic cancer cells of Bxpc-3 cells. *P < 0.05, **P <
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0.01 versus the blank control (0 µM celecoxib group). Figure 2. L1CAM immunohistochemistry staining of paraffin embedded 5 µm thick sections of pancreatic cancer tissue specimens and matched adjacent tissues (100 ×). Compared to the control
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(cancer surrounding tissue), the pancreatic cancer tissue showed: A, strong positive L1CAM expression;
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B, positive L1CAM expression; C, weak positive L1CAM expression.
Figure 3. Suppression of L1CAM/STAT3/NF-κB signaling cascade in celecoxib-induced anti-cancer efficacy in pancreatic cancer cells
A, expression of STAT3, p-STAT3, NF-κB and p-NF-κB of pancreatic cancer cells after exposure to
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celecoxib. Panc-1 and Bxpc-3 cells were treated with celecoxib (0, 20, 60, 100 µM) for 24 h. Total proteins were extracted and β-actin was used as a loading control. B, L1CAM expression in the total protein of Panc-1 and Bxpc-3 cells after exposure to celecoxib (0, 20, 60, 100 µM) for 24 h.
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Figure 4. The influence of L1CAM on migration of pancreatic cancer cells detected by the wound
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healing assay. A) Western Blot analyses of Panc-1 and Bxpc-3 cells after overexpression or silencing of L1CAM compared to the control. B) Distances between Panc-1 and Bxpc-3 cell formations in wound healing assays after L1CAM overexpression and L1CAM silencing compared to the controls. Figure 5. The influence of L1CAM on the levels of p-STAT3 and p-NF-κB in pancreatic cancer cells detected by immunofluorescence assay. Panc-1-Co or Bxpc-3-Co; Bxpc-3-Ov; Panc-1-Ov.
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S1424-3903(18)30030-9
DOI:
10.1016/j.pan.2018.02.006
Reference:
PAN 839
To appear in:
Pancreatology
Received Date: 16 August 2017 Revised Date:
31 January 2018
Accepted Date: 12 February 2018
Please cite this article as: Zuo C, Hong Y, Qiu X, Yang D, Liu N, Sheng X, Zhou K, Tang B, Xiong S, Ma M, Liu Z, Celecoxib suppresses proliferation and metastasis of pancreatic cancer cells by downregulating STAT3 / NF-kB and L1CAM activities, Pancreatology (2018), doi: 10.1016/j.pan.2018.02.006. 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 proof before it is published in its final 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.
ACCEPTED MANUSCRIPT Celecoxib suppresses proliferation and metastasis of pancreatic cancer cells by down-regulating STAT3 / NF-kB and L1CAM activities
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Running title: Celecoxib inhibit pancreatic cancer via L1CAM/NFkB
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Bo Tang2, Shuhan Xiong4, Min Ma1, Zhuo Liu1
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Chaohui Zuo1*#, Yuan Hong2*, Xiaoxin Qiu2, Darong Yang3, Nianli Liu3, Xinyi Sheng2, Kunyan Zhou1,
Department of Gastroduodenal and Pancreatic Surgery, Hunan Cancer Hospital and The Affiliated
Cancer Hospital of Xiangya School of Medicine, Central South University. No 283 Tongzipo Road, Changsha 410013, China.
Graduates School, University of South China, 28 West Changsheng Road, Hengyang 421001, China.
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Department of Molecular Medicine, College of Biology, State Key Laboratory of Chemo / Biosensing
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and Chemometrics, Hunan University. No 2 Lushan South road, Changsha 410082, China
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School of Public Health, Jilin University. No 2699 Qianjin Street, Changchun, 130021, China
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* Chaohui Zuo and Yuan Hong contribute equally in this study.
#
Correspondence to
Chaohui Zuo Department of Gastroduodenal and Pancreatic Surgery, Hunan Cancer Hospital and The Affiliated Cancer Hospital of Xiangya School of Medicine, Central South University. No 283 Tongzipo Road,
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ACCEPTED MANUSCRIPT Changsha 410013, China. Tel: +86-13875911328 Fax: +86-731-89762142
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Email: [email protected]
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ACCEPTED MANUSCRIPT Abstract Objective: To explore the molecular mechanisms of celecoxib-induced pancreatic cancer suppression in vivo and in vitro. Methods: The anti-pancreatic cancer activities of celecoxib (0, 20, 60 and 100
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µmol/L) were investigated by cell viability and migration of Panc-1 and Bxpc-3 cells in vitro. The expression of L1CAM in pancreatic cancer and adjacent tissues was compared using immunohistochemistry. The expressions of L1CAM, STAT3, p-STAT3, NF-κB, p-NF-κB were
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determined by western blotting, and cell invasive ability was determined by wound healing assay in
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L1CAM-silenced and over-expressed Panc-1and Bxpc-3 cells. Results: The expression of L1CAM in pancreatic carcinoma was stronger than that in the adjacent tissues and L1CAM could increase the growth and invasion of pancreatic cancer cells. Over-expression of L1CAM activated the STAT3/NF-κB signaling pathway in Panc-1 and Bxpc-3 pancreatic cancer cells and celecoxib inhibited
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their viability and the expressions of STAT3, p-STAT3, NF-κB, p-NF-κB as well as full length L1CAM in a concentration dependent manner. Conclusions: L1CAM was highly expressed in pancreatic cancer tissue and positively correlated with age, TNM staging and tumor differentiation. L1CAM activated the
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STAT/NF-κB signaling pathway and celecoxib could inhibit the activity of L1CAM, STAT3 and the
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NF-κB signaling pathway resulting in decreased growth and invasion of pancreatic cancer cells.
Keywords: pancreatic cancer; celecoxib; L1CAM; STAT3/NF-kB
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ACCEPTED MANUSCRIPT Introduction Pancreatic cancer is a most aggressive gastrointestinal malignancy and the 4th leading cause of carcinoma-related mortality in the US. In recent years, the incidence of pancreatic cancer has increased
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significantly. There were 48,960 new cases estimated in 2015, and about 40,560 cases die of this disease in America. Regardless of decades of endeavor, pancreatic cancer is still has a poor prognosis with the mortality being comparable to the morbidity 1. The incidence and mortality of pancreatic
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cancer is also increasing in China. In 2015, the incidence of pancreatic cancer in China was
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90.1/100,000, and the mortality was 79.4/100,000 people ranking it as the 6th leading cause of cancer-related death 2. Even though only resection can provide the possibility of cure, most pancreatic cancer patients miss the chance due to no early specific and sensitive clinical diagnostic technique, which results in just 15% of pancreatic cancer patients being diagnosed at an early stage so the surgery
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can be performed 3. Consequently, systemic chemotherapy has grown to be a most essential therapeutic intervention for advanced pancreatic cancer patients. It has been reported that COX-2 is highly expressed and closely related to the development of pancreatic cancers
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and thus COX-2 inhibitors
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such as celecoxib have the potential to suppress their invasion and metastasis.
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STAT3 is a transcriptional factor, which is intimately involved in cell proliferation, differentiation and apoptosis, and abnormal activation is associated with poor prognosis of solid tumors 5. NF-κB is up-regulated in over 70% of pancreatic cancer cell lines and tumor tissues 6 and has been proven to be associated with angiogenesis, invasion, and metastasis of pancreatic cancer 7. Both NF-κB and STAT3 have been reported to participate in the invasion, metastasis, angiogenesis and immune escape of cancer cells, and STAT3/NF-κB signaling is a major regulator of tumor angiogenesis and invasiveness 8. In addition, it has been reported that COX-2 activates the STAT3/NF-κB signaling pathway to promote
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ACCEPTED MANUSCRIPT invasive growth and metastasis of pancreatic cancer 9. L1 cell adhesion molecule (L1CAM) is a glycoprotein with a molecular weight of 200-220 KDa, and belongs to the immune protein superfamily. It is expressed in neurons to play a role in neuronal migration, axonal growth, bundles and elongation, 10
.
A 200 kDa soluble
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whereas in tumors it has been associated with cell invasion and motility
ectodomain of L1CAM can be cleaved proximal to the plasma membrane by specific metalloproteinases
11, 12
. In pancreatic cancer, it has been reported that L1CAM is up-regulated
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and
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closely related with invasive growth, metastasis and prognosis of pancreatic cancer 10, 14 and is believed
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to be a good target for treatment 15. In addition, expression of COX-2 and L1CAM in pancreatic cancer tissues were both enhanced, suggesting that COX-2 and L1CAM could be targets for pancreatic cancer therapy
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. However, the involvement of L1CAM in celecoxib-induced anti-cancer efficacy in
pancreatic cancer remains unclear.
Compounds and reagents
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Materials and Methods
Antibodies against STAT3 and NF-κB were purchased from Wuhan Boster Biological Technology, Ltd.
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(Wuhan, China); antibodies against p-STAT3 and p-NF-κB were obtained from Cell Signaling
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Technology (Danvers, USA); the antibody against L1CAM was bought from Santa-Cruz (Santa-Cruz, USA); the MTT kit came from Sigma-Aldrich (St. Louis, USA); celecoxib was purchased from Pfizer Inc. (New York, USA); L1CAM plasmid was purchased from Beijing Yi Qiao Shenzhou Biotechnology Co., Ltd.(Beijing, China); L1CAM siRNA was purchased from GenePharma Co., Ltd (Shanghai, China). The used transfection reagent was HiPerFect (Qiagen, Hilden, Germany). Cell lines and culture The human pancreatic cancer cells, Panc-1 and Bxpc-3, were obtained from the cell bank of the cancer research institute in the central south university. Both cell lines were cultured in DMEM medium 5
ACCEPTED MANUSCRIPT containing 10% FBS, 20 mmol/L NaHCO3, 100 U/mL penicillin and 100 µg/mL streptomycin, at 37°C in a 5% CO2 atmosphere incubator. Panc-1 and Bxpc-3 cells were treated with various concentrations of celecoxib.
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Cancer and adjacent tissue of pancreatic cancer patients A total of 56 pancreatic cancer tissue specimens (TNM stages II-III), and the matched adjacent tissue (5 cm from the cancerous tissue) were collected by stomach and duodenum surgical resection from
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patients in the Hunan Provincial Tumor Hospital from 2010 to 2015, and stored at -80°C. All of the
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patients did not accept preoperative chemotherapy or radiotherapy. Western blot
Pancreatic cancer cells in 6-well plates pretreated with PBS or different concentration of celecoxib were washed twice with cold PBS, and then re-suspended in RIPA cell lysis buffer (50mM Tris-HCl,
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PH7.4, 150mMNaCl, 1% NP40, 0.5% Sodium deoxycholate, 0.1% SDS) with proteinase inhibitors (100:1) and incubated on ice for 15 min followed by scratch. The supernatant of the cell lysate was collected by centrifugation at 13,000 g and 4°C for 5 min. The supernatant was transferred into new 1.5
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mL EP tubes, and the protein concentrations were determined, using the bicinchoninic acid (BCA)
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method according to the manufacturer’s protocol. A total of 25 µg of proteins mixed with the same volume of 2 × loading buffer were run on 10% SDS-PAGE gels and subsequently transferred to polyvinylidene fluoride (PVDF) membranes (120 V × 90 min). Membranes were blocked with 5% nonfat milk for 1 h at room temperature, and then incubated overnight at 4°C or 1 h at room temperature with primary antibodies as indicated. The membranes were washed with TBST 3 times × 5 min and further incubated with an appropriate second antibody for 1 h at room temperature. Signals were developed using an enhanced chemiluminescent (ECL) detection kit (Pierce, Rockford, USA) at
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ACCEPTED MANUSCRIPT room temperature for 5 min after further washing with TBST 3 times × 5 min. The bands of the target proteins were obtained and the gray value of each band was calculated using Image J software. Cell viability assay
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Cell viability was measured using a MTT assay. Briefly, the logarithmic phase pancreatic cancer cells were seeded at a density of 5,000 cells/100 µL/well, in 96-well plates; the marginal wells were filled with sterile PBS. The cells were incubated at 37°C in a 5% CO2 incubator overnight until the cells were
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attached to the bottom (i.e. a monolayer of cells covered the bottom of the wells), and treated with 0, 20, 60 or 100 µM of celecoxib with 5 duplicates of each concentration for 24 h. Then, 20 µL of MTT (5
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mg/mL) was added to each well and cultured for 3-4 h. The wells with the same volume of serum free medium, MTT and DMSO as the test wells were designated as the blank control group. The wells with the same number of pancreatic cancer cells and the same volume of serum free medium, MTT and
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DMSO as in the test wells were the control group. After termination of the reaction, the optical density (O.D.) value of each well was measured with a microplate reader at a wavelength of 570 nm. The cell viability and inhibition rate were calculated according to the following equations, respectively: cell
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viability = (OD value of test drug group - OD value of blank group) / (OD value of control group - OD
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value of blank group) × 100%; inhibition rate = (1- OD value of test drug group) / OD value of control group.
Wound healing assay
The migration abilities of wild type pancreatic cancer cells, L1CAM silenced and over-expressed pancreatic cancer cells were determined using the wound healing assay. Briefly, logarithmic phase pancreatic cancer cells were seeded at a density of 8 × 105 cells/mL/well, in 6-well plates, in triplicate. The cells were pretreated as described above for the indicated groups. Five straight lines at intervals of
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ACCEPTED MANUSCRIPT 1 cm were marked using a pen on the back of the well with pretreated cells. A straight line, perpendicular to the 5 lines on the back of each well at the center, was gently scraped using the tip of a disposable pipette, when the pancreatic cancer cells were grown to 95% confluence. The wells were
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then rinsed with sterile PBS 3 times and filled with serum-free DMEM medium. The wound healing was observed and photographed at 0, 6 and 24 h. Fluorescence microscope observations
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The coverslips were soaked in 75% alcohol and placed in the wells of 6 well-plates with 1 mL
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PBS/well. Cells were seeded onto the coverslips at a concentration of 5 × 105 cells/mL/well after washing the coverslips twice with 2 mL of PBS. The medium was discarded and plates washed once with 2 mL of PBS. A total of 2 mL of acetone was added into each well, which were then placed in a freezer at -20°C for 8 min, and then the acetone was discarded. The coverslip was moved to the
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microscope slide with cells upward and washed 3 times × 5min with PBS, followed by the addition of 250 µL sheep serum to block for 30 min. Immediately, 250 µL of primary antibody was added and cells incubated at room temperature for 60 min after discarding the sheep serum, and then washed 3 times ×
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5min with PBS. Then, 250 µL of the second antibody was added and the cells incubated at room
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temperature for 60 min in the dark, and then washed 3 times × 5 min with PBS. The air-dried coverslips with treated cells were placed carefully, face down on a new microscope slide with 10 µL of DAPI so that DAPI could be expanded slowly without bubbles. Specimens were observed and photographed under a fluorescence microscope at a 360~400 nm excitation wavelength. Cell transfection with L1CAM expressing plasmid and L1CAM siRNA Pancreatic cancer cells were seeded in 6-well plates at a concentration of 6 × 105/well and incubated overnight at 37°C in a 5% CO2 incubator. The target plasmid and siRNA were diluted with Opti-MEM 8
ACCEPTED MANUSCRIPT serum-free medium and transfected with HiPerFect reagent before transfection, gently mixed, and allowed to stand at room temperature for 20 min. The solution was added to the cell culture dish and shaken back and forth to mix it with the cell culture medium. Cells transfected with scrambled siRNA
indicated time points according to experimental requirements. Immunohistochemistry
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or empty plasmid were used as the corresponding controls. Gene expression was measured at the
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Tumor tissue was fixed in 10% neutral buffered paraformaldehyde at 4°C for 24 h. Sections (5 µm thick) were deparaffinized, rehydrated with PBS (pH 7.4), treated with aqueous 3% H2O2 for 10 min,
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and the antigen retrieved in 0.1% trypsin (M/V) at 37°C for 10 min. Sections were blocked with 5% normal goat serum at room temperature for 30 min, then 1:100 anti-L1CAM antibody was applied and sections incubated at 4°C overnight. Next, after being washed 2 times × 5 min with PBS sections were
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incubated with a second antibody for 1 h at 37°C. Then they were incubated with diaminobenzidine (DAB) for 5 min and counterstained with hematoxylin after being washed in running water. After gradient ethanol dehydration and xylene for 3 min, the sections were then mounted with neutral gum
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after being made transparent with xylene.
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Observation and evaluation criteria (using the quartile method) Images were acquired with an optical microscope (Olympus IX70, Japan). PBS was used as the negative control instead of primary antibody. The expression level of the target protein in the tissues was determined according to the positive cell numbers in the visual field 17. First five areas with the highest number of L1CAM -positive cells were selected. Then, these cells were counted (per mm2) at higher (×400) magnification. Finally, L1CAM positive cells were scored into four categories: 1 (< 25%); 2 (25% to 50%); 3 (50% to 75%); and 4 (> 75%) and the staining intensity of positive cells was
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ACCEPTED MANUSCRIPT scored as weak, moderate and strong. The final score was the product of the intensity and the percentages of L1CAM expressing cells. Ethics statement
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The protocol of this study was approved by the ethics committee of the Hunan Cancer Hospital and The Affiliated Cancer Hospital of Xiangya School of Medicine, Central South University and all participants provided written informed consent. Statistical analysis
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All statistical analyses were performed with SSPS for Windows (version 18.0. Chicago: SPSS Inc). The measurement data were analyzed using a paired t-test. The count data was expressed as a percentage
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and compared using a χ2 test. Comparisons between multiple groups were performed using variance analysis of repeated measurement data. P < 0.05 was taken to indicate a statistically significant difference. Results
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Celecoxib inhibited cell viability of pancreatic cancer cells Panc-1 and Bxpc-3 To investigate the inhibitory effect of celecoxib on pancreatic cancer cell viability, the Panc-1 and Bxpc-3 cells were challenged with celecoxib for 24, 36 and 48 h at final concentrations of 0, 20, 60 or
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100 µM, and the relative viable cell numbers were analyzed by MTT assay. As shown in Figure 1A
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and 1B, celecoxib significantly inhibited the viability of both Panc-1 (Figure 1A) and Bxpc-3 (Figure 1B) cells in a dose- and time-dependent manner, and the difference between each celecoxib treatment group and the blank control group was statistically significant (P < 0.05). The inhibition rate of celecoxib at final concentrations of 0, 20, 60 or 100 µM on the Panc-1 cells at 24 h treatment was 0%, 12%, 28% and 61% respectively; after 36 h treatment the inhibition rate was 0%, 19%, 35% and 78% respectively, and after 48 h 0%, 23%, 41% and 86%. The inhibition rate of celecoxib at the final concentrations of 0, 20, 60 or 100 µM on the Bxpc-3 cells after 24 h treatment was 0%, 8%, 14% and
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ACCEPTED MANUSCRIPT 36% respectively; after 36 h 0%, 4%, 26% and 58%; and after 48 h treatment 0%, 19%, 37% and 76%. These data reveal the anticancer activity of celecoxib in pancreatic cancer cells, with Panc-1 cells being more sensitive to celecoxib than Bxpc-3 cells.
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Up-regulation of L1CAM in human pancreatic cancer cells A total of 56 pancreatic cancer tissue specimens and matched adjacent tissues were collected from patients in the Hunan Provincial Tumor Hospital from 2008 to 2014. The 56 pancreatic cancer patients
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included 36 males and 20 females; the median age was 59 years (39 to 78 years) at pathological
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diagnosis. There were 48 cases of ductal adenocarcinoma and 8 cases of mucinous cystadenocarcinoma; 3 cases of stage I, 8 cases of stage II, 18 cases of stage III and 27 cases of stage IV, according to the TNM staging
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. All these patients received radical surgery (radical pancreaticoduodenectomy or
pancreatic tail resection), without radiotherapy and chemotherapy before the surgery.
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The expression of L1CAM in 56 pancreatic cancer tissue specimens and matched adjacent tissues was detected by immunohistochemistry. Our results showed that L1CAM was strongly positively expressed in 21 pancreatic cancer tissue specimens, positively expressed in 10, and weakly positively expressed
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in 25 versus the matched adjacent tissues, which showed negative expression of L1CAM (Figure 2).
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The expression of L1CAM was negatively correlated with age, the TNM stage and tumor differentiation status (Table 1). Down-regulation of L1CAM, STAT3 and NF-κB in Panc-1 and Bxpc-3 cells by celecoxib To demonstrate whether the anti-cancer effect of celecoxib was associated with L1CAM and related mechanisms in pancreatic cancer cells, the total protein of Panc-1 and Bxpc-3 cells, treated with different concentrations of celecoxib (0, 20, 60, 100 µM) for 24 h, was subjected to western blot analysis to determine the expression levels of L1CAM, STAT3, p-STAT3, NF-κB and p-NF-κB. The
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ACCEPTED MANUSCRIPT results showed that the expression of STAT3, p-STAT3, NF-κB and p-NF-κB decreased with an increase in the concentration of celecoxib in Panc-1 and Bxpc-3 cells (Figure 3 A); the appearance of full length L1CAM (230 kDa) was also reduced by celecoxib in a concentration-dependent manner,
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which was most obvious in Bxpc-3 cells at a concentration of 100 µM (Figure 3B). Effects of over-expression of L1CAM and L1CAM silencing on migration and invasion of pancreatic cancer cells.
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To demonstrate further that L1CAM promotes the migration of pancreatic cancer cells, the L1CAM
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gene was over-expressed or silenced in Panc-1 and Bxpc-3 cells (Figure 4A) and the migration abilities of the L1CAM over-expressing Panc-1 (Panc-1-Ov) cells, L1CAM over-expressed Bxpc-3 (Bxpc-3-Ov)cells, L1CAM silenced Panc-1 cells (Panc-1-Si) and L1CAM silenced Bxpc-3 (Bxpc-3-Si) cells were measured using wound healing assays and compared with the corresponding control cells
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(Panc-1-Co or Bxpc-3-Co), which were the cells transfected with scrambled siRNA. Figure 4B shows the sizes of the gaps in wound healing assays at the indicated time points. Our results showed that compared with the corresponding control cells, the migration abilities of Panc-1-Si and
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Bxpc-3-Si were decreased, while the migration abilities of Panc-1-Ov and Bxpc-3-Ov cells were
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increased, which was reflected in more closed gaps in the wound healing assays. These results further confirmed the importance of L1CAM in promoting pancreatic cancer metastasis. L1CAM promotes phosphorylated STAT3 and NF-κB To confirm further that the effect of L1CAM in promoting pancreatic cancer viability and metastasis is related to STAT3/NF-κB, the levels of p-STAT3 and p-NF-κB in Panc-1-Ov and Bxpc-3-Ov cells were detected using an immunofluorescence assay. The results revealed that the expression of p-STAT3 and p-NF-κB were increased in Panc-1-Ov and Bxpc-3-Ov cells, which indicated that L1CAM could
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ACCEPTED MANUSCRIPT stimulate the phosphorylation level of STAT3 and NF-κB (Figure 5). This finding was consistent with the results of our western blot assays. The results confirmed that L1CAM could activate the STAT3/NF-κB signaling pathway by increasing the expression of p-STAT3 and p-NF-κB. Discussion
cancer cells, which is in agreement with the literature
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In the present study we found that L1CAM, NF-κB and STAT3 were enhanced expressed in pancreatic . In addition, we found that
down-regulation of L1CAM led to reduced migration of Bxpc-3 and Panc-1 cells, which is in line with
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previous reports that L1CAM triggers cell migration and invasion 20. Moreover, celecoxib could inhibit the expressions of STAT3, p-STAT3, NF-κB and p-NF-κB in Panc-1 and Bxpc-3 cells in a
constitutive NF-κB activity
22, 23
21
and leads to
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concentration-dependent manner. L1CAM has been reported to activate STAT3
, while interactions between STAT3 and NF-κB promote the
development and progression cancers 24. In addition, previous research revealed that COX-2 inhibitors downregulated STAT3 and NF-κB activities
9, 25
thereby exhibiting anti-cancer properties. However,
though a previous study reported common over-expression of L1CAM, COX-2 and epidermal growth factor receptor (EGFR) in anaplastic pancreatic cancer, a link between their activation has not been 16
. L1CAM can be cleaved by the metalloproteinases ADAM10 and ADAM17
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further described
leading to the release of a 200 kDa soluble ectodomain with the 32 kDa transmembrane stump retaining in the plasma membrane 11. The results of our western blot analysis suggested that celecoxib
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might induce enhanced ectodomain shedding, but further studies are necessary, since there are no information in the literature about transcriptional regulations of L1CAM.
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Taken together, our data showed that LICAM activates the STAT3/NF-kB signaling pathway and enhances the proliferation and invasion of pancreatic cancer cells, which is inhibited by silencing of L1CAM. Celecoxib reduced the expression of L1CAM, STAT3 and NF-kB in pancreatic cancer cells.
Acknowledgments This study was supported by a grant from the Development and reform commission of Hunan Province Industry Research Project (2015-99), and the Hunan Province Tumor Hospital Science and Technology 13
ACCEPTED MANUSCRIPT Project (K2013).
Conflict of interests
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The authors declare no conflict of interests for this research.
Authors’ contribution
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CZ was responsible for the conception and design of the study. XQ, MM, BT, YH and SX were
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responsible for acquisition of data and analyzed the data; furthermore, KZ was in charge of statistical analysis. DY ZL and NL drafted the manuscript; XS and CZ revised and commented the draft. All
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authors approved the final version of the manuscript.
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ACCEPTED MANUSCRIPT Tables Table 1. Correlation of L1CAM expression in human pancreatic cancer tissues and the clinical characteristics of patients
Characteristics
Case number Low (-/+)
High (++/+++)
Age
0.005
15
> 55
34
10
UICC stage I+II
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III+IV
45
3
17
28
15
11
4
41
14
27
0.038
0.001
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Histologic differentiation
7
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≤ 55
High/Moderate
P-value
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Expression of L1CAM
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ACCEPTED MANUSCRIPT Figure legends Figure 1. A). Inhibition rate of celecoxib on the viability of pancreatic cancer cells of Panc-1. B). Inhibition rate of celecoxib on the viability of pancreatic cancer cells of Bxpc-3 cells. *P < 0.05, **P <
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0.01 versus the blank control (0 µM celecoxib group). Figure 2. L1CAM immunohistochemistry staining of paraffin embedded 5 µm thick sections of pancreatic cancer tissue specimens and matched adjacent tissues (100 ×). Compared to the control
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(cancer surrounding tissue), the pancreatic cancer tissue showed: A, strong positive L1CAM expression;
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B, positive L1CAM expression; C, weak positive L1CAM expression.
Figure 3. Suppression of L1CAM/STAT3/NF-κB signaling cascade in celecoxib-induced anti-cancer efficacy in pancreatic cancer cells
A, expression of STAT3, p-STAT3, NF-κB and p-NF-κB of pancreatic cancer cells after exposure to
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celecoxib. Panc-1 and Bxpc-3 cells were treated with celecoxib (0, 20, 60, 100 µM) for 24 h. Total proteins were extracted and β-actin was used as a loading control. B, L1CAM expression in the total protein of Panc-1 and Bxpc-3 cells after exposure to celecoxib (0, 20, 60, 100 µM) for 24 h.
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Figure 4. The influence of L1CAM on migration of pancreatic cancer cells detected by the wound
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healing assay. A) Western Blot analyses of Panc-1 and Bxpc-3 cells after overexpression or silencing of L1CAM compared to the control. B) Distances between Panc-1 and Bxpc-3 cell formations in wound healing assays after L1CAM overexpression and L1CAM silencing compared to the controls. Figure 5. The influence of L1CAM on the levels of p-STAT3 and p-NF-κB in pancreatic cancer cells detected by immunofluorescence assay. Panc-1-Co or Bxpc-3-Co; Bxpc-3-Ov; Panc-1-Ov.
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