Mol Cancer Ther. 2005;4:487-493
© 2005 American Association for Cancer Research
The conventional nonsteroidal anti-inflammatory drug sulindac sulfide arrests ovarian cancer cell growth via the expression of NAG-1/MIC-1/GDF-15
Jong-Sik Kim1,
Seung Joon Baek2,
Tina Sali1 and
Thomas E. Eling1
1 Laboratory of Molecular Carcinogenesis, National Institute of Environmental Health Sciences, NIH, Research Triangle Park, North Carolina and 2 Laboratory of Environmental Carcinogenesis, Department of Pathobiology, College of Veterinary Medicine, University of Tennessee, Knoxville, Tennessee
Requests for reprints: Thomas E. Eling, Laboratory of Molecular Carcinogenesis, National Institute of Environmental Health Sciences, NIH, MD: E4-09, P.O. Box 12233, 111 TW Alexander Drive, Research Triangle Park, NC 27709. Phone: 919-541-3911; Fax: 919-541-0146. E-mail: eling{at}niehs.nih.gov
 |
Abstract
|
|---|
Although the chemopreventive and antitumorigenic activities of nonsteroidal anti-inflammatory drug (NSAID) against colorectal cancer are well established, the molecular mechanisms responsible for these properties in ovarian cancer have not been elucidated. Therefore, there is an urgent need to develop mechanism-based approaches for the management of ovarian cancer. To this end, the effect of several NSAIDs on ovarian cancer cells was investigated as assessed by the induction of NAG-1/MIC-1/GDF-15, a proapoptotic gene belonging to the transforming growth factor-ß superfamily. Sulindac sulfide was the most significant NSAID activated gene 1 (NAG-1) inducer and its expression was inversely associated with cell viability as determined by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay. This growth suppression by sulindac sulfide was recovered by transfection of NAG-1 small interfering RNA. These results indicate that NAG-1 is one of the genes responsible for growth suppression by sulindac sulfide. Furthermore, we observed down-regulation of p21 WAF1/CIP1 by introduction of NAG-1 small interfering RNA into sulindac sulfide–treated cells. In addition, to elucidate other potential molecular mechanisms involved in sulindac sulfide treatment of ovarian cancer cells, we did a membrane-based microarray experiment. We found that cyclin D1, MMP-1, PI3KR1, and uPA were down-regulated by sulindac sulfide. In conclusion, a novel molecular mechanism is proposed to explain the experimental results and provide a rationale for the chemopreventive activity of NSAIDs in ovarian cancer.
Key Words: NAG-1 • p21 • NSAID • sulindac sulfide • ovarian cancer
|
Introduction
|
|---|
Ovarian cancer has the highest mortality rate of any of the gynecologic cancers; it is the fourth leading cause of cancer-related deaths among women in the United States and causes over 100,000 deaths annually worldwide (1, 2). Although intensive treatments for ovarian cancer, such as radical surgery, radiation therapy, and chemotherapy have improved survival rates, cure rates have stayed essentially the same over the last 20 years. Thus, early intervention with chemopreventive agents has been considered as a desirable alternative treatment for this invasive disease.
Three major chemopreventive agents, nonsteroidal anti-inflammatory drugs (NSAID), retinoids, and oral contraceptives, have been studied by many researchers for ovarian cancer (3–5). Some of the most promising pharmaceutical agents described to date for the prevention of cancer are the NSAIDs. Of all the various NSAIDs reported to inhibit the development of tumors, sulindac seems to be one of the most effective in experimental animal models (6). Sulindac itself does not inhibit cyclooxygenase (COX), but its metabolite, sulindac sulfide, is a potent nonselective inhibitor of COX. Whereas some reports suggest that some NSAIDs induce apoptosis and cell cycle arrest in human ovarian cancer cells (3, 7), the exact molecular mechanism by which NSAIDs induce antitumorigenic activity has not been investigated.
In contrast to colorectal cancer, no one has reported whether sulindac sulfide has any effect on apoptosis and cell cycle arrest in ovarian cancer cells. Epidemiologic findings regarding NSAID effects on ovarian cancer are rather conflicting. For example, an Italian study found that NSAID use was not related to ovarian cancer risk (8), whereas a hospital-based case-control study reported that long-term use of NSAIDs was associated with a reduced risk (9). In the latter case, however, there has been no reported molecular mechanism to explain the effect of NSAIDs on ovarian cancer inhibition. Therefore, it is important to know whether NSAIDs, such as sulindac sulfide, induce growth suppression in ovarian carcinoma cells, and to further investigate the molecular mechanism by which such NSAIDs may arrest ovarian cancer cell growth.
NSAID activated gene (NAG-1), also known as MIC-1, GDF-15, PTGFß, and PLAB, was identified in our laboratory as a gene regulated by NSAIDs, cyclooxygenase inhibitors based on PCR-based subtractive hybridization of indomethacin-treated human colorectal cells (10). The human NAG-1 cDNA encodes a secreted protein with homology to members of the transforming growth factor-ß superfamily and a plethora of data support the finding that NAG-1 is linked to apoptosis and that its reduced expression may enhance tumorigenesis (11–13). NAG-1 expression is up-regulated in a prostaglandin-independent manner in human colorectal cancer cells by several NSAIDs (10). Other antitumorigenic compounds, such as resveratrol (14), genistein (15), diallyl disulfide (16), catechins (17), proliferator-activated receptor-
ligands (18), 5F-203 (19), and retinoid 6-[3-(20)-4-hydroxyphenyl]-2-naphthalene carboxylic acid (20), also up-regulate NAG-1. Whereas some dietary factors, including resveratrol and genistein, induce NAG-1 expression through the p53 tumor suppressor protein (14, 15), NSAIDs and anoxia induce NAG-1 in a p53-independent manner (14, 21). Recently, Yamguchi et al. (22) reported that expression of NAG-1 is regulated by phosphatidylinositol 3-kinase/AKT/glycogen synthase kinase-3ß pathway, the signalings of which are deregulated/inappropriately activated with high frequency in human tumors. Thus, several pathways may affect NAG-1 expression.
Although NAG-1 has been well studied in colorectal cancer as an antitumorigenic protein, the expression and regulation of NAG-1 in ovarian cancer remains to be characterized. In this report, we have found that the sulindac sulfide induces NAG-1 expression in three different ovarian cancer cell lines, which have different COX-1/COX-2 and p53 status. Therefore, we propose that NAG-1 represents a novel protein target for sulindac sulfide treatment in ovarian cancer, and that sulindac sulfide induces the expression of NAG-1 in a COX- and p53-independent manner. NAG-1 promotes growth suppression and mediates the antitumorigenic effects of NSAIDs.
|
Materials and Methods
|
|---|
Cell Lines and Reagents
Human ovarian cancer cells, SKOV3, OVCAR3, and PA-1, were purchased from American Type Culture Collection (Manassas, VA) and maintained in DMEM supplemented with 10% fetal bovine serum and penicillin-streptomycin (Invitrogen, Carlsbad, CA). NSAIDs (indomethacin, NS-398, and SC-560) were purchased from Sigma (St. Louis, MO), whereas sulindac, sulidac sulfone, and sulindac sulfide were purchased from Calbiochem (San Diego, CA). Antibodies to p21 WAF1/CIP1 and actin were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), whereas NAG-1 antibody was described previously (10).
Western Blot Analysis
Cells were lysed in radioimmunoprecipitation assay buffer [50 mmol/L Tris-Cl (pH 8.0), 150 mmol/L NaCl, 1% NP40, 0.1% SDS, and 0.5% sodium deoxycholate]. Twenty micrograms of whole cell extracts were separated in a 4% to 12% gel (Invitrogen) and transferred onto a nitrocellulose membrane (Invitrogen). Membranes were blocked in 0.05 mmol/L Tris, 0.138 mmol/L NaCl, 0.0027 mmol/L KCl, 0.05% Tween 20 (pH 8.0) and 5% skim milk. Protein bands were probed with primary antibody followed by labeling with horseradish peroxidase–conjugated anti-mouse, anti-rabbit (Amersham, Piscataway, NJ), or anti-goat secondary antibody (Santa Cruz Biotechnology). Bands were visualized by enhanced chemiluminescence using the ECL kit (Amersham) according to manufacturer's instruction.
Cell Viability Assay
The cell growth of the stable cell lines was measured using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium colorimetric assay kit purchased from Promega (Madison, WI). Approximately 3 x 103 cells were seeded and grown in each well of 96-well plates overnight. Cell viability was measured daily for 4 days at 490 nm in an ELISA reader plate following the addition of 20 µL 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium solution and incubation at 37°C for 4 hours.
NAG-1 Promoter and Luciferase Assay
SKOV3 cells were plated in six-well plates at 1 x 105 cells/well in DMEM supplemented with 10% fetal bovine serum. After growth for 24 hours, 1 µg NAG1 promoter containing plasmid (pNAG133/LUC) and 0.05 µg pRL-null (Promega) were transfected with FuGene6 (Roche, Indianapolis, IN) according to the manufacturer's instruction. NAG-1 promoter (pNAG133/LUC) was described previously (23). After 24 hours of transfection, cells were treated with DMSO or sulindac sulfide. After 24 hours of sulindac sulfide treatment, cells were harvested in 1x luciferase assay buffer, and luciferase activity was measured and normalized to the pRL-null luciferase activity using the Dual Luciferase Assay kit (Promega).
NAG-1 Small Interfering RNA Construct and Trypan Blue Exclusion Assay
NAG-1 small interfering RNA (siRNA) construct was kindly provided by Dr. Jim Lambert (University of Colorado, Denver, CO). Cells were transfected with pcDNA3.1 or NAG-1 siRNA before 24-hour treatment with sulindac sulfide. After 24-hour treatment with sulindac sulfide, cells were collected with trypsinization in 300 µL PBS. Cells were stained with trypan blue solution and then the viable cells were counted in a hemocytometer. Each sample was counted as three independent determinations from three independent experiments.
Reverse Transcription-PCR
Total cellular RNAs were extracted from cell lines using TRIzol reagent (Life Technologies, Gaithersburg, MD). Ten micrograms total RNA were reverse transcribed and a one tenth volume of synthesized cDNA was then added to a 50 µL PCR reaction mixture with each set of gene-specific primers (Table 1). The thermal cycling conditions used consisted of initial denaturation at 94°C for 4 minutes, followed by 25 cycles of 94°C for 30 seconds, 58°C for 30 seconds and 72°C for 45 seconds, and a final extension for 10 minutes at 72°C. The final PCR products were electrophoresed on a 1% agarose gel and photographed under UV light.
Microarray Experiment
The procedure for biotinylated cDNA probe synthesis was done using the Ampho-LPR labeling kit (Superarray, Inc., Frederick, MD). Briefly, 5 µg total RNA were used as a template for cDNA synthesis and then the cDNA was labeled with biotin-dUTP (Roche) during a PCR reaction. The reaction was stopped and denatured at 94°C for 2 minutes and the resulting DNA probe was applied to a prehybridized GEArray membrane. The hybridization was done at 60°C for 12 hours in a hybridization oven. After two-step washing at 60°C, the membrane was blocked and treated with alkaline phosphatase–conjugated streptavidin and finally exposed to CDP-Star alkaline phosphatase chemiluminescent substrate. The membrane was exposed to an X-ray film. The intensity of the spot was compared using glyceraldehyde-3-phosphate dehydrogenase as a positive control.
|
Results
|
|---|
Sulindac Sulfide Induces NAG-1 Expression in Ovarian Cancer Cell
Previously, we have reported that some NSAIDs induce NAG-1 expression in colorectal cancer cells (10, 24). However, there is no information regarding NAG-1 expression by NSAIDs in ovarian cancer cells. To determine whether NSAIDs increase NAG-1 expression, conventional nonselective NSAIDs (sulindac, sulindac sulfone, sulindac sulfide, and indomethacin) that inhibit COX-1 and COX-2, as well as selective COX-1 (SC-560) or COX-2 inhibitors (NS-398), were incubated with SKOV3 cells (COX-1/-2 and p53-negative cells). Cells were treated with various NSAIDs at different concentrations shown in Fig. 1A for 24 hours, and Western blot analysis was done using a NAG-1–specific antibody. Among the NSAIDs tested in this study, sulindac sulfide dramatically induced NAG-1 expression, whereas SC-560 barely induced NAG-1 expression. In agreement with previous studies with human colorectal cancer cells, sulindac and sulindac sulfone were ineffective, whereas sulindac sulfide was an excellent inducer of NAG-1 expression. Therefore, sulindac sulfide is the best NAG-1 inducer among NSAIDs tested here and, thus, was used for further studies. In addition, two other ovarian cancer cells, OVCAR3 (COX-1/-2 positive) and PA-1 (p53 positive), were examined. Interestingly, dramatic NAG-1 induction was detected in all cell lines (Fig. 1B), suggesting that sulindac sulfide induces NAG-1 expression possibly independent of COX and p53 status.
View larger version (44K):
[in this window]
[in a new window]
|
Figure 1. NAG-1 protein expression in NSAID-treated ovarian cancer cells. A, SKOV3 cells were treated with either vehicle (0.2% DMSO), sulindac (30 µmol/L), sulindac sulfone (S. Sulfone, 30 µmol/L), sulindac sulfide (S. Sulfide, 30 µmol/L), indomethacin (100 µmol/L), SC-560 (30 µmol/L), or NS-398 (100 µmol/L) for 24 h. Cell lysates were prepared and subjected to Western blot analysis using NAG-1 and actin antibodies. B, three ovarian cancer cell lines, SKOV3, OVCAR3, and PA-1, were treated with vehicle or sulindac sulfide (SS, 30 µmol/L) for 24 h, and NAG-1 or actin expression was detected by Western blot analysis.
|
|
Sulindac Sulfide Suppresses Ovarian Cancer Cell Growth and Up-Regulates NAG-1 and p21 Expression
NAG-1 expression is linked to cell growth arrest and apoptosis (10, 11). To investigate the effects of sulindac sulfide on the growth of ovarian cancer cells in culture, SKOV3 cells were grown and treated with different concentration of sulindac sulfide (10, 20, and 30 µmol/L) for 3 days and the cell growth rate was measured by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay at 490 nm absorbance. As shown in Fig. 2, sulindac sulfide treatment resulted in the complete inhibition of cell growth, whereas vehicle treatment did not affect any significant inhibition in SKOV3 cells, suggesting that sulindac sulfide induces cell growth arrest in SKOV3 cells in a dose-dependent manner. This data is consistent with our previous report that sulindac sulfide inhibit cell growth in HCT-116 human colorectal cancer cells (10). Based on this result, we evaluated NAG-1 expression, induced at different time and concentrations by sulindac sulfide, with Western blot analysis. Figure 3A and B shows that NAG-1 expression is dependent upon both the concentration and time of treatment. Highest expression was detected at 48 hours. In addition, a luciferase assay with the NAG-1 promoter (pNAG133/LUC) shows a similar concentration dependency for NAG-1 expression (Fig. 3C). Finally, we evaluated expression of p21 WAF1/CIP1 by Western blot analysis because Han et al. (25) previously reported sulindac sulfide–up-regulated p21 WAF1/CIP1 in human breast cancer cells. As shown in Fig. 3D, sulindac sulfide up-regulate p21 WAF1/CIP1 at the protein level in a time-dependent manner. This result indicates that p21 WAF1/CIP1 may also be one of the genes responsible for the growth arrest induced by sulindac sulfide.
View larger version (13K):
[in this window]
[in a new window]
|
Figure 2. Effect of sulindac sulfide on SKOV3 cell growth. SKOV3 cells were plated at 3,000 cells/well in a 96-well plate and incubated with vehicle (DMSO) or various concentrations of sulindac sulfide. Cell growth was measured using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium cell proliferation kit (Promega). Points, mean of six replicate experiments; bars, SD.
|
|
View larger version (31K):
[in this window]
[in a new window]
|
Figure 3. NAG-1 and p21 expression by sulindac sulfide. A, SKOV3 cells were treated with three different concentrations of sulindac sulfide, and treated cells were collected for Western blot analysis using NAG-1 and actin antibodies. B, SKOV3 cells were treated with 30 µmol/L sulindac sulfide at three different time points. Cell lysates were subjected to Western blot analysis with NAG-1 and actin antibodies. C, a NAG-1 promoter construct, pNAG133/LUC (1 µg), was cotransfected with 0.1 µg pRL-null vector into SKOV3 cells using FuGene6. After 24-h transfection, cells were treated with 10, 20, or 30 µmol/L sulindac sulfide or DMSO (vehicle, VEH). The promoter activity was measured by luciferase activity after 24-h treatment. Transfection efficiency for luciferase activity was normalized to the Renilla luciferase activity. X-axis, relative luciferase unit (RLU, luciferase activity/Renilla unit). Columns, mean of three independent transfections; bars, SD. D, SKOV3 cells were treated with 20 µmol/L sulindac sulfide and cells were collected at different time points. Cell lysates were prepared and Western blot analysis was done using p21 and actin antibodies.
|
|
Reversal of Sulindac Sulfide–Induced Apoptosis by NAG-1 siRNA
The correlations observed between the induction of NAG-1 and cell growth arrest necessitated construction of a NAG-1 siRNA to directly assess the biological activities of sulindac sulfide and NAG-1 in ovarian cancer cells. The NAG-1 siRNA was prepared and SKOV3 cells were transiently transfected with either empty vector (pcDNA3.1) or the NAG-1 siRNA vector, followed by the treatment of sulindac sulfide. The cells transfected with the NAG-1 siRNA construct showed a repression of sulindac sulfide–induced NAG-1 expression (Fig. 4A). Next, we examined whether the suppression of NAG-1 by siRNA may attenuate sulindac sulfide–induced cell growth arrest. The empty vector and NAG-1 siRNA constructs were transiently transfected into SKOV3 cells and then the cells incubated with sulindac sulfide for 24 hours. As shown in Fig. 4B, NAG-1 siRNA transfection restored cell viability to SKOV3 cells treated with sulindac sulfide, indicating that the sulindac sulfide–induced NAG-1 expression is responsible, at least in part, for the sulindac sulfide–induced cell growth arrest observed in SKVO3 cells. These results also indicate that NAG-1 expression is directly associated with growth suppression by sulindac sulfide. To explain the molecular mechanisms of growth recovery by NAG-1 siRNA transfection, we analyzed NAG-1 and p21 expression after introduction of pcDNA3.1 or NAG-1 siRNA construct, followed by sulindac sulfide treatment. As shown in Fig. 4A, NAG-1 siRNA–transfected cells showed reduced protein expression of NAG-1, as well as p21 expression, compared with pcDNA3.1-transfected cells. These results indicate that NAG-1 is directly associated with the growth suppression caused by sulindac sulfide and p21 may be one of genes downstream of NAG-1. However, the direct molecular mechanism connecting NAG-1 and p21 remains to be elucidated.
View larger version (29K):
[in this window]
[in a new window]
|
Figure 4. NAG-1 siRNA recovers cell growth arrest induced by sulindac sulfide. A, SKOV3 cells were transfected with pcDNA3.1 or the NAG-1 siRNA construct before 24-h treatment of 30 µmol/L sulindac sulfide. Cell lysates were prepared and subjected to Western blot using NAG-1, p21, and actin antibodies. B, either 1 µg NAG-1 siRNA construct or 1 µg pcDNA3.1 (control) was transfected into SKOV3 cells. After 24 h, cells were treated with DMSO (vehicle) or 30 µmol/L sulindac sulfide. After 24-h treatment, cells were collected in PBS solution, stained with trypan blue, and live cells were counted using a hemocytometer. Columns, mean of three independent experiments of three independent determinations; bars, SD (*P = 0.007, **P = 0.019).
|
|
Analysis of Gene Expression Changes by Sulindac Sulfide with Membrane-Based Microarray
To provide other potential mechanisms to explain the antitumorigenic effect of sulindac sulfide in ovarian cancer, we did a membrane-based microarray (Superarray) analysis. The Cancer Pathway Finder microarray was chosen because it contains 96 key genes representing the six cancer-related biological pathways. We found that 4 of the 96 genes were dramatically down-regulated by sulindac sulfide, whereas we could not detect up-regulated genes. The four down-regulated genes are cyclin D1, MMP-1, PI3KR1, and uPA. Down-regulation of those genes was also evaluated by reverse transcription-PCR and the result agreed with the microarray data (Fig. 5). The characteristics of those genes will be described in the following section.
View larger version (63K):
[in this window]
[in a new window]
|
Figure 5. Changes in gene expression by sulindac sulfide. SKOV3 cells were treated either with 30 µmol/L sulindac sulfide (SS) or DMSO (vehicle, V) for 48 h and then superarray analysis was done according to the manufacturer's instructions. The GEArray Series Human Cancer Pathway Finder Gene Array (HS-060) was used. The spots represent scanned images of each spot for down-regulated genes, whereas glyceraldehyde-3-phosphate dehydrogenase was used as a control image (left). Total RNAs used for superarray experiment were subjected to reverse transcription-PCR with gene-specific primers (right).
|
|
|
Discussion
|
|---|
In this study, we have shown that the NSAID sulindac sulfide exerts a potential antitumorigenic effect in ovarian cancer cells through the up-regulation of the antitumorigenic protein NAG-1. Our results support the conclusion that NAG-1 has an antiproliferative activity against ovarian cancer cells. First, up-regulation of NAG-1 by sulindac sulfide treatment is inversely associated with ovarian cancer cell growth. Second, blocking of NAG-1 expression by NAG-1 siRNA resulted in recovery from the growth arrest caused by sulindac sulfide. Third, down-regulation of NAG-1 and p21 by NAG-1 siRNA points to a suitable molecular mechanism of growth recovery. These results indicate the NSAID sulindac sulfide arrests ovarian cancer cell growth via up-regulation of the NAG-1 and p21 genes. However, the exact molecular mechanism by which NAG-1 and p21 affect cell growth arrest remains to be elucidated.
It has been reported that 9 of 10 primary cultures of ovarian cancer cells isolated from ascite fluid remained sensitive to transforming growth factor-ß growth inhibition and that these cells express all components of the transforming growth factor-ß/Smad signaling pathway (26). It is notable that NAG-1 belongs to the transforming growth factor-ß superfamily. Therefore, it is very interesting to examine the induction of NAG-1 by drugs including NSAIDs, retinoids, and oral contraceptives. In this report, we showed that sulindac sulfide induced NAG-1 expression. However, the other two compounds are also promising for chemoprevention against ovarian cancer. Oral contraceptives have been shown to reduce the subsequent risk of ovarian carcinoma in observation studies (27). An innovative study that supports the use of progestins as a chemopreventive agent in ovarian cancer was published (28). In addition, epidemiologic and laboratory data suggest that retinoids may have a role as preventive or therapeutic agents for ovarian cancer (29). For example, fenretinide or 4-HPR induces apoptosis in ovarian cancer cells and has few side effects compared with other vitamin A derivatives. In addition, 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid (AHPN/CD437), a conformationally restricted retinoid engineered to bind selectively to retinoic acid receptor-, also shares the apoptotic profile of 4-HPR. In ovarian cancer cells, exposure to AHPN was associated with increased expression of BAX and decreased expression of Bcl-2 (30). Indeed, we have reported that AHPN induces NAG-1 expression in lung carcinoma cells (20).
To identify other potential mechanisms, we analyzed gene expression changes in sulindac sulfide–treated SKOV3 cells compared with vehicle-treated cells by membrane microarray. We found that four genes were dramatically down-regulated by sulindac sulfide treatment—cyclin D1, MMP-1, PI3KR1, and uPA. Increased cyclin D1 expression was detected in ovarian cancer (31) and was associated with malignancy and recurrence of ovarian cancer (32). Therefore, down-regulation of cyclin D1 by sulindac sulfide may have a significant meaning. Nishikawa et al. (33) provided evidence that MMP-1 and MMP-2 were important genes in the invasive activity of several cancer cell lines. However, in this microarray experiment, we did not detect any change in either MMP-2 or MMP-9. Therefore, MMP-1 may be one of the important targets of sulindac sulfide among the metalloproteinases. PI3KR1 is a regulatory subunit of phosphoinositide 3-kinase. Philp et al. (34) reported that PI3KR1 and serine kinase AKT were activated in SKOV3 cells and NAG-1 was identified as a downstream target of phosphoinositide 3-kinase (22). Therefore, sulindac sulfide could block the AKT pathway by down-regulation of PI3KR1. The blockage of phosphoinositide 3-kinase pathway by sulindac sulfide is under investigation. In ovarian cancer, elevated expression of uPA and PAI-1 have been reported (35, 36). Microarray data and reverse transcription-PCR data shows sulindac sulfide dramatically reduces uPA mRNA expression (Fig. 5). However, expression of those genes was not affected by NAG-1 siRNA transfection (data not shown). Taken together, the conventional NSAID sulindac sulfide has a chemopreventive effect in ovarian cancer and modulates the expression such genes as NAG-1, p21, cyclin D1, MMP-1, PI3KR1, and uPA. However, additional studies are needed to determine the relationship between the regulation of these genes and sulindac sulfide.
|
Acknowledgments
|
|---|
We thank Dr. Jeanelle Martinez and Dr. Minako Ishibashi for their helpful suggestions and comments.
|
Footnotes
|
|---|
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 8/ 9/04;
revised 1/ 4/05;
accepted 1/ 5/05.
|
References
|
|---|
Landis SH, Murray T, Bolden S, Wingo PA. Cancer statistics, 1999. CA Cancer J Clin 1999;49:8–31.[Abstract/Free Full Text]
Wingo PA, Cardinez CJ, Landis SH, et al. Long-term trends in cancer mortality in the United States, 1930-1998. Cancer 2003;97:3133–275.[CrossRef][Medline]
Rodriguez-Burford C, Barnes MN, Oelschlager DK, et al. Effects of nonsteroidal anti-inflammatory agents (NSAIDs) on ovarian carcinoma cell lines: preclinical evaluation of NSAIDs as chemopreventive agents. Clin Cancer Res 2002;8:202–9.[Abstract/Free Full Text]
Um SJ, Lee SY, Kim EJ, et al. Antiproliferative mechanism of retinoid derivatives in ovarian cancer cells. Cancer Lett 2001;174:127–34.[CrossRef][Medline]
Bosetti C, Negri E, Trichopoulos D, et al. Long-term effects of oral contraceptives on ovarian cancer risk. Int J Cancer 2002;102:262–5.[CrossRef][Medline]
Rao CV, Rivenson A, Simi B, et al. Chemoprevention of colon carcinogenesis by sulindac, a nonsteroidal anti-inflammatory agent. Cancer Res 1995;55:1464–72.[Abstract/Free Full Text]
Drake JG, Becker JL. Aspirin-induced inhibition of ovarian tumor cell growth. Obstet Gynecol 2002;100:677–82.[Abstract/Free Full Text]
Tavani A, Gallus S, La Vecchia C, Conti E, Montella M, Franceschi S. Aspirin and ovarian cancer: an Italian case-control study. Ann Oncol 2000;11:1171–3.[Abstract/Free Full Text]
Rosenberg L, Palmer JR, Rao RS, et al. A case-control study of analgesic use and ovarian cancer. Cancer Epidemiol Biomarkers Prev 2000;9:933–7.[Abstract/Free Full Text]
Baek SJ, Kim KS, Nixon JB, Wilson LC, Eling TE. Cyclooxygenase inhibitors regulate the expression of a TGF-ß superfamily member that has proapoptotic and antitumorigenic activities. Mol Pharmacol 2001;59:901–8.[Abstract/Free Full Text]
Li PX, Wong J, Ayed A, et al. Placental transforming growth factor-ß is a downstream mediator of the growth arrest and apoptotic response of tumor cells to DNA damage and p53 overexpression. J Biol Chem 2000;275:20127–35.[Abstract/Free Full Text]
Kim KS, Baek SJ, Flake GP, Loftin CD, Calvo BF, Eling TE. Expression and regulation of nonsteroidal anti-inflammatory drug-activated gene (NAG-1) in human and mouse tissue. Gastroenterology 2002;122:1388–98.[CrossRef][Medline]
Liu T, Bauskin AR, Zaunders J, et al. Macrophage inhibitory cytokine 1 reduces cell adhesion and induces apoptosis in prostate cancer cells. Cancer Res 2003;63:5034–40.[Abstract/Free Full Text]
Baek SJ, Wilson LC, Eling TE. Resveratrol enhances the expression of non-steroidal anti-inflammatory drug-activated gene (NAG-1) by increasing the expression of p53. Carcinogenesis 2002;23:425–34.[Abstract/Free Full Text]
Wilson LC, Baek SJ, Call A, Eling TE. Nonsteroidal anti-inflammatory drug-activated gene (NAG-1) is induced by genistein through the expression of p53 in colorectal cancer cells. Int J Cancer 2003;105:747–3.[CrossRef][Medline]
Bottone FG Jr, Baek SJ, Nixon JB, Eling TE. Diallyl disulfide (DADS) induces the antitumorigenic NSAID-activated gene (NAG-1) by a p53-dependent mechanism in human colorectal HCT 116 cells. J Nutr 2002;132:773–8.[Abstract/Free Full Text]
Baek SJ, Kim JS, Jackson FR, Eling TE, McEntee MF, Lee SH. Epicatechin gallate-induced expression of NAG-1 is associated with growth inhibition and apoptosis in colon cancer cells. Carcinogenesis 2004;25:2425–32.[Abstract/Free Full Text]
Baek SJ, Kim JS, Nixon JB, DiAugustine RP, Eling TE. Expression of NAG-1, a transforming growth factor-ß superfamily member, by troglitazone requires the early growth response gene EGR-1. J Biol Chem 2004;279:6883–92.[Abstract/Free Full Text]
Monks A, Harris E, Hose C, Connelly J, Sausville EA. Genotoxic profiling of MCF-7 breast cancer cell line elucidates gene expression modifications underlying toxicity of the anticancer drug 2-(4-amino-3-methylphenyl)-5-fluorobenzothiazole. Mol Pharmacol 2003;63:766–72.[Abstract/Free Full Text]
Newman D, Sakaue M, Koo JS, et al. Differential regulation of nonsteroidal anti-inflammatory drug-activated gene in normal human tracheobronchial epithelial and lung carcinoma cells by retinoids. Mol Pharmacol 2003;63:557–64.[Abstract/Free Full Text]
Albertoni M, Shaw PH, Nozaki M, et al. Anoxia induces macrophage inhibitory cytokine-1 (MIC-1) in glioblastoma cells independently of p53 and HIF-1. Oncogene 2002;21:4212–9.[CrossRef][Medline]
Yamaguchi K, Lee SH, Eling TE, Baek SJ. Identification of nonsteroidal anti-inflammatory drug-activated gene (NAG-1) as a novel downstream target of phosphatidylinositol 3-kinase/AKT/GSK-3{ß} pathway. J Biol Chem 2004;279:49617–23.[Abstract/Free Full Text]
Baek SJ, Horowitz JM, Eling TE. Molecular cloning and characterization of human nonsteroidal anti-inflammatory drug-activated gene promoter. Basal transcription is mediated by Sp1 and Sp3. J Biol Chem 2001;276:33384–92.[Abstract/Free Full Text]
Baek SJ, Kim JS, Moore SM, Lee SH, Martinez JM, Eling TE. Cyclooxygenase inhibitors induce the expression of the tumor suppressor gene EGR1, which results in the up-regulation of NAG-1, an antitumorigenic protein. Mol Pharmacol 2005;67:356–64.[Abstract/Free Full Text]
Han EK, Arber N, Yamamoto H, et al. Effects of sulindac and its metabolites on growth and apoptosis in human mammary epithelial and breast carcinoma cell lines. Breast Cancer Res Treat 1998;48:195–203.[CrossRef][Medline]
Dunfield LD, Dwyer EJ, Nachtigal MW. TGF ß-induced Smad signaling remains intact in primary human ovarian cancer cells. Endocrinology 2002;143:1174–81.[Abstract/Free Full Text]
Narod SA, Risch H, Moslehi R, et al. Oral contraceptives and the risk of hereditary ovarian cancer. Hereditary Ovarian Cancer Clinical Study Group. N Engl J Med 1998;339:424–8.[Abstract/Free Full Text]
Rodriguez GC, Walmer DK, Cline M, et al. Effect of progestin on the ovarian epithelium of macaques: cancer prevention through apoptosis? J Soc Gynecol Investig 1998;5:271–6.[CrossRef][Medline]
Formelli F, Cleris L. Synthetic retinoid fenretinide is effective against a human ovarian carcinoma xenograft and potentiates cisplatin activity. Cancer Res 1993;53:5374–6.[Abstract/Free Full Text]
Wu S, Zhang D, Donigan A, Dawson MI, Soprano DR, Soprano KJ. Effects of conformationally restricted synthetic retinoids on ovarian tumor cell growth. J Cell Biochem 1998;68:378–88.[CrossRef][Medline]
Worsley SD, Ponder BA, Davies BR. Overexpression of cyclin D1 in epithelial ovarian cancers. Gynecol Oncol 1997;64:189–95.[CrossRef][Medline]
Barbieri F, Cagnoli M, Ragni N, et al. Increased cyclin D1 expression is associated with features of malignancy and disease recurrence in ovarian tumors. Clin Cancer Res 1999;5:1837–42.[Abstract/Free Full Text]
Nishikawa A, Iwasaki M, Akutagawa N, et al. Expression of various matrix proteases and Ets family transcriptional factors in ovarian cancer cell lines: correlation to invasive potential. Gynecol Oncol 2000;79:256–63.[CrossRef][Medline]
Philp AJ, Campbell IG, Leet C, et al. The phosphatidylinositol 3'-kinase p85
gene is an oncogene in human ovarian and colon tumors. Cancer Res 2001;61:7426–9.[Abstract/Free Full Text]
Casslen B, Bossmar T, Lecander I, Astedt B. Plasminogen activators and plasminogen activator inhibitors in blood and tumour fluids of patients with ovarian cancer. Eur J Cancer 1994;30A:1302–9.
van der Burg ME, Henzen-Logmans SC, Berns EM, van Putten WL, Klijn JG, Foekens JA. Expression of urokinase-type plasminogen activator (uPA) and its inhibitor PAI-1 in benign, borderline, malignant primary and metastatic ovarian tumors. Int J Cancer 1996;69:475–9.[CrossRef][Medline]
This article has been cited by other articles:
|
|
|
|
 
S. Chintharlapalli, S. Papineni, and S. Safe
1,1-Bis(3'-Indolyl)-1-(p-substitutedphenyl)methanes Inhibit Growth, Induce Apoptosis, and Decrease the Androgen Receptor in LNCaP Prostate Cancer Cells through Peroxisome Proliferator-Activated Receptor {gamma}-Independent Pathways
Mol. Pharmacol.,
February 1, 2007;
71(2):
558 - 569.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. R. Bauskin, D. A. Brown, T. Kuffner, H. Johnen, X. W. Luo, M. Hunter, and S. N. Breit
Role of macrophage inhibitory cytokine-1 in tumorigenesis and diagnosis of cancer.
Cancer Res.,
May 15, 2006;
66(10):
4983 - 4986.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Chintharlapalli, S. Papineni, and S. Safe
1,1-Bis(3'-indolyl)-1-(p-substituted phenyl)methanes inhibit colon cancer cell and tumor growth through PPAR{gamma}-dependent and PPAR{gamma}-independent pathways
Mol. Cancer Ther.,
May 1, 2006;
5(5):
1362 - 1370.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Chintharlapalli, S. Papineni, S. J. Baek, S. Liu, and S. Safe
1,1-Bis(3'-indolyl)-1-(p-substitutedphenyl)methanes Are Peroxisome Proliferator-Activated Receptor {gamma} Agonists but Decrease HCT-116 Colon Cancer Cell Survival through Receptor-Independent Activation of Early Growth Response-1 and Nonsteroidal Anti-Inflammatory Drug-Activated Gene-1
Mol. Pharmacol.,
December 1, 2005;
68(6):
1782 - 1792.
[Abstract]
[Full Text]
[PDF]
|
|
|
Top
Abstract
Introduction
