![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
CHEMOTHERAPY, ANTIBIOTICS, AND GENE THERAPY
Laboratories of Molecular Toxicology (J.M.M.) and Toxicology Operations Branch (N.J.W.) and Molecular Carcinogenesis (T.S., R.O., C.A., S.J.B., T.E.E.) National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina; SAIC-Frederick, Inc., Screening Technologies Branch, Laboratory of Functional Genomics, National Cancer Institute, Frederick, Maryland (M.H.,C.H., A.M.); National Decontamination Team, Office of Solid Waste and Emergency Response, Environmental Protection Agency, Cincinnati, Ohio (J.M.M.); Department of Pathobiology, College of Veterinary Medicine, University of Tennessee, Knoxville, Tennessee (S.J.B.); and Department of Radiation Biology and Health, School of Medicine, University of Occupational and Environmental Health Kitakyushu, Japan (R.O.)
Received December 16, 2005; accepted May 15, 2006.
 |
Abstract |
|---|
 Top Abstract Materials and MethodsResultsDiscussionReferences |
|---|
). NAG-1 is a member of the transforming growth factor (TGF)-
(TGF-1) superfamily and has proapoptotic and antitumorigenic activities. NAG-1 is also known as macrophage inhibitory cytokine (MIC)-1, growth/differentiation factor-15, prostate-derived factor (PDF), and placental TGF- (Baek and Eling, 2006). Not only is NAG-1 regulated by Cox inhibitors but also by an increasing number of chemopreventive and antitumorigenic chemicals. Transcriptional regulation of NAG-1 is complex, and the promoter sequence has many different cis- and trans-acting promoter elements (Baek et al., 2001a). In addition, NAG-1 seems to be a potentially important downstream target of three tumor suppressor genes. NAG-1 expression is regulated by the tumor suppressor genes p53 (Baek et al., 2002a, 2004a) and early growth response (EGR) gene-1 (Baek et al., 2004b). Recently, we have identified NAG-1 as a novel negative downstream target of the phosphatidylinositol 3-kinase/protein kinase B (AKT)/glycogen synthase kinase (GSK)-3 pathway (Yamaguchi et al., 2004). Thus, NAG-1 seems to be a central and possibly key protein regulated by these tumor suppressor pathways.
The mechanism by which this protein acts to suppress tumor growth is not clear. Compelling evidence in the literature supports the hypothesis that NAG-1 regulates apoptosis in a number of cells, including human colorectal and breast cells. Since most of the data on the biological activity and expression are based on in vitro experiments, it is important to confirm the activity in vivo and to confirm that drug-targeted, NAG-1 up-regulation in an intact animal can be correlated with inhibition of tumor development. A potent drug with characterized anticancer activity in vivo would be useful to obtain these results. One candidate drug is 2-(4-amino-3-methylphenyl)-5-fluorobenzothiazole (5F203). 5F203 is the active moiety of Phortress (NSC 710305), a drug that is in the early phase I clinical studies in cancer patients. The antitumor activity of this drug is based on the growth inhibitory and cytocidal activity in specific tumor cell lines and on the inhibition of a variety of xenografts, including MCF-7 (Bradshaw et al., 2002). The mechanism for the antitumor activity is not clearly known, but the drug does offer the advantage of not being a Cox inhibitor and thus is devoid of toxic side effects associated with this class of drugs. MCF-7 breast cancer cells are sensitive to the antiproliferative activity of 5F203, and cDNA microarray analysis revealed NAG-1 as the most highly 5F203-induced gene in the MCF-7 cell line (Monks et al., 2003). If the activity of this potent antitumorigenic drug is dependent on NAG-1, it may be a useful model to show that the increase in NAG-1 expression in vivo can be correlated with the in situ NAG-1 expression and reduced tumor growth.
The goals of this study were to 1) determine whether NAG-1 expression is increased by 5F203 and ascertain the regulatory mechanism underlying the increase, 2) determine whether the biological activity of 5F203 is dependent on NAG-1, 3) determine whether NAG-1 inhibits MCF-7 tumor development in mouse xenographs, and 4) demonstrate in an animal model whether 5F203 can exert its antitumorigenic activity by targeting the up-regulation of NAG-1 in vivo.
|
Materials and Methods |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Cell Culture Conditions. The MCF-7 cells, MDA-MB-435 cells, and HCT-116 cells were acquired from American Type Culture Collection (Manassas, VA). HCT-116 cells null for p53 were obtained from Dr. Bert Vogelstein (Johns Hopkins University, Baltimore, MD).
Cell Proliferation. On day 0, 50,000 cells were plated in a 96-well tissue culture plate (Nunclon; NUNC A/S, Roskilde, Denmark). Cells were incubated for various indicated times. On day 1, 50 µl of medium was added for further incubations up to 72 h. The number of viable cells was determined daily by a 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) nonradioactive cell proliferation assay (Promega, Madison, WI). In brief, 25 µl of freshly prepared MTS mixture was added, and after 1 h absorbance at 490 nm was measured. Each experiment was repeated at least three times.
Cycloheximide Treatment. Cells grown to 60 to 80% confluence were washed with PBS, and then treated with serum-free medium containing DMSO plus or minus 5 µg/ml cycloheximide (Sigma-Aldrich, St. Louis, MO). After 30 min, 5F203 or vehicle was added directly to the medium. Cells were harvested 24 h after the addition of 500 nM 5F203 and analyzed by real-time PCR, as described below.
MCF-7 Orthotopic Tumors. In nude mice, a 1- to 1.5-cm ventral midline incision was made through the skin in the abdominal region, and a flap of skin was bluntly dissected from the abdominal wall, exposing the ventral aspect of the number 4 mammary gland. A syringe with a 27-gauge needle was used to make the injection of 7 x 106 MCF-7 breast cancer cells in 0.1 ml of PBS. Mice were given 3 mg/kg estradiol every 7 days subcutaneously throughout the experiment. The mice were treated with Phortress starting on day 20. Control mice were treated with the vehicle. Growth curves for xenograft tumors were determined by external measurements from two dimensions. Tumor measurements began when the size was more than 3 mm in diameter (around 14 days after injection). Tumor volume or weight was determined, respectively, by the equations V = [(L + W) 0.5] x L x W x 0.5236 and weight (in milligrams) = [(length) x width x width)]/2.
Ectopic Expression of NAG-1 in MCF-7 Cells. MCF-7 cells lines were engineered to overexpress NAG-1 or empty vector. The stable cell lines were grown in Dulbecco's modified Eagle's medium supplemented with 250 µg/ml G418 (Invitrogen, Carlsbad, CA). Log-phase NAG-1 was resuspended in PBS and then injected into mice at a concentration of 1 x 107 cells in 50 µl as described above.
In Vivo Analysis of NAG-1. Samples of the mouse tumors were obtained by fine needle aspirates from the mammary fat pad of nude mice 6 and 24 h after 15- or 10-mg/kg treatment with Phortress. RNA samples were isolated using a QIAGEN RNeasy mini kit (QIAGEN, Valencia, CA). NAG-1 expression in the samples was measured using Applied Biosystems (Foster City, CA) TaqMan real-time PCR. glyceraldehyde-3-phosphate dehydrogenase was used as the endogenous control.
Western Blot Analysis. Anti-human-NAG-1 antibody that recognizes both the precursor and secreted forms of NAG-1 (Baek et al., 2001b) developed in this laboratory was used. Actin antibody was obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). ERK1/2 and phosphorylated ERK1/2 was obtained from Cell Signaling Technology Inc. (Beverly, MA). Phospho-GSK-3, AKT, and phospho-AKT (Ser 473) antibodies were purchased from Cell Signaling Technology Inc. Cells were grown to 60 to 80% confluence in 10-cm plates followed by 24 h of additional growth in the absence of serum. Cells were treated with indicated compounds, and total cell lysates were isolated using 0.1 M Tris, pH 8.0, containing proteinase inhibitors (Sigma-Aldrich). After sonication of samples, proteins (30 µg) were separated by SDS-polyacrylamide gel electrophoresis and transferred for 1 h to nitrocellulose membrane (Whatman Schleicher and Schuell, Keene, NH). The blots were blocked overnight with 5% skim milk in Tris-buffered saline/0.05% Tween, and probed with each antibody for 2 h at room temperature. After washing with Tris-buffered saline/0.05% Tween, the blots were treated with horseradish peroxidase-conjugated secondary antibody for 1 h and washed several times. Protein bands were detected by the enhanced chemiluminescence system (GE Healthcare, Arlington Heights, IL).
NAG-1 Small Interfering RNA and Apoptosis. The NAG-1 siRNA construct and NAG-1 scrambled sequence siRNA construct were gifts from Jim Lambert (Department of Pathology, University of Colorado Health Sciences Center, Denver, CO). The oligonucleotides used for generating NAG-1 siRNA were 5'-GGGACCCTCAGAGTTGCACTCAAGCTTGAGTGCAACTCTGAGGGTCCCTTTTTG-3' and 5'-AATTCAAAAAGGGACCCTCAGAGTTGCACTCAAGCTTGAGTGCAACTCTGAGGGTCCC-3'; the oligonucleotides used for generating the control siRNA containing a scrambled sequence were 5'-GGGTTCCTACAGCACGCTCAAAGCTTTGAGCGTGCTGTAGGAACCCCTTTTTG-3' and 5'-AATTCAAAAAGGGGTTCCTACAGCACGCTCAAAGCTTTGAGCGTGCTTGTAGGAACCC-3'. Cells at 60 to 80% confluence were transfected using FuGENE 6 (Roche Diagnostics, Indianapolis, IN) with NAG-1 siRNA (2 µg/well) or with the scrambled NAG-1 siRNA (2 µg/well) overnight. Medium was removed, and the cells were washed with PBS and then treated with 100 to 500 nM 5F203 or vehicle in medium containing reduced serum (1%) for up to 48 h. Cells were harvested at 24 h and analyzed by Western blot as described above and harvested at 48 h for apoptosis analysis. For apoptosis, cells were washed with PBS, trypsinized, collected, washed in ice-cold PBS, and fixed by the slow addition of 75% ethanol to a total of 5 ml and stored at 4°C overnight. Fixed cells were pelleted and then washed with PBS and stained with 20 µg/ml propidium iodide and 1 mg/ml RNase in PBS for 20 min. Cells were examined by flow cytometry using Becton Dickinson FAC Sort and CellQuest software (BD Biosciences, San Jose, CA). Apoptosis was measured by the level of subdiploid DNA in cells.
NAG-1 Promoter Activity. The NAG-1 promoter constructs were prepared as described previously (Baek et al., 2001a). Specific constructs included the following promoter regions: -133/+70, -966/+41, -966/+70, -5700/+70, and -133/+70 with NAG-1 intron. HCT-116 or MCF-7 cells were cotransfected with the NAG-1 promoter cloned into the pgal basic vector (BD Biosciences) or into the luciferase vector and with the pRL null vector (Promega) to control for transfection efficiency. Lipofectamine (Invitrogen) was used to transfect the cells for 5 h. Medium containing serum was then added, and cells were incubated at 37°C overnight. To treat cells, serum-free medium containing either 5F203, negative control (DMSO), or positive control (sulindac sulfide or troglitazone) was added. After 24 h of drug treatment, cells were lysed using passive lysis buffer (Promega), and -galactosidase activity was measured using a luminometer and normalized using the -galactosidase/pRL null ratio for comparison of the various samples. After 24 h of transfection, luciferase activity was determined and normalized to the pRL-null luciferase activity with a dual luciferase assay kit (Promega).
RNA Stability in the Presence of Actinomycin D. MCF-7 or HCT-116 cells were plated into six-well plates at 2 x 105 cells per well, grown to 60 to 80% confluence, and then treated with either DMSO or 5F203 in serum-free medium. After 24 h, 5 µg/ml actinomycin D (Sigma-Aldrich) was added for the indicated length of time. Cells were lysed in QIAGEN RNA isolation buffer, and RNA was purified using the QIAGEN RNeasy kit. Total RNA was reverse-transcribed using Moloney murine leukemia virus reverse transcriptase, and real-time PCR was performed using a SYBR Green PCR Master Mix (Applied Biosystems) according to the manufacturer's instructions. Actin was used as the endogenous control. A negative control containing all the reagents and cDNA in the absence of the reverse transcriptase enzyme (no reverse transcriptase control) was routinely performed. Primers used for NAG-1 were 5'-TGCCCGCCAGCTACAATC-3' and 5'-TCTTTGGCTAACAAGTCATCATAGGT-3' and for actin were 5'-CCTGGCACCCAGCACAAT-3' and 5'-GCTGATCCACATCTGCTGGAA-3'. The relative level of normalized NAG-1 mRNA was calculated, and the results were plotted as the ratio of the mRNA level present at time 0 of actinomycin D treatment.
ERK1/2 Inhibitor Studies. Cells at 60 to 80% confluence were treated with DMSO, PD98059 (Calbiochem, San Diego, CA), or U0126 (Cell Signaling Technology Inc.) in serum-free medium. After 30 min, either DMSO or 5F203 was added directly to the medium. After 24 h, cells were both harvested and analyzed by Western blot, as described above, or treated with 5 µg/ml actinomycin D (Sigma-Aldrich) for the indicated times. Then, cells were harvested and analyzed by real-time PCR, as described above.
|
Results |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
) and to compare NAG-1 induction by chemopreventive chemicals. Therefore, initial studies in this well defined model focused on the response of NAG-1 to 5F203. Robust induction of NAG-1 was observed following treatment with 5F203, and this induction was dependent on incubation time and the drug concentration (Fig. 1, A and B). The time course of NAG-1 protein expression was similar to the course observed previously (Monks et al., 2003) in that the highest expression was observed after 24 h (Fig. 1A). An increase in NAG-1 expression was observed at 10 nM 5F203, the lowest concentration tested. Thus, 5F203 was more potent than any other drug we have tested in its ability to increase NAG-1 expression in HCT-116 cells (Baek et al., 2001b).
|
). Phase I cancer clinical studies using the prodrug form of 5F203 (Phortress) are based, in part, on inhibition of tumor growth with the breast cancer cell lines in the nude mouse xenograft model and other biochemical data (Bradshaw and Westwell, 2004). Thus, it was important to determine whether 5F203 increased NAG-1 expression in human breast cancer cells. Two human tumor cell lines were used: the MCF-7 cells, which are responsive to 5F203, and MDA-MB-435, which are nonresponsive to 5F203 (Bradshaw et al., 2002). To measure the expression of NAG-1 protein in MCF-7 cells, cells were incubated with 1 µM 5F203 for up to 96 h (Fig. 1C). A time-dependent increase in NAG-1 protein expression was observed in the MCF-7 cells (Fig. 1C). 5F203 was also a potent NAG-1 inducer in MCF-7 cells with induction observed at as low as 10 nM, and maximal induction with 100 to 500 nM (Fig. 1D). In addition, NAG-1 induction by 5F203 treatment was observed in the human prostate cancer cells LNCaP and PC3, but we did not observe an increase in the expression of NAG-1 in MDA-MB-435 cells (data not shown). To determine the optimal concentration of 5F203 to alter cell growth of MCF-7, cell proliferation was measured after treatment with 5F203, which was a potent inhibitor of cell proliferation (Table 2). Both a time- and concentration-dependent inhibition of cell growth was observed, which was similar to the time and concentration dependence of NAG-1 induction. The growth of MDA-MB-435 cells has been shown not to be inhibited by 5F203 (Bradshaw et al., 2002).
|
|
Effect of 5F203 on NAG-1 Transcription. NAG-1 has been reported to be regulated by many chemicals at the transcriptional level (Li et al., 2000; Baek et al., 2001a,b, 2002a, 2004a,b, 2005) and some of which are dependent on de novo protein synthesis (Newman et al., 2003; Baek et al., 2005). Regulation of NAG-1 at the transcriptional level was examined with various promoter constructs designed with different regions of the 5-kilobase 5' flanking region and inserted into luciferase and -galactosidase reporting plasmids in HCT-116 and MCF-7 cells. However, despite incubation with several concentrations of 5F203, we could not detect any drug-induced increase in the promoter activity. Furthermore, incubation with 5F203 also inhibited the basal activity of the promoters. The 5F203 seems to inhibit luciferase and -galactosidase activities or is toxic to the promoter constructs; thus, this approach cannot be used to investigate the transcriptional regulation (data not shown).
The expression of NAG-1 is regulated by antitumorigenic chemicals at the transcriptional level and is mediated by tumor suppressor gene p53, by EGR-1, and by GSK-3/AKT, three pathways linked to tumor growth. HCT-116 and MCF-7 cells were incubated with either 1 µM 5F203 or 30 µM sulindac sulfide, and the expression of EGR-1 was measured by Western analysis. As shown in Fig. 2A, for HCT-116 cells, sulindac sulfide, a positive control (Baek et al., 2005), increased the expression of EGR-1, but 5F203 was ineffective at increasing the expression of EGR-1. Similar results were observed in MCF-7 cells (data not shown). These results indicate that 5F203 does not increase NAG-1 by altering EGR-1 expression.
|
and phosphorylated GSK-3, AKT, and phosphorylated AKT expression (Fig. 2C). However, we did not observe changes in the expression of these proteins after treatment with 5F203. Thus, 5F203 did not seem to alter the expression of NAG-1 by established transcriptional mechanisms examined, but we cannot rule out other transcriptional mechanisms. 5F203 Stimulates ERK Phosphorylation. The effect of 5F203 on the phosphorylation of several regulatory proteins was examined in HCT-116 and MCF-7 cells. 5F203 did not alter p38 expression or p38 phosphorylation (data not shown), but it did alter the phosphorylation of ERK1/2 in HCT-116 cells (Fig. 3A). The expression of total ERK1/2 was not altered by 5F203. We next determined whether the expression of NAG-1 was dependent on ERK1/2 phosphorylation. MCF-7 and HCT-116 cells were incubated with 5F203 in the presence and absence of PD98059, an inhibitor of ERK1/2 phosphorylation. As shown in Fig. 3B, the inhibitor PD98509 suppressed the 5F203 induction of NAG-1 protein. Similar results were obtained with the ERK inhibitor U0216 (data not shown).
|
Effect of 5F203 on RNA Stability. Because 5F203 apparently does not affect NAG-1 expression by the known transcriptional factors, we suspected that 5F203 may affect NAG-1 expression at the post-transcriptional level. 5F203 could act post-transcriptionally by increasing mRNA stability. MCF-7 cells were treated with 5F203 or vehicle for 24 h, and then transcription was inhibited by the addition of actinomycin. RNA was harvested at different time points after RNA transcription was halted, and levels of NAG-1 mRNA were determined by real-time RT-PCR. Incubation with 5F203 increased the half-life of NAG-1 mRNA in the MCF-7 cell line from 
18 to 54 min (Fig. 4). This increase in NAG-1 mRNA was subsequently confirmed by Northern analysis in the HCT-116 cell line (data not shown). Thus, 5F203 increased the expression of NAG-1 protein via a post-transcriptional mechanism, in turn increasing mRNA stability.
|
We next examined the effect of the ERK inhibitor on 5F203-mediated increases in the half-life of NAG-1 mRNA. Inhibition of ERK phosphorylation inhibited the 5F203-dependent increase in the half-life of mRNA from 54 to 35 min (Fig. 4), but it did not alter the mRNA stability in the absence of 5F203 (16 min). These results suggest that ERK1/2 kinase may play an important role in the 5F203-induced NAG-1 expression.
5F203 Induction of Apoptosis Is Blocked by NAG-1 siRNA. The controlled expression of NAG-1 in MCF-7 and HCT-116 cells induces apoptosis, and it may, in part, be responsible for the anticancer activity of NAG-1 as measured in mouse xenograph models (Baek et al., 2001b). To link the antitumorigenicity of 5F203 to NAG-1 proapoptotic activity, we transfected MCF-7 cells with NAG-1 siRNA or scrambled NAG-1 siRNA (vector) and then incubated cells with 100 to 500 nM 5F203 and measured apoptosis and NAG-1 expression (Fig. 5). A large induction of NAG-1 was observed at 24 h in cells transfected with the scrambled siRNA and treated with 5F203. In contrast, cells transfected with NAG-1 siRNA and then treated with 5F203 showed minimal induction of NAG-1 protein (Fig. 5A). Incubation of the scrambled siRNA-transfected cells with 5F203 increased apoptosis compared with control, DMSO-treated cells (Fig. 5B). In contrast, cells transfected with NAG-1 siRNA and then treated with 5F203 showed a significantly reduced apoptosis. These data support the conclusion that 5F203-induced apoptosis in MCF-7 cells is mediated, in part, by the increased expression of NAG-1.
|
|
|
Discussion |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
superfamily (Baek et al., 2002a,b, 2004a, 2005; Bottone et al., 2002; Wilson et al., 2003; Lee et al., 2005). Considerable evidence obtained from in vitro experiments and biochemical data indicated that this protein has a proapoptotic activity. The antitumor activity is based solely on the inhibition of tumor growth of human colorectal cells in a nude mouse xenograph model (Baek et al., 2001b). In this report, we have confirmed the antitumor activity of NAG-1 and extended these findings to inhibition of mammary tumors formed from human breast cancer cells. The pharmacological agent 5F203 has well documented anticancer activity and has lead to the development of the prodrug Phortress that is entering phase 1 clinical trials in the United Kingdom (Bradshaw et al., 2002). Of the drugs and chemicals this laboratory has so far tested in a colon carcinoma cell culture model system, 5F203 is by far the most potent drug to induce NAG-1 in vitro. Thus, 5F203 was selected as a model drug to determine whether it is possible for a drug to alter the in vivo expression of NAG-1 and then to test for a correlation of NAG-1 expression with reduction of tumor growth. The mechanisms responsible for the anticancer activity of 5F203 are not fully understood, and most likely several possible mechanisms are responsible for the activity.
Induction of NAG-1 protein by 5F203 has been confirmed in several cell lines, including the MCF-7 breast cancer cell line, which has been used to establish the anticancer activity of 5F203 in mouse models of cancer (Bradshaw et al., 2002; Monks et al., 2003). 5F203 inhibits cell growth of MCF-7 cells and stimulates apoptosis of MCF-7 cells in culture. The controlled expression of NAG-1 in these cells stimulates apoptosis in vitro, and MCF-7 cells engineered to express NAG-1 form smaller tumors than the vector control cells. In support of the role of NAG-1 as a key proapoptotic stimuli induced by 5F203, siRNA for NAG-1 blocks the transcription of NAG-1 and leads to inhibition of 5F203-induced apoptosis and NAG-1 protein expression. Furthermore, the correlation of NAG-1 expression with the reduction of tumor growth by 5F203 treatment is supported by studies with MDA-435 cells. Mammary tumors derived from MDA-MB-435 cells in the mouse orthotopic model do not respond to treatment with the prodrug Phortress (Bradshaw and Westwell, 2004), nor does 5F203 cause an increase in NAG-1 expression in MDA-435 cells. These data indicate that the expression of NAG-1 is an important component in the antitumorigenic activity of 5F203.
Treatment of mice with the prodrug Phortress inhibited growth of MCF-7-derived tumors in the mouse mammary breast cancer model and increased the in situ expression of NAG-1 RNA in the tumors. In addition, there seems to be an inverse relationship between tumor size and in vivo NAG-1 expression. Treatment of mice with 5F203 increased the expression of NAG-1 in situ and inhibited tumor growth. The results clearly support the hypothesis that the antitumorigenic effect of 5F203 is mediated, in part, by the induction of NAG-1. Modlich et al. (2004) recently examined gene expression in biopsies from primary breast cancer before and after neoadjuvant chemotherapy, which is associated with improved survival. Two genes p21 and NAG-1 were up-regulated most prominently in the post-treatment biopsy samples. Thus, an increase in the expression of NAG-1 is observed after drug treatment in both a mouse tumor model and in patients with primary cancer. These findings raise the possibility for the development of other chemopreventive or anticancer chemicals that act by increasing the expression of NAG-1 in the tumor.
Regulation of NAG-1 seems to be complex, involving both transcriptional and post-transcriptional mechanisms. NAG-1 is regulated via a number of different cis- and trans-acting promoter elements present in the promoter sequence, and most of the chemicals and drugs we have studied increase NAG-1 by transcriptional mechanisms. In contrast, 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid, a retinoid X receptor agonist, increased NAG-1 expression by a post-transcriptional mechanism that altered NAG-1 mRNA stability, and for this retinoid the increase in mRNA stability was regulated independently of ERK1/2 (Newman et al., 2003). Although we cannot rule out a transcriptional mechanism without additional data, we show here that the increased NAG-1 protein expression from 5F203 treatment is mediated by increasing the stability of NAG-1 mRNA. In contrast to 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid, the increased stability is partially dependent on ERK1/2. 5F203-induced ERK1/2 phosphorylation and NAG-1 expression can be effectively inhibited by an ERK1/2 inhibitor. The ERK pathway is also linked to post-transcriptional regulation. Indeed, the ERK pathway controls tumor necrosis factor-
, interleukins, and -adrenergic receptor expressions at the post-transcriptional levels (Rutault et al., 2001). The 3'-untranslated region of NAG-1 mRNA contains four AU-rich elements that could determine the stability of NAG-1 mRNA. Other evidence points to the mitogen-activated protein kinase pathway as a regulator of mRNA stability (Headley et al., 2004). The suppression of 5F203 induction of NAG-1 by cycloheximide suggests the de novo synthesis of proteins is responsible for the stabilization of the mRNA. This finding was unexpected and points to a novel mechanism for the regulation of NAG-1 by this drug. The results illustrate the complex nature of the regulation of NAG-1 expression and a mechanism that is not fully understood.
In summary, we have presented evidence demonstrating that the expression of NAG-1 attenuates tumor growth, supporting NAG-1 as an important mediator for the activity of 5F203, an antitumorigenic drug in clinical trials for cancer. 5F203 increases the expression by a novel post-transcriptional mechanism that mediates an increase in the stability of NAG-1 mRNA, resulting in an increase in protein expression. The in vivo expression of NAG-1 is increased in the mammary tumors by drug treatment, and this increased expression correlates with reduced mammary tumor growth. We propose that NAG-1 is a key modulator in tumor suppression by a number of chemopreventive and antitumorigenic chemicals and may provide a common pathway to explain the anticancer activity of these structurally and chemically diverse xenobiotics.
|
Acknowledgements |
|---|
|
Footnotes |
|---|
ABBREVIATIONS: Cox, cyclooxygenase; NAG-1, nonsteroidal anti-inflammatory drug-activated gene; TGF, transforming growth factor; MIC, macrophage inhibitory cytokine; PDF, prostate-derived factor; EGR, early growth response; AKT, protein kinase B; GSK, glycogen synthase kinase; 5F203, 2-(4-amino-3-methylphenyl)-5-fluorobenzothiazole; RT-PCR, reverse transcription-polymerase chain reaction; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; DMSO, dimethyl sulfoxide; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; ERK, extracellular signal-regulated kinase; siRNA, small interfering RNA; PD98059, 2'-amino-3'-methoxyflavone; U0126, 1,4-diamino-2,3-dicyano-1,4-bis(methylthio)butadiene; ANOVA, analysis of variance.
Address correspondence to: Dr. Thomas E. Eling, Laboratory of Molecular Carcinogenesis, 111 T.W. Alexander Dr., P.O. Box 12233, MD E4-09, Research Triangle Park, NC 27709. E-mail: eling{at}niehs.nih.gov
|
References |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Baek SJ and Eling TE (2006) Changes in gene expression contribute to cancer prevention by COX inhibitors. Prog Lipid Res 45: 1-16.[CrossRef][Medline]
Baek SJ, Horowitz JM, and Eling TE (2001a) Molecular cloning and characterization of human nonsteroidal anti-inflammatory drug-activated gene promoter. Basal transcription is mediated by Sp1 and Sp3. J Biol Chem 276: 33384-33392.
Baek SJ, Kim JS, Jackson FR, Eling TE, McEntee MF, and Lee SH (2004a) Epicatechin gallate-induced expression of NAG-1 is associated with growth inhibition and apoptosis in colon cancer cells. Carcinogenesis 25: 2425-2432.
Baek SJ, Kim JS, Moore SM, Lee SH, Martinez J, and Eling TE (2005) Cyclooxygenase inhibitors induce the expression of the tumor suppressor gene EGR-1, which results in the up-regulation of NAG-1, an antitumorigenic protein. Mol Pharmacol 67: 356-364.
Baek SJ, Kim JS, Nixon JB, DiAugustine RP, and Eling TE (2004b) Expression of NAG-1, a transforming growth factor-beta superfamily member, by troglitazone requires the early growth response gene EGR-1. J Biol Chem 279: 6883-6892.
Baek SJ, Kim KS, Nixon JB, Wilson LC, and Eling TE (2001b) Cyclooxygenase inhibitors regulate the expression of a TGF- superfamily member that has proapoptotic and antitumorigenic activities. Mol Pharmacol 59: 901-908.
Baek SJ, Wilson LC, and Eling TE (2002a) Resveratrol enhances the expression of non-steroidal anti-inflammatory drug-activated gene (NAG-1) by increasing the expression of p53. Carcinogenesis 23: 425-432.
Baek SJ, Wilson LC, Lee CH, and Eling TE (2002b) Dual function of nonsteroidal anti-inflammatory drugs (NSAIDs): inhibition of cyclooxygenase and induction of NSAID-activated gene. J Pharmacol Exp Ther 301: 1126-1131.
Bottone FG Jr, Baek SJ, Nixon JB, and Eling TE (2002) Diallyl disulfide (DADS) induces the antitumorigenic NSAID-activated gene (NAG-1) by a p53-dependent mechanism in human colorectal HCT 116 cells. J Nutr 132: 773-778.
Bradshaw TD, Bibby MC, Double JA, Fichtner I, Cooper PA, Alley MC, Donohue S, Stinson SF, Tomaszewjski JE, Sausville EA, et al. (2002) Preclinical evaluation of amino acid prodrugs of novel antitumor 2-(4-amino-3-methylphenyl)benzothiazoles. Mol Cancer Ther 1: 239-246.
Bradshaw TD and Westwell AD (2004) The development of the antitumour benzothiazole prodrug, Phortress, as a clinical candidate. Curr Med Chem 11: 1009-1021.[CrossRef][Medline]
Headley VV, Tanveer R, Greene SM, Zweifach A, and Port JD (2004) Reciprocal regulation of beta-adrenergic receptor mRNA stability by mitogen activated protein kinase activation and inhibition. Mol Cell Biochem 258: 109-119.[CrossRef][Medline]
Lee SH, Kim JS, Yamaguchi K, Eling TE, and Baek SJ (2005) Indole-3-carbinol and 3,3'-diindolylmethane induce expression of NAG-1 in a p53-independent manner. Biochem Biophys Res Commun 328: 63-69.[CrossRef][Medline]
Li PX, Wong J, Ayed A, Ngo D, Brade AM, Arrowsmith C, Austin RC, and Klamut HJ (2000) Placental transforming growth factor-beta is a downstream mediator of the growth arrest and apoptotic response of tumor cells to DNA damage and p53 overexpression. J Biol Chem 275: 20127-20135.
Modlich O, Prisack HB, Munnes M, Audretsch W, and Bojar H (2004) Immediate gene expression changes after the first course of neoadjuvant chemotherapy in patients with primary breast cancer disease. Clin Cancer Res 10: 6418-6431.
Monks A, Harris E, Hose C, Connelly J, and Sausville EA (2003) 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 63: 766-772.
Newman D, Sakaue M, Koo JS, Kim KS, Baek SJ, Eling T, and Jetten AM (2003) Differential regulation of nonsteroidal anti-inflammatory drug-activated gene in normal human tracheobronchial epithelial and lung carcinoma cells by retinoids. Mol Pharmacol 63: 557-564.
Rutault K, Hazzalin CA, and Mahadevan LC (2001) Combinations of ERK and p38 MAPK inhibitors ablate tumor necrosis factor-alpha (TNF-) mRNA induction. Evidence for selective destabilization of TNF- transcripts. J Biol Chem 276: 6666-6674.
Wilson LC, Baek SJ, Call A, and Eling TE (2003) Nonsteroidal anti-inflammatory drug-activated gene (NAG-1) is induced by genistein through the expression of p53 in colorectal cancer cells. Int J Cancer 105: 747-753.[CrossRef][Medline]
Yamaguchi K, Lee SH, Eling TE, and Baek SJ (2004) Identification of nonsteroidal anti-inflammatory drug-activated gene (NAG-1) as a novel downstream target of phosphatidylinositol 3-kinase/AKT/GSK-3 pathway. J Biol Chem 279: 49617-49623.
This article has been cited by other articles:
|
|
 |
|
  M. Shim and T. E. Eling Vitamin E succinate induces NAG-1 expression in a p38 kinase-dependent mechanism Mol. Cancer Ther., April 1, 2008; 7(4): 961 - 971. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y.-L. Chen, P.-C. Lin, S.-P. Chen, C.-C. Lin, N.-M. Tsai, Y.-L. Cheng, W.-L. Chang, S.-Z. Lin, and H.-J. Harn Activation of Nonsteroidal Anti-Inflammatory Drug-Activated Gene-1 via Extracellular Signal-Regulated Kinase 1/2 Mitogen-Activated Protein Kinase Revealed a Isochaihulactone-Triggered Apoptotic Pathway in Human Lung Cancer A549 Cells J. Pharmacol. Exp. Ther., November 1, 2007; 323(2): 746 - 756. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C.-Y. Huang, T. M. Beer, C. S. Higano, L. D. True, R. Vessella, P. H. Lange, M. Garzotto, and P. S. Nelson Molecular Alterations in Prostate Carcinomas that Associate with In vivo Exposure to Chemotherapy: Identification of a Cytoprotective Mechanism Involving Growth Differentiation Factor 15 Clin. Cancer Res., October 1, 2007; 13(19): 5825 - 5833. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. S. Selander, D. A. Brown, G. B. Sequeiros, M. Hunter, R. Desmond, T. Parpala, J. Risteli, S. N. Breit, and A. Jukkola-Vuorinen Serum Macrophage Inhibitory Cytokine-1 Concentrations Correlate with the Presence of Prostate Cancer Bone Metastases Cancer Epidemiol. Biomarkers Prev., March 1, 2007; 16(3): 532 - 537. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
 |
|
 |
 |
 |
