© The Author 2006. Published by Oxford University Press.
ARTICLE |
Tolfenamic Acid and Pancreatic Cancer Growth, Angiogenesis, and Sp Protein Degradation
Affiliations of authors: Institute of Biosciences and Technology, Health Science Center, Texas A&M University, Houston, TX (MA, SS); Department of Cancer Biology (CHB), Department of Gastrointestinal Medical Oncology (JLA), University of Texas M. D. Anderson Cancer Center, Houston, TX; Department of Veterinary Physiology and Pharmacology, Texas A&M University, College Station, TX (SS)
Correspondence to: Stephen Safe, DPhil, Department of Veterinary Physiology and Pharmacology, Texas A&M University, 4466 TAMU, Vet. Res. Bldg. 410, College Station, TX 77843-4466 (e-mail: ssafe{at}cvm.tamu.edu).
 |
ABSTRACT |
|---|
 TopNotes Abstract IntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Background: Sp1, Sp3, and Sp4 are transcription factors that regulate cell proliferation and vascular endothelial growth factor (VEGF) expression and are overexpressed in many cancer cell lines. For some cancers, Sp1 overexpression is associated with poor survival. Cyclooxygenase inhibitors decrease Sp1 expression in cancer cells, and therefore different structural classes of nonsteroidal anti-inflammatory drugs (NSAIDs) were screened for their ability to decrease levels of Sp1, Sp3, and Sp4 and to decrease pancreatic tumor growth and metastasis in an in vivo model. Methods: Levels of Sp1, Sp3, Sp4, and VEGF proteins in pancreatic cancer cell lines were assessed by immunoblot analysis. mRNA was assessed by reverse transcription–polymerase chain reaction. Panc-1 pancreatic cancer cells transfected with VEGF promoter constructs were used to assess VEGF promoter activation. Pancreatic tumor weight and size and liver metastasis were assessed in an orthotopic mouse model of pancreatic cancer (groups of 10 mice). Protein expression in tumors was assessed immunohistochemically. Results: Tolfenamic acid and structurally related biaryl derivatives induced degradation of Sp1, Sp3, and Sp4 in pancreatic cancer cells. Tolfenamic acid also inhibited VEGF mRNA and protein expression in pancreatic cancer cells; this inhibition was associated with the decreased Sp-dependent activation of the VEGF promoter. In the mouse model for pancreatic cancer, treatment with tolfenamic acid (50 mg/kg of body weight), compared with control treatment, statistically significantly decreased tumor growth and weight (P = .005), liver metastasis (P = .027), and levels of Sp3 and VEGF (P = .009) and Sp1 and Sp4 (P = .006) proteins in tumors. For example, tumors from mice treated with tolfenamic acid (50 mg/kg) had statistically significantly lower VEGF levels (45%, 95% confidence interval = 39% to 51%; P = .009) than tumors from control mice. Conclusions: Tolfenamic acid is a new antipancreatic cancer NSAID that activates degradation of transcription factors Sp1, Sp3, and Sp4; reduces VEGF expression; and decreases tumor growth and metastasis.
|
INTRODUCTION |
|---|
|
TopNotesAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Nonsteroidal anti-inflammatory drugs (NSAIDs) inhibit cyclooxygenase (COX)-dependent synthesis of prostaglandins that mediate inflammatory responses in multiple tissues (1–3). NSAIDs also induce cell growth inhibition, apoptosis, and antiangiogenesis; activation of the pathways involved in these effects is critical for the reported anticarcinogenic activities of NSAIDs and related COX-2 inhibitors, such as celecoxib (3–7). Extensive epidemiologic studies of the role of NSAIDs in the prevention and treatment of colon cancer indicate that the use of NSAIDs, such as aspirin and some COX-2 inhibitors, is associated with a decrease in the incidence and/or mortality of colon cancer (8–13). Patients with familial adenomatous polyposis coli are highly susceptible to development of colon cancer, and these individuals have been successfully treated with the COX-1 and COX-2 inhibitor sulindac to repress colonic polyp formation (11,13). Laboratory animal and cell culture studies also confirm the efficacy of NSAIDs for inhibiting growth of colon cancers and tumors derived from other tissues (3–7,14–17).
Although aspirin use has not been associated with decreased incidence of pancreatic cancer (16–19), several studies have found that NSAIDs and COX-2 inhibitors alone and in combination with chemotherapeutic drugs can inhibit pancreatic cancer cell and tumor growth (20–28). For example, celecoxib statistically significantly increased gemcitabine-induced growth inhibition and apoptosis in BxPC-3 pancreatic cancer cells, which express high levels of COX-2 (27). Other studies (26–28) reported growth inhibition with combinations of NSAIDs and chemotherapeutic drugs, through both COX-2–dependent and –independent mechanisms.
Growth and metastasis of pancreatic cancer depend on activation of vascular endothelial growth factor (VEGF) and other angiogenic factors. Sp1, Sp3, and Sp4 are transcription factors that are overexpressed in many cancer cell lines. For some cancers, Sp1 overexpression is associated with poor survival. There is strong evidence that Sp1 and other Sp proteins regulate VEGF expression in pancreatic cancer cells (29,30). The COX-2 inhibitor celecoxib decreases the expression of Sp1 and VEGF by inducing degradation of Sp1 in pancreatic cancer cells (31), and studies from this laboratory (32) show that COX-2 inhibitors decrease VEGF expression in colon cancer cells by decreasing the level of Sp1 and Sp3.
Because VEGF expression in pancreatic cancer cells depends on expression of Sp1, Sp3, and Sp4 proteins (30), an ideal antiangiogenic agent for pancreatic cancer would induce degradation of all three Sp proteins. In this study, we screened 14 NSAIDs or inhibitors of COX-1 and/or COX-2 for their ability to reduce the levels of Sp1, Sp3, and Sp4 proteins in pancreatic cancer cells. We also investigated the mechanism of action of the most active NSAID in Panc-1, L3.6pl, and Panc-28 pancreatic cancer cell lines and in an orthotopic model for pancreatic cancer. The purpose of this study was to identify NSAIDs that decrease levels of Sp1, Sp3, and Sp4, resulting in decreased pancreatic tumor growth and metastasis in an in vivo model.
|
MATERIALS AND METHODS |
|---|
|
TopNotesAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Cell Lines, Cell Culture, Constructs, Antibodies, NSAIDs, and Oligonucleotides
The human pancreatic cell line Panc-1 was obtained from the American Type Culture Collection (Manassas, VA). The human pancreatic cell lines Panc-28 and L3.6pl (33,34) were obtained from the University of Texas M. D. Anderson Cancer Center (Houston). DME/F12 medium with or without phenol red, a 100x antibiotic/antimycotic solution, and lactacystin were purchased from Sigma Chemical Co. (St. Louis, MO). Fetal bovine serum was purchased from Intergen (Purchase, NY). [
-32P]ATP (300 Ci/mmol) was obtained from Perkin Elmer Life Sciences (Boston, MA). Poly(dI-dC) and T4 polynucleotide kinase were purchased from Roche Molecular Biochemicals (Indianapolis, IN). Antibodies against Sp1 (PEP2), Sp3 (D-20), Sp4 (V-20), histone deacetylase (H-51), 
-tubulin (H-235), and VEGF (A-20) proteins and goat anti–rabbit immunoglobulin G F secondary antibody were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Celecoxib (32) was obtained from the University of Texas M. D. Anderson Cancer Center. The NSAIDs valeryl salicylate, acemetacin, ampiroxicam, diclofenac, diflunisal, naproxen, fenbufen, ibuprofen, ketoprofen, tolmetin, letrozole, and tolfenamic acid (i.e., Clotam) were purchased from LKT Laboratories, Inc. (St. Paul, MN). Lysis buffer and luciferase reagent were obtained from Promega Corp. (Madison, WI). VEGF promoter constructs have previously been described (30,32).
Transfection of Pancreatic Cancer Cells, Proteasome Inhibitor Experiments, and Preparation of Nuclear Extracts
Panc-1 and L3.6pl cells were cultured in six-well plates, with each well containing 2 mL of DME/F12 medium supplemented with 5% fetal bovine serum. After 16–20 hours of incubation when cells were 50%–60% confluent, reporter gene constructs were transfected by use of the Lipofectamine reagent as described by the manufacturer (Invitrogen, Carlsbad, CA). The pVEGF1 and pVEGF2 constructs contain VEGF promoter inserts (positions –2018 to +50 and positions –131 to +54, respectively) linked to luciferase (a reporter gene), as previously described (30,32). The effects of NSAIDs that induced Sp protein degradation (tolfenamic acid and diclofenac) and did not affect Sp protein levels (ampiroxicam) on luciferase activity were investigated in Panc-1 and L3.6pl cells cotransfected with 500 ng of the pVEGF1 or pVEGF2 constructs. Cells were treated with 0.1% dimethyl sulfoxide (DMSO; control vehicle) or with the indicated concentration of NSAIDs for 24 or 48 hours, and then the effects on transactivation (luciferase activity, which reflects the activity of the VEGF promoter insert) in whole cell lysates (relative to -galactosidase activity) was determined. For nuclear extracts, cells were washed twice in phosphate-buffered saline (PBS), scraped into 1 mL of 1x lysis buffer (50 mM HEPES, 0.5 M NaCl, 1.5 mM MgCl2, 1 mM EGTA, 10% [vol/vol] glycerol, 1% Triton X-100, and 5 µL of protease inhibitor cocktail [product P8340; Sigma] per 1 mL of buffer), incubated at 4 °C for 15 minutes, and centrifuged at 14 000g for 1 minute at 20 °C. Cell pellets were initially washed three times in 1 mL of lysis buffer; lysis buffer supplemented with 500 mM KCl was then added to the cell pellet and incubated with it for 45 minutes at 4 °C with frequent vortex mixing. Nuclei were pelleted by centrifugation at 14 000g for 1 minute at 4 °C, and aliquots of supernatant were stored at –80 °C for use in Western blot analyses and gel shift assays. For proteasome inhibitor experiments, cells were cotreated with 2 µM lactacystin, a proteasome inhibitor that would inhibit NSAID-induced activation of this protein breakdown pathway. For the electrophoretic mobility shift assay (see below), nuclear extracts from Panc-1 and L3.6pl cells were isolated as previously described (30,32), and aliquots were stored at –80 °C.
Immunoblot Analysis and Laser Scanning Densitometry
Panc-1 cells were washed once with PBS and collected by scraping cells from the culture plate in 200 µL of lysis buffer. The cell lysates were incubated on ice for 1 hour with intermittent vortex mixing and then centrifuged at 40 000g for 10 minutes at 4 °C. Equal amounts of protein (60 µg per lane) from cells treated with 0.1% DMSO or individual NSAIDs, at the concentrations indicated, were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis on 10%–12.5% gels. For VEGF immunoblots, 100 µg of protein per lane was used. Samples were electrophoresed and transferred to a membrane (30,32), and proteins were detected by incubation with primary polyclonal antibodies against Sp1, Sp3, Sp4, histone deacetylase, VEGF, or -tubulin, followed by incubation with an appropriate horseradish peroxidase–conjugated goat anti-rabbit immunoglobulin G F secondary antibody, as previously described (30). Band intensities were determined by scanning laser densitometry (Sharp Electronics Corporation, Mahwah, NJ) and Zero-D Scanalytics software (Scanalytics Corporation, Billerica, MA).
Electrophoretic Mobility Shift Assay
Sense and antisense VEGF oligonucleotides derived from the VEGF promoter (positions –66 to –47) were synthesized and annealed to each other, and 5-pmol aliquots were 5' end-labeled by use of T4 kinase and [-32P]ATP. A 30-µL electrophoretic mobility shift assay reaction mixture contained approximately 100 mM KCl, 3 µg of crude nuclear protein from Panc-1 or L3.6p1 cells treated with 0.1% DMSO, 50 µM ampiroxicam, or 50 µM tolfenamic acid for 48 hours, 1 µg of poly(dI-dC), and 10 fmol of radiolabeled probe. After incubation for 20 minutes on ice, antibodies against Sp1, Sp3, or Sp4 protein were added, and the mixture was incubated another 20 minutes on ice. Protein–DNA complexes were resolved by polyacrylamide gel electrophoresis on 5% gels, as previously described (30,32). Specific DNA–protein and antibody-supershifted complexes were observed as retarded bands in the gel by autoradiography.
Cell Proliferation Assay
Panc-1, Panc-28, and L3.6pl cells were plated in DME/F12 medium with 5% fetal bovine serum and treated on the next day with vehicle (0.1% DMSO) or various concentrations of ampiroxicam, tolfenamic acid, diclofenac, or naproxen, as indicated. Cells were counted at the indicated times with a Coulter Z1 cell counter (Beckman-Coulter, Fullerton, CA). Each experiment was done in triplicate, and results are expressed as means, with error bars representing 95% confidence intervals (CIs).
Immunocytochemistry and Immunohistochemistry
Panc1 cells were cultured on Lab-Tek Chamber slides (Nalge Nunc International, Naperville, IL) at 100 000 cells per well in DME/F12 medium supplemented with 5% fetal bovine serum. Cells were then treated with the indicated NSAID for 48 hours, the medium chamber was detached from the slide, and the glass slide was washed in PBS. VEGF was detected by immunostaining essentially as previously described (30). In brief, cells on the glass slides were fixed in methanol at –20 °C for 10 minutes and then washed for two 5-minute periods in 0.3% PBS–Tween 20 before blocking with 5% goat serum in antibody dilution buffer (stock solution = 100 mL of PBS–Tween, 1 g of bovine serum albumin, and 45 mL of glycerol, pH 8.0) for 1 hour at 20 °C. After removal of the blocking solution, rabbit anti-VEGF polyclonal antibodies (1 : 200 dilution) were added in antibody dilution buffer and incubated for 12 hours at 4 °C. Slides were washed for three 10-minute periods with 0.3% Tween–PBS and incubated with fluorescein isothiocyanate–conjugated goat anti-rabbit antibodies (1 : 1000 dilution) for 2 hours at 20 °C. Slides were then washed for four 10-minute periods in 0.3% Tween–PBS. Slides were mounted in ProLonged antifading medium with 4',6-deamidino-2-phenylindole for nuclear counterstaining (Molecular Probes, Eugene, OR), and cover slips were sealed with Nailslicks nail polish (Noxell Corp., Hunt Valley, MD). Fluorescence imaging was performed with a Carl Zeiss Axiophoto 2 (Carl Zeiss, Thornwood, NY), and Adobe Photoshop version 5.5 was used to capture the images.
For immunohistochemistry and histologic staining procedures using mouse tumors, one part of the tumor tissue was fixed in formalin and embedded in paraffin and the other part of the tumor was embedded in OCT compound (Miles, Inc., Elkhart, IN), snap-frozen in liquid nitrogen, and stored at –70 °C. Paraffin-embedded tissues were used for identification of VEGF and CD31. Sections (4–6 µm thick) were mounted on positively charged Superfrost slides (Fischer Scientific, Co., Houston, TX) and dried overnight. Sections were deparaffinized in xylene, treated with a graded series of alcohol (100%, 95%, and 80% ethanol [vol/vol] in double distilled H2O), and rehydrated in PBS (pH 7.5). Tissues were then treated with pepsin (Biomedia Corp., Foster City, CA) for 15 minutes at 37 °C and washed with PBS (35). A positive reaction was visualized by incubating the slides with stable 3,3'-diaminobenzidine (Research Genetics, Huntsville, AL) for 10–20 minutes. The sections were rinsed with distilled water, counter-stained with Gill's hematoxylin (Sigma) for 1 minute, and mounted with Universal Mount (Research Genetics). Control samples exposed to secondary antibody alone showed no specific staining.
Semiquantitative Reverse Transcription–Polymerase Chain Reaction Analysis
Panc-1 cells were treated with 0.1% DMSO (control) or the NSAID as indicated for 48 hours, and then total RNA was collected (32) for reverse transcription-polymerase chain reaction (RT-PCR). For VEGF mRNA stability studies, Panc-1 cells were treated with actinomycin D (5 mg/mL) alone or in combination with 50 µM tolfenamic acid, as indicated. Total RNA was obtained with RNAzol B (Tel-Test, Friendswood, TX) according to the manufacturer's protocol. The RNA concentration was determined by use of the 260 nm/280 nm absorption ratio, and RNA at 200 ng/µL was used in each reaction mixture for RT-PCR. RNA was reverse transcribed at 42 °C for 25 minutes with oligo(dT) primers (Promega), and then the reverse transcription product was amplified by PCR in a reaction mixture containing MgCl2 (2 mmol/L), each gene-specific primer at 1 µmol/L, all four deoxynucleoside triphosphates (each at 1 mmol/L), and 2.5 units of AmpliTaq DNA polymerase (Promega). Gene products were amplified for 22–25 cycles of 95 °C for 30 seconds, 56 °C for 30 seconds, and 72 °C for 30 seconds. Sequences of the oligonucleotide primers used in this study were as follows: VEGF forward = 5'-CCATGAACTTTCTGCTGTCTT-3'; VEGF reverse = 5'-ATCGCATCAGGGGCACACAG-3'; glyceraldehyde-3-phosphate dehydrogenase (GAPDH) forward = 5'-AATCCCATCACCATCTTCCA–3'; and GAPDH reverse = 5'-GTCATCATATTTGGCAGGTT-3'. After amplification in a Peltier thermal cycler (Hybaid US, Franklin, MA), 20 µL of each reaction mixture was loaded on a 2% agarose gel containing ethidium bromide. Electrophoresis was performed at 80 V in 1x TAE buffer (48.4 g of Tris base, 11.4 mL of acetic acid, and 20 mL of 0.5 M EDTA in 1 L of water; diluted 1 : 10) for 1 hour, and the gel was photographed by UV transillumination on Polaroid film (Waltham, MA). The intensity of VEGF and GAPDH bands was obtained by scanning the Polaroid film with a Sharp JX-330 scanner (Sharp Electronics, Mahwah, NJ); and a densitometric analysis was performed on the inverted image with Zero-D software (Scanalytics). Results for VEGF band intensity were normalized to GAPDH band intensity values and then the normalized values from three determinations for each treatment group were averaged.
Animals and Orthotopic Implantation of Tumor Cells
Male athymic nude mice (NCr-nu/nu) were purchased from the Animal Production Area of the National Cancer Institute Frederick Cancer Research and Development Center (Frederick, MD). The mice were housed and maintained under specific pathogen-free conditions in facilities approved by the American Association for Accreditation of Laboratory Animal Care and in accordance with current regulations and standards of the United States Department of Agriculture, United States Department of Health and Human Services, and the National Institutes of Health. The mice were used in accordance with institutional guidelines when they were 8–12 weeks old.
To produce tumors, L3.6pl cells were harvested from subconfluent cultures by a brief exposure to 0.25% trypsin and 0.02% EDTA. Trypsinization was stopped with minimal essential medium containing 10% fetal bovine serum, and the harvested cells were washed once in serum-free medium and resuspended in Hanks' balanced salt solution. Only single-cell suspensions with greater than 90% viability were used for the injections. Injection of 1x106 cells into the pancreas was performed as described previously (34). Each treatment group contained 10 mice. The mice were killed when moribund (5–6 weeks after injection). The size and weight of each primary pancreatic tumor and the incidence of liver metastases were recorded. Histopathologic studies confirmed the nature of the disease. For immunohistochemical and histologic staining procedures, one part of the tumor tissue was fixed in formalin and embedded in paraffin, and the other part of the tumor was embedded in OCT compound (Miles), snap-frozen in liquid nitrogen, and stored at –70 °C.
Treatment of Established Human Pancreatic Carcinoma Tumors Growing in the Pancreas of Nude Mice
Seven days after implantation of 106 L3.6p1 tumor cells into the pancreas of each mouse (10 mice per treatment group, for a total of 40 mice); five mice were killed to confirm the presence of tumor lesions. Tumor volumes were calculated by using the following formula: 0.5 x length x width2. At 7 days after injection of tumor cells, the median tumor volume was 21 mm3 (interquartile range of 18–25 mm3). Histologic examination confirmed that the lesions were actively growing pancreatic cancers. Mice were randomly assigned to receive one of the following treatments: 1) thrice weekly oral administrations of vehicle solution for tolfenamic acid in corn oil (control group); 2) thrice weekly oral administrations of tolfenamic acid (25 mg/kg of body weight) in corn oil; 3) thrice weekly oral administrations of tolfenamic acid (50 mg/kg) in corn oil; or 4) twice weekly intraperitoneal injections of gemcitabine (50 mg/kg) alone, which is the most commonly used drug for treatment of pancreatic cancer (33,34). Treatments were continued for 4 weeks, and the mice were killed by CO2 asphyxiation on day 35, weighed, and subjected to necropsy. The volume of pancreatic tumors and the incidence of liver metastases were recorded (33,34).
Necropsy Procedures and Histologic Studies
Mice were killed and body weight was recorded. Primary tumors in the pancreas were excised, measured, and weighed. For immunohistochemical and hematoxylin–eosin staining procedures, one part of the tumor tissue was fixed in formalin and embedded in paraffin, and another part was embedded in OCT compound, rapidly frozen in liquid nitrogen, and stored at –70 °C. Visible liver metastases were counted with the aid of a dissecting microscope, and the tissues were processed for hematoxylin–eosin staining.
Statistical Analysis
Statistical significance of differences in protein levels, luciferase activity, cell and tumor growth, and liver metastasis between NSAID-treated cells or mice and solvent control cells or mice, respectively, was determined by an analysis of variance and Scheffe's test, and the levels of probability were noted. The results of cell culture studies are expressed as means (95% confidence intervals) for at least three separate (replicate) experiments for each treatment, and statistical significance of differences between values in the treated and solvent control cells (P<.05) was determined by an analysis of variance and Scheffe's tests. All statistical tests were two-sided.
|
RESULTS |
|---|
|
TopNotesAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Structurally Diverse NSAIDs and the Inhibition of Sp1, Sp3, and Sp4 Expression in Pancreatic Cancer Cells
Previous RNA interference studies have reported that Sp1, Sp3, and Sp4 proteins regulate VEGF expression in pancreatic cancer cells and that celecoxib decreased the expression of Sp1, Sp3, and VEGF in both in vitro and in vivo models (30,31). To investigate the effects of NSAID structure on expression of Sp proteins, we screened biphenyl or biphenylamine carboxylic acids (tolfenamic acid, diclofenac, and diflunisal), oxicams (ampiroxicam), acetic acid derivatives (acemetacin and tolmetin), and propionic acid (ibuprofen, naproxen, fenbufen, and ketoprofen) derivatives. We initially screened compounds at 50 and 100 µM, including compounds from different structural classes of NSAIDs, celecoxib (a COX-2 inhibitor), and valeryl salicylate (a COX-1 inhibitor), because most studies (20–30) found that growth was inhibited at COX-1 or COX-2 inhibitor concentrations between 25 µM and 100 µM. We used Panc-1 cells and immunoblot analysis to screen COX-1 or COX-2 inhibitor compounds for changes in the expression of Sp1, Sp3, and Sp4 proteins after 48 hours of treatment. Treatment with 50 µM celecoxib decreased levels of Sp1 and Sp4 proteins (Fig. 1, A), as previously described in colon cancer cells (32). Treatment with acemetacin, ampiroxicam, naproxen, fenbufen ibuprofen, tolmetin, letrozole, or valeryl salicylate (each at 50 µM) had no effect. Treatment with diphenyl- and diphenylamine-derived carboxylic acid compounds (each at 50 µM) decreased expression of Sp1, Sp3, and Sp4 proteins in Panc-1 cells. The most potent compound of the third group was tolfenamic acid (Fig. 1, B). Similar data for each compound were obtained at 100 µM (data not shown). A 48-hour treatment with 50 µM tolfenamic acid also induced a time-dependent decrease in the expression of Sp1, Sp3, and Sp4 proteins in Panc-1 cells (a >80% decrease in levels of all three proteins), compared with those in solvent (0.1% DMSO)-treated cells (Fig. 1, C) and in highly metastatic L3.6pl pancreatic cancer cells (a >65% decrease in the levels of all three proteins) (Fig. 1, D). In contrast, treatment with 50 µM of the NSAID ampiroxicam (used as a negative control) did not affect the expression of Sp proteins. Thus, the effects of tolfenamic acid on VEGF expression were investigated further in Panc-1 cells transfected with constructs (pVEGF1 and pVEGF2) containing VEGF promoter inserts.
|
Tolfenamic Acid and Transactivation of Luciferase Activity in Pancreatic Cancer Cells Transfected With VEGF Promoter Constructs
Because previous studies (29–32) showed that COX-2 inhibitors or Sp small inhibitory RNAs decreased VEGF expression in colon and pancreatic cancer cells, we investigated the effect of 0.1% DMSO, 50 µM tolfenamic acid, or 50 µM ampiroxicam on transactivation in Panc-1 cells transfected with pVEGF1 or pVEGF2 (Fig. 2, A and B). pVEGF1 contains a VEGF promoter insert from positions –2018 to +50, and pVEGF2 contains a VEGF promoter insert from positions –131 to +54. These experiments were intended to determine the effects of tolfenamic acid on the full-length VEGF promoter (pVEGF1) and on the Sp-binding, GC-rich proximal region of the VEGF promoter (pVEGF2). Tolfenamic acid, but not ampiroxicam, statistically significantly decreased the expression of luciferase by at least 50% in cells transfected with either construct. For example, in Panc1 cells, relative luciferase activity in cells treated with 0.1% DMSO and transfected with pVEGF1 (Fig. 2, A) or pVEGF2 (Fig. 2, B) was statistically significantly higher (0.214, 95% CI = 0.200 to 0.228, or 0.037, 95% CI = 0.023 to 0.051, respectively) than that in transfected cells treated with 50 µM tolfenamic acid (0.070, 95% CI = 0.061 to 0.079, and 0.010, 95% CI = 0.0004 to 0.0196, respectively) (P = .003). In parallel experiments in Panc-1 cells, we investigated the effect of diclofenac, which also induces Sp protein degradation (Fig. 1, A) and found that treatment with diclofenac decreased transactivation of luciferase activity in Panc-1 cells transfected with pVEGF1 (Fig. 2, C) or with pVEGF2 (Fig. 2, D). Decreased luciferase activity after treatment with tolfenamic acid or diclofenac is consistent with previous studies showing that activation of these constructs depended on interactions of Sp1, Sp3, and Sp4 proteins with proximal GC-rich motifs (29–32). A comparable experiment was carried out in L3.6pl cells (Fig. 2, E and F), and the results were similar to those observed in Panc-1 cells. These data are consistent with the decreased levels of Sp1, Sp3, and Sp4 protein in L3.6pl and Panc-1 cells treated with tolfenamic acid, as shown in Fig. 1. A concentration-dependent decrease in transactivation of luciferase activity was detected in Panc-1 cells transfected with pVEGF2 and treated with 20–80 µM tolfenamic acid (Fig. 2, G), but ampiroxicam (Fig. 2, G) and the COX-1 inhibitor valeryl salicylate (data not shown) had no effect.
|
Tolfenamic Acid and Sp Protein Binding to GC-rich VEGF Promoter Oligonucleotides
VEGF is regulated by the binding of Sp proteins to proximal GC-rich promoter sequences (29–32). To determine the effects of tolfenamic acid and ampiroxicam on the binding of Sp proteins to such DNA sequences, we used gel electrophoretic mobility shift assays (Fig. 3, A and B). Panc-1 or L3.6pl cells were treated with 0.1% DMSO, 50 µM tolfenamic acid, or 50 µM ampiroxicam for 48 hours. Nuclear extracts from these cells were isolated and incubated with 32P-labeled VEGF oligonucleotide, and then the presence of Sp–oligonucleotide complexes was assessed in gel mobility shift assays. The 32P-labeled oligonucleotide contained a GC-rich region (positions –66 to –47) from the VEGF promoter that binds Sp proteins. In extracts from Panc-1 cells or from L3.6pl cells treated with 0.1% DMSO or ampiroxicam, the retarded bands containing Sp3 or Sp1–Sp4 (overlapping) had similar intensities; however, in extracts from Panc-1 cells or from L3.6pl cells treated with tolfenamic acid, intensities of both retarded bands decreased compared with bands from extracts of cells treated with 0.1% DMSO. No retarded bands were observed in reaction mixtures containing 32P-labeled VEGF oligonucleotides alone. To confirm that the complexes contained the indicated Sp protein, antibodies against Sp1, Sp3, or Sp4 proteins were added to the reaction mixtures described above, so that the band of the corresponding Sp–oligonucleotide–antibody complex would be retarded even more or supershifted, as shown in Fig. 3, B. Thus, tolfenamic acid decreased the binding of Sp1, Sp3, and Sp4 to GC-rich oligonucleotides containing the VEGF promoter sequence between positions –66 and –47, a result that is consistent with decreased transactivation observed in transient transfection studies with the pVEGF1 and pVEGF2 constructs.
|
refers to antibodies.Tolfenamic Acid, VEGF mRNA and Protein Expression, and Activation of Proteasome-Dependent Degradation of Sp1, Sp3, and Sp4
We next investigated the effects of tolfenamic acid on the expression of VEGF mRNA and protein. We first treated Panc-1 cells with 0.1% DMSO, 50 µM ampiroxicam, or 50 µM tolfenamic acid for 48 hours and then determined VEGF protein levels (relative to that of -tubulin) (Fig. 4, A). Treatment with tolfenamic acid decreased VEGF protein expression by more than 60%, compared with that of solvent (0.1% DMSO) control. The effects of tolfenamic acid on VEGF mRNA levels were also investigated in Panc-1 cells by RT-PCR and compared with levels in solvent (0.1% DMSO)- or ampiroxicam-treated cells. A 24-hour tolfenamic acid treatment decreased VEGF mRNA levels by more than 60%, compared with that of 0.1% DMSO treatment (Fig. 4, B). The possibility that tolfenamic acid decreased VEGF mRNA stability was ruled out because VEGF mRNA levels decreased at similar rates in Panc-1 cells treated with actinomycin D alone or with 50 µM tolfenamic acid. In Panc-1 cells treated with 0.1% DMSO or 50 µM ampiroxicam, VEGF protein was secreted, and extracellular VEGF staining was observed (Fig. 4, D). However, in Panc-1 cells treated for 48 hours with 50 µM tolfenamic acid, no secreted VEGF protein was detected, indicating that VEGF protein was not secreted. Thus, the tolfenamic acid–dependent decrease in the expression of Sp1, Sp3, and Sp4 proteins appears to lead to decreased levels of VEGF mRNA and protein, a result that is consistent with the reported Sp-dependent regulation of VEGF (29–32).
|
In a previous study (32), we reported that the COX-2 inhibitor nimesulide decreased levels of Sp1 and Sp4 (but not Sp3) in colon cancer cells by activating the proteasome pathway and that this response was blocked by the proteasome inhibitor gliotoxin. Treatment with tolfenamic acid decreased the levels of Sp1, Sp3, and Sp4 proteins (Fig. 4, E), in contrast to treatment with nimesulfide (32) or celecoxib (Fig. 1, A), both of which induced degradation of Sp1 and Sp4 but not Sp3. However, when Panc-1 cells were treated with a combination of 50 µM tolfenamic acid and 2 µM lactacystin (a proteasome inhibitor), Sp1, Sp3, and Sp4 protein levels were not decreased. Thus, like the COX-2 inhibitors celecoxib and nimesulide, tolfenamic acid appeared to activate proteasome-dependent degradation of Sp1 and Sp4 in Panc-1 cells, but unlike those compounds, tolfenamic acid also induced degradation of Sp3. Also, decreased transactivation of luciferase activity in Panc-1 cells transfected with pVEGF2 and treated with tolfenamic acid was also blocked with cotreatment of lactacystin (Fig. 4, F), and similar results were obtained for gliotoxin (data not shown). Ampiroxicam had no effect in the presence or absence of lactacystin. These results show that inhibition of tolfenamic acid-induced Sp protein degradation by proteasome inhibitors blocks decreased VEGF expression and also confirmed the role of Sp proteins as key mediators of VEGF expression.
Tolfenamic Acid and the Proliferation of Pancreatic Cancer Cells
Because Sp proteins regulate genes involved in cancer cell proliferation and angiogenesis, we investigated the mechanism by which tolfenamic acid and other NSAIDs inhibit the proliferation of pancreatic cancer cells. Ampiroxicam, naproxen, diclofenac, and tolfenamic acid decreased the proliferation of Panc-1 cells, with diclofenac and tolfenamic acid having the highest potencies (Fig. 5, A–D). Because the differences in the effects of NSAIDs at 50 µM observed for Sp protein degradation (Fig. 1, A) may be related, in part, to their relative growth-inhibitory activities, we compared the effects of tolfenamic acid, ampiroxicam, and naproxen on Sp protein expression by use of concentrations that induced comparable inhibition of Panc-1 cell proliferation. Thus, Panc-1 cells were treated with 50 µM tolfenamic acid, 150 µM naproxen, or 150 µM ampiroxicam for 48 hours and subjected to Western blot analysis. Tolfenamic acid was the only compound tested that induced protein degradation and poly(ADP-ribose)polymerase cleavage (Fig. 5). These data support results of the initial screening assay (Fig. 1, A) showing that only NSAIDs containing the diphenyl or diphenylamine carboxylic acid structure induced Sp protein degradation. When we used a similar range of concentrations in L3.6pl (Fig. 6, A and B) and Panc-28 (Fig. 6, C and D) cells, we also found that tolfenamic acid was a more potent inhibitor of pancreatic cancer cell proliferation than ampiroxicam. Thus, the growth inhibitory effects of tolfenamic acid in pancreatic cancer cells appear to be associated, in part, with degradation of Sp proteins.
|
|
Tolfenamic Acid and Pancreatic Tumor Growth and Metastasis in an Orthotopic Model
Potential antitumorigenic and antiangiogenic activities of tolfenamic acid against pancreatic tumors were next investigated in an orthotopic athymic nude mouse model. L3.6pl cells were used for this study because of their aggressive growth and production of liver metastases (33,34). Treatment with tolfenamic acid (at 25 or 50 mg/kg/day) for 4 weeks decreased median tumor weights and volumes (Fig. 7, A and B) and also decreased the percent incidence of liver metastasis (Table 1). Moreover, at these doses, changes in body or organ weights or organ toxic effects were not observed. At comparable doses, tolfenamic acid appeared to be a more effective tumor growth inhibitor than gemcitabine; gemcitabine, however, did not affect the incidence of liver metastasis.
|
|
We also assessed levels of Sp1, Sp3, Sp4, and VEGF proteins in pancreatic tumors from tolfenamic acid–treated versus corn oil–treated mice. Tolfenamic acid treatment statistically significantly decreased expression of Sp1, Sp3, Sp4, and VEGF proteins in pancreatic tumors (Fig. 7, C), as observed in pancreatic cancer cells treated with tolfenamic acid (Figs. 1 and 4). For example, the VEGF protein level in tumors from mice treated with tolfenamic acid (50 mg/kg) was statistically significantly lower (45%, 95% confidence interval = 39% to 51%; P = .009) than that in tumors from control mice treated with corn oil (i.e., 100%). Immunostaining for VEGF in tumor sections from control animals and animals treated with gemcitabine (50 mg/kg) or tolfenamic acid (25 and 50 mg/kg) also indicated relatively high concentrations of VEGF in tumors from control and gemcitabine-treated animals but relatively low concentrations of VEGF in pancreatic tumors from mice treated with tolfenamic acid (Fig. 7, D).
In parallel studies, staining with CD31 to determine microvessel density indicated lower microvessel density in tumors from mice treated with tolfenamic acid than in tumors from vehicle (corn oil)-treated or gemcitabine-treated animals (Fig. 7, E). Thus, tolfenamic acid had antitumorigenic and antiangiogenic activities in pancreatic cancer cells and tumors in vivo through the degradation of Sp proteins, which leads to decreased VEGF expression.
|
DISCUSSION |
|---|
|
TopNotesAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Tolfenamic acid induced degradation of Sp proteins and decreased expression of VEGF in pancreatic cancer cells and tumors, inhibited cell or tumor growth and decreased metastasis to the liver in a mouse orthotopic pancreatic tumor model. Sp proteins play a critical role in growth and metastasis of cancer (36–38), and Sp1 expression may be negatively associated with survival in some cancer patients (29,39–43). The important role of Sp proteins in tumor growth and metastasis are not surprising because Sp1 and other Sp proteins are transcription factors that regulate sets of genes responsible for cancer cell proliferation and angiogenesis (29–32,36–38). Development of anticancer drugs that specifically inhibit Sp-dependent transactivation have been reported, including oligonucleotides, peptide nucleic acid–DNA chimeras, drugs such as mithramycin that disrupt Sp protein–DNA binding (44–48), and agents such as COX-2 inhibitors that induce Sp protein degradation (31,32). Wei et al. (31) first reported that celecoxib decreased cell and tumor growth and the expression of Sp1 and VEGF in pancreatic cancer cells and in tumors from nude mice bearing FG pancreatic cancer cells (orthotopic and xenograft models). In colon cancer cells, the COX-2 inhibitors celecoxib, nimesulide, and NS-398 induced proteasome-dependent degradation of Sp1 and Sp4 proteins but not Sp3 protein, and this degradation also resulted in decreased VEGF expression (32).
In several pancreatic cancer cell lines, we used RNA interference to knock down the expression of Sp proteins (30) and showed that Sp1, Sp3, and Sp4 are important transcription factors for constitutive expression of VEGF. We have also found (Kelly Higgins, Maen Abdelrahim, and Stephen Safe, unpublished results) that Sp proteins are important for regulation of VEGF receptors in pancreatic cancer cells. Therefore, an optimal antiangiogenic agent for treating pancreatic cancer should modulate expression of all three Sp proteins. In our initial screening of pancreatic cancer cells with COX-2 inhibitors, such as celecoxib (Fig. 1, A), we found that celecoxib induced degradation of Sp1 and Sp4, as previously reported in colon cancer cells (32). However, on the bases of previous reports of the antiangiogenic activity of NSAIDs (4–6), we further screened various NSAIDS as potential inducers of Sp protein degradation (Fig. 1, A). Our initial screening approach was to examine the effects of different structural classes of NSAIDs, including substituted diphenylamines (diclofenac and tolfenamic acid), diphenyls (diflunisal and fenbufen), diphenylmethanes (letrozole), indoles (acemetacin), 1,2-benzothiazines (ampiroxicam), benzophenones (ketoprofen), phenyl/pyrrole ketones (tolmetin), naphthyl and phenyl acetic acids (ibuprofen and naproxen), celecoxib, and valeryl salicylate, a COX-1 inhibitor. Only the substituted diphenyl and diphenylamine carboxylic acid derivatives induced degradation of Sp1, Sp3, and Sp4 proteins (Fig. 1, A), and the most active compound was tolfenamic acid. This is, to our knowledge, the first report of a compound that induces degradation of all three Sp proteins.
We then investigated the antiangiogenic and antitumorigenic activity of tolfenamic acid, which induced the parallel degradation of Sp1, Sp3, and Sp4 proteins. Ampiroxicam, an NSAID that does not affect Sp protein expression, was used as a negative control for these studies. Tolfenamic acid treatment reduced Sp protein levels by inducing degradation of Sp proteins (Fig. 1) by activation of the proteasome pathway (Fig. 5), as previously reported for COX-2 inhibitors (32), and the proteasome inhibitor lactacystin reversed the effects of tolfenamic acid. Degradation of Sp proteins after treatment of pancreatic cancer cells with tolfenamic acid was accompanied by decreased transactivation of luciferase activity in pancreatic cancer cells transfected with VEGF promoter constructs (Fig. 2), decreased expression of VEGF protein and mRNA in cells (Fig. 4), and decreased pancreatic cancer cell growth (Figs. 5–7).
A recent study (49) reported that prostaglandin E2 induced VEGF in smooth muscle cells and that this induction was accompanied by increased phosphorylation of Sp1. It is unlikely that tolfenamic acid acts by inhibiting COX-2–dependent prostaglandin E2 synthesis because most other COX inhibitors do not affect Sp protein expression (Fig. 1) and because Panc-1 cells do not express COX-2 (50). Thus, our results coupled with the decreased or nondetectable activity of the NSAID ampiroxicam in the same assays support the role of Sp protein degradation as a major pathway for the growth-inhibitory and/or antiangiogenic activity of tolfenamic acid and the direct linkage of this degradation of Sp protein to decreased VEGF expression. Our results, however, do not exclude a role for other Sp-dependent genes in mediating effects of tolfenamic acid.
Results of our in vitro studies in pancreatic cancer cells clearly demonstrate that tolfenamic acid exhibits growth-inhibitory and antiangiogenic activities through the activation of proteasome-dependent degradation of Sp1, Sp3, and Sp4. The in vivo effects of tolfenamic acid were further investigated in an orthotopic model of pancreatic cancer by use of L3.6pl cells, which not only form pancreatic tumors in mice but also metastasize to the liver (33,34). Gemcitabine (50 mg/kg) was included as a control in this model because it is widely used for treatment of pancreatic cancer (51). Treatment with gemcitabine at 50 mg/kg decreased tumor volume and weight but did not affect metastasis to the liver (Table 1). In contrast, tolfenamic acid (50 mg/kg) statistically significantly decreased the median tumor volume and weight (Fig. 7, A, and Table 1) and liver metastasis (Table 1). The expression of Sp1, Sp3, Sp4, and VEGF proteins in individual tumors was decreased by tolfenamic acid treatment (Fig. 7, C). Moreover, the expression of VEGF was decreased in pancreatic tumors (Fig. 7, D) and cells (Fig. 4, D) by treatment with tolfenamic acid, and this decreased VEGF expression corresponded to decreased CD31 expression in tumors from mice treated with tolfenamic acid (Fig. 7, E). These effects of tolfenamic acid in pancreatic cells and tumors demonstrate that Sp transcription factors may be important targets for pancreatic cancer chemotherapy. Moreover, among intercalating agents, COX-2 inhibitors, and Sp oligonucleotide decoys used for blocking Sp-mediated transcription and downstream responses (30–32,44–48), tolfenamic acid and structurally related NSAIDs appear to be among the most effective.
This study has several limitations. It is also possible that structural classes of NSAIDs other than tolfenamic acid may exhibit similar activities, and this possibility will require more extensive screening of these compounds. Sp protein degradation is accompanied by a parallel decrease in levels of VEGF mRNA and protein (Figs. 4 and 7); however, identity of other key growth regulatory and angiogenic factors that are also affected by these NSAIDs has not been determined. Moreover, although it is clear that tolfenamic acid has antipancreatic cancer activity in cells and in an orthotopic model for pancreatic cancer, the different effects of these NSAIDs on other tumor and nontumor tissues, from mice and other species (including humans), require further investigation.
In summary, two structurally related classes of NSAIDs (i.e., diphenyl and diphenylamine carboxylic acids) were potent inhibitors of pancreatic cell and tumor growth and angiogenesis. The mechanisms of action for tolfenamic acid appeared to result, at least in part, from activation of proteasome-dependent degradation of Sp1, Sp3, and Sp4. The use of tolfenamic acid for treatment of migraines can require daily doses of 7.5–10 mg/kg, which is less than the doses used in this study, and so development of more active analogs may be required for clinical treatment of pancreatic cancer. The prospect that tolfenamic acid, alone or in combination with other drugs, may be useful in the treatment of pancreatic cancer—which is often not diagnosed in humans until the tumors are well established and have already metastasized—is tantalizing. Future studies should focus on the development of more potent derivatives of tolfenamic acid, on determining the mechanism by which these compounds activate proteasomes to selectively degrade Sp transcription factors, and on investigating the effects of tolfenamic acid on expression of other key factors involved in tumor growth and angiogenesis.
|
NOTES |
|---|
|
TopNotesAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Supported by grants from the National Institutes of Health (CA108178 and ES09106), University of Texas M. D. Anderson Cancer Center Pancreatic Spore grant (P20-CA-10193), and the Texas Agricultural Experiment Station. The financial support was used for salaries of the coauthors and supplies required for the research. The funding agencies had no role in the design of the study, collection of data, analysis and interpretation of the results, the preparation of this manuscript, or the decision to submit a manuscript.
Present address: Cheryl H. Baker, M. D. Anderson Cancer Center, Orlando Cancer Research Institute, Orlando, FL 32806.
|
REFERENCES |
|---|
|
TopNotesAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
(1) Vane JR. Inhibition of prostaglandin synthesis as a mechanism of action for aspirin-like drugs. Nat New Biol 1971;231:232–5.[Web of Science][Medline]
(2) DuBois RN, Abramson SB, Crofford L, Gupta RA, Simon LS, Van De Putte LB, et al. Cyclooxygenase in biology and disease. FASEB J 1998;12:1063–73.
(3) Williams CS, Mann M, DuBois RN. The role of cyclooxygenases in inflammation, cancer, and development. Oncogene 1999;18:7908–16.[CrossRef][Web of Science][Medline]
(4) Tarnawski AS, Jones MK. Inhibition of angiogenesis by NSAIDs: molecular mechanisms and clinical implications. J Mol Med 2003;81:627–36.[CrossRef][Web of Science][Medline]
(5) Taketo MM. Cyclooxygenase-2 inhibitors in tumorigenesis (Part I). J Natl Cancer Inst 1998;90:1529–36.
(6) Taketo MM. Cyclooxygenase-2 inhibitors in tumorigenesis (Part II). J Natl Cancer Inst 1998;90:1609–20.
(7) Gately S, Li WW. Multiple roles of COX-2 in tumor angiogenesis: a target for antiangiogenic therapy. Semin Oncol 2004;31(2 Suppl 7):2–11.[Web of Science][Medline]
(8) Giovannucci E, Egan KM, Hunter DJ, Stampfer MJ, Colditz GA, Willett WC, et al. Aspirin and the risk of colorectal cancer in women. N Engl J Med 1995;333:609–14.
(9) Martinez ME, McPherson RS, Levin B, Annegers JF. Aspirin and other nonsteroidal anti-inflammatory drugs and risk of colorectal adenomatous polyps among endoscoped individuals. Cancer Epidemiol Biomarkers Prev 1995;4:703–7.[Abstract]
(10) Peleg II, Lubin MF, Cotsonis GA, Clark WS, Wilcox CM. Long-term use of nonsteroidal antiinflammatory drugs and other chemopreventors and risk of subsequent colorectal neoplasia. Dig Dis Sci 1996;41:1319–26.[CrossRef][Web of Science][Medline]
(11) Labayle D, Fischer D, Vielh P, Drouhin F, Pariente A, Bories C, et al. Sulindac causes regression of rectal polyps in familial adenomatous polyposis. Gastroenterology 1991;101:635–9.[Web of Science][Medline]
(12) Chiu CH, McEntee MF, Whelan J. Sulindac causes rapid regression of preexisting tumors in Min/+ mice independent of prostaglandin biosynthesis. Cancer Res 1997;57:4267–73.
(13) Nugent KP, Farmer KC, Spigelman AD, Williams CB, Phillips RK. Randomized controlled trial of the effect of sulindac on duodenal and rectal polyposis and cell proliferation in patients with familial adenomatous polyposis. Br J Surg 1993;80:1618–9.[Web of Science][Medline]
(14) Pereira MA, Barnes LH, Rassman VL, Kelloff GV, Steele VE. Use of azoxymethane-induced foci of aberrant crypts in rat colon to identify potential cancer chemopreventive agents. Carcinogenesis 1994;15:1049–54.
(15) Boolbol SK, Dannenberg AJ, Chadburn A, Martucci C, Guo XJ, Ramonetti JT, et al. Cyclooxygenase-2 overexpression and tumor formation are blocked by sulindac in a murine model of familial adenomatous polyposis. Cancer Res 1996;56:2556–60.
(16) Schernhammer ES, Kang JH, Chan AT, Michaud DS, Skinner HG, Giovannucci E, et al. A prospective study of aspirin use and the risk of pancreatic cancer in women. J Natl Cancer Inst 2004;96:22–8.
(17) Jacobs EJ, Connell CJ, Rodriguez C, Patel AV, Calle EE, Thun MJ. Aspirin use and pancreatic cancer mortality in a large United States cohort. J Natl Cancer Inst 2004;96:524–8.
(18) Anderson KE, Johnson TW, Lazovich D, Folsom AR. Association between nonsteroidal anti-inflammatory drug use and the incidence of pancreatic cancer. J Natl Cancer Inst 2002;94:1168–71.
(19) Menezes RJ, Huber KR, Mahoney MC, Moysich KB. Regular use of aspirin and pancreatic cancer risk. BMC Public Health 2002;2:18.[CrossRef][Medline]
(20) Molina MA, Sitja-Arnau M, Lemoine MG, Frazier ML, Sinicrope FA. Increased cyclooxygenase-2 expression in human pancreatic carcinomas and cell lines: growth inhibition by nonsteroidal anti-inflammatory drugs. Cancer Res 1999;59:4356–62.
(21) Takahashi M, Furukawa F, Toyoda K, Sato H, Hasegawa R, Imaida K, et al. Effects of various prostaglandin synthesis inhibitors on pancreatic carcinogenesis in hamsters after initiation with N-nitrosobis(2-oxopropyl)amine. Carcinogenesis 1990;11:393–5.
(22) Perugini RA, McDade TP, Vittimberga FJ Jr, Duffy AJ, Callery MP. Sodium salicylate inhibits proliferation and induces G1 cell cycle arrest in human pancreatic cancer cell lines. J Gastrointest Surg 2000;4:24–32.
(23) Yip-Schneider MT, Sweeney CJ, Jung SH, Crowell PL, Marshall MS. Cell cycle effects of nonsteroidal anti-inflammatory drugs and enhanced growth inhibition in combination with gemcitabine in pancreatic carcinoma cells. J Pharmacol Exp Ther 2001;298:976–85.
(24) Kokawa A, Kondo H, Gotoda T, Ono H, Saito D, Nakadaira S, et al. Increased expression of cyclooxygenase-2 in human pancreatic neoplasms and potential for chemoprevention by cyclooxygenase inhibitors. Cancer 2001;91:333–8.[CrossRef][Web of Science][Medline]
(25) Schuller HM, Zhang L, Weddle DL, Castonguay A, Walker K, Miller MS. The cyclooxygenase inhibitor ibuprofen and the FLAP inhibitor MK886 inhibit pancreatic carcinogenesis induced in hamsters by transplacental exposure to ethanol and the tobacco carcinogen NNK. J Cancer Res Clin Oncol 2002;128:525–32.[CrossRef][Web of Science][Medline]
(26) Eibl G, Reber HA, Wente MN, Hines OJ. The selective cyclooxygenase-2 inhibitor nimesulide induces apoptosis in pancreatic cancer cells independent of COX-2. Pancreas 2003;26:33–41.[CrossRef][Medline]
(27) El-Rayes BF, Ali S, Sarkar FH, Philip PA. Cyclooxygenase-2-dependent and -independent effects of celecoxib in pancreatic cancer cell lines. Mol Cancer Ther 2004;3:1421–6.
(28) Yip-Schneider MT, Nakshatri H, Sweeney CJ, Marshall MS, Wiebke EA, Schmidt CM. Parthenolide and sulindac cooperate to mediate growth suppression and inhibit the nuclear factor-kappa B pathway in pancreatic carcinoma cells. Mol Cancer Ther 2005;4:587–94.
(29) Shi Q, Le X, Abbruzzese JL, Peng Z, Qian CN, Tang H, et al. Constitutive Sp1 activity is essential for differential constitutive expression of vascular endothelial growth factor in human pancreatic adenocarcinoma. Cancer Res 2001;61:4143–54.
(30) Abdelrahim M, Smith III R, Burghardt R, Safe S. Role of Sp proteins in regulation of vascular endothelial growth factor expression and proliferation of pancreatic cancer cells. Cancer Res 2004;64:6740–9.
(31) Wei D, Wang L, He Y, Xiong HQ, Abbruzzese JL, Xie K. Celecoxib inhibits vascular endothelial growth factor expression in and reduces angiogenesis and metastasis of human pancreatic cancer via suppression of Sp1 transcription factor activity. Cancer Res 2004;64:2030–8.
(32) Abdelrahim M, Safe S. Cyclooxygenase-2 inhibitors decrease vascular endothelial growth factor expession in colon cancer cells by enhanced degradation of Sp1 and Sp4 proteins. Mol Pharmacol 2005;68:317–29.
(33) Baker CH, Solorzano CC, Fidler IJ. Blockade of vascular endothelial growth factor receptor and epidermal growth factor receptor signaling for therapy of metastatic human pancreatic cancer. Cancer Res 2002;62:1996–2003.
(34) Bruns CJ, Harbison MT, Kuniyasu H, Eue I, Fidler IJ. In vivo selection and characterization of metastatic variants from human pancreatic adenocarcinoma by using orthotopic implantation in nude mice. Neoplasia 1999;1:50–62.[CrossRef][Medline]
(35) Weidner N, Semple JP, Welch WR, Folkman J. Tumor angiogenesis and metastasis—correlation in invasive breast carcinoma. N Engl J Med 1991;324:1–8.[Abstract]
(36) Bouwman P, Philipsen S. Regulation of the activity of Sp1-related transcription factors. Mol Cell Endocrinol 2002;195:27–38.[CrossRef][Web of Science][Medline]
(37) Black AR, Black JD, Azizkhan-Clifford J. Sp1 and Krüppel-like factor family of transcription factors in cell growth regulation and cancer. J Cell Physiol 2001;188:143–60.[CrossRef][Web of Science][Medline]
(38) Safe S, Kim K. Nuclear receptor-mediated transactivation through interaction with Sp proteins. Prog Nucleic Acid Res Mol Biol 2004;77:1–36.[Web of Science][Medline]
(39) Wang L, Wei D, Huang S, Peng Z, Le X, Wu TT, et al. Transcription factor Sp1 expression is a significant predictor of survival in human gastric cancer. Clin Cancer Res 2003;9:6371–80.
(40) Yao JC, Wang L, Wei D, Gong W, Hassan M, Wu TT, et al. Association between expression of transcription factor Sp1 and increased vascular endothelial growth factor expression, advanced stage, and poor survival in patients with resected gastric cancer. Clin Cancer Res 2004;10(12 Pt 1):4109–17.
(41) Zannetti A, Del VS, Carriero MV, Fonti R, Franco P, Botti G, et al. Coordinate up-regulation of Sp1 DNA-binding activity and urokinase receptor expression in breast carcinoma. Cancer Res 2000;60:1546–51.
(42) Chiefari E, Brunetti A, Arturi F, Bidart JM, Russo D, Schlumberger M, et al. Increased expression of AP2 and Sp1 transcription factors in human thyroid tumors: a role in NIS expression regulation? BMC Cancer 2002;2:35.[CrossRef][Medline]
(43) Hosoi Y, Watanabe T, Nakagawa K, Matsumoto Y, Enomoto A, Morita A, et al. Up-regulation of DNA-dependent protein kinase activity and Sp1 in colorectal cancer. Int J Oncol 2004;25:461–8.[Medline]
(44) Xu X, Hamhouyia F, Thomas SD, Burke TJ, Girvan AC, McGregor WG, et al. Inhibition of DNA replication and induction of S phase cell cycle arrest by G-rich oligonucleotides. J Biol Chem 2001;276:43221–30.
(45) Borgatti M, Lampronti I, Romanelli A, Pedone C, Saviano M, Bianchi N, et al. Transcription factor decoy molecules based on a peptide nucleic acid (PNA)-DNA chimera mimicking Sp1 binding sites. J Biol Chem 2003;278:7500–9.
(46) Martin B, Vaquero A, Priebe W, Portugal J. Bisanthracycline WP631 inhibits basal and Sp1-activated transcription initiation in vitro. Nucleic Acids Res 1999;27:3402–9.
(47) Ishibashi H, Nakagawa K, Onimaru M, Castellanous EJ, Kaneda Y, Nakashima Y, et al. Sp1 decoy transfected to carcinoma cells suppresses the expression of vascular endothelial growth factor, transforming growth factor beta1, and tissue factor and also cell growth and invasion activities. Cancer Res 2000;60:6531–6.
(48) Chae YM, Park KK, Magae J, Lee IS, Kim CH, Kim HC, et al. Sp1-decoy oligodeoxynucleotide inhibits high glucose-induced mesangial cell proliferation. Biochem Biophys Res Commun 2004;319:550–5.[CrossRef][Web of Science][Medline]
(49) Bradbury D, Clarke D, Seedhouse C, Corbett L, Stocks J, Knox A. Vascular endothelial growth factor induction by prostaglandin E2 in human airway smooth muscle cells is mediated by E prostanoid EP2/EP4 receptors and SP-1 transcription factor binding sites. J Biol Chem 2005;280:29993–30000.
(50) Yip-Schneider MT, Barnard DS, Billings SD, Cheng L, Heilman DK, Lin A, et al. Cyclooxygenase-2 expression in human pancreatic adenocarcinomas. Carcinogenesis 2000;21:139–46.
(51) Li D, Xie K, Wolff R, Abbruzzese JL. Pancreatic cancer. Lancet 2004;363:1049–57.[CrossRef][Web of Science][Medline]
Manuscript received November 3, 2005; revised April 13, 2006; accepted May 4, 2006.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  S. Papineni, S. Chintharlapalli, M. Abdelrahim, S.-o. Lee, R. Burghardt, A. Abudayyeh, C. Baker, L. Herrera, and S. Safe Tolfenamic acid inhibits esophageal cancer through repression of specificity proteins and c-Met Carcinogenesis, July 1, 2009; 30(7): 1193 - 1201. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 X. Liu, M. Abdelrahim, A. Abudayyeh, P. Lei, and S. Safe The nonsteroidal anti-inflammatory drug tolfenamic acid inhibits BT474 and SKBR3 breast cancer cell and tumor growth by repressing erbB2 expression Mol. Cancer Ther., May 1, 2009; 8(5): 1207 - 1217. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Konduri, J. Colon, C. H. Baker, S. Safe, J. L. Abbruzzese, A. Abudayyeh, Md. R. Basha, and M. Abdelrahim Tolfenamic acid enhances pancreatic cancer cell and tumor response to radiation therapy by inhibiting survivin protein expression Mol. Cancer Ther., March 1, 2009; 8(3): 533 - 542. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S.-H. Lee, J. H. Bahn, C. K. Choi, N. C. Whitlock, A. E. English, S. Safe, and S. J. Baek ESE-1/EGR-1 pathway plays a role in tolfenamic acid-induced apoptosis in colorectal cancer cells Mol. Cancer Ther., December 1, 2008; 7(12): 3739 - 3750. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. Chadalapaka, I. Jutooru, S. Chintharlapalli, S. Papineni, R. Smith III, X. Li, and S. Safe Curcumin Decreases Specificity Protein Expression in Bladder Cancer Cells Cancer Res., July 1, 2008; 68(13): 5345 - 5354. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Sun, Z. Estrov, Y. Ji, K. R. Coombes, D. H. Harris, and R. Kurzrock Curcumin (diferuloylmethane) alters the expression profiles of microRNAs in human pancreatic cancer cells Mol. Cancer Ther., March 1, 2008; 7(3): 464 - 473. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. U. Mertens-Talcott, S. Chintharlapalli, X. Li, and S. Safe The Oncogenic microRNA-27a Targets Genes That Regulate Specificity Protein Transcription Factors and the G2-M Checkpoint in MDA-MB-231 Breast Cancer Cells Cancer Res., November 15, 2007; 67(22): 11001 - 11011. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S.-W. Cho, I.-K. Kim, S. H. Bhang, B. Joung, Y. J. Kim, K. J. Yoo, Y.-S. Yang, C. Y. Choi, and B.-S. Kim Combined therapy with human cord blood cell transplantation and basic fibroblast growth factor delivery for treatment of myocardial infarction Eur J Heart Fail, October 1, 2007; 9(10): 974 - 985. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Abdelrahim, C. H. Baker, J. L. Abbruzzese, D. Sheikh-Hamad, S. Liu, S. D. Cho, K. Yoon, and S. Safe Regulation of Vascular Endothelial Growth Factor Receptor-1 Expression by Specificity Proteins 1, 3, and 4 in Pancreatic Cancer Cells Cancer Res., April 1, 2007; 67(7): 3286 - 3294. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Chintharlapalli, S. Papineni, S. K. Ramaiah, and S. Safe Betulinic Acid Inhibits Prostate Cancer Growth through Inhibition of Specificity Protein Transcription Factors Cancer Res., March 15, 2007; 67(6): 2816 - 2823. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||