p21 Expression by Curcumin in U-87MG Human Glioma Cells: Role of Early Growth Response-1 Expression

Curcumin, a natural compound, is a well-known chemopreventive agent with potent anticarcinogenic activity in a wide variety of tumor cells. Curcumin inhibits cancer cell proliferation in part by suppressing cyclin D1 and inducing expression of the cyclin-dependent kinase inhibitor p21. Both p53-dependent and p53-independent mechanisms regulate p21 expression, but the mechanism by which curcumin regulates p21 expression remains unknown. Here, we report that transcription of the p21 gene is activated by early growth response-1 (Egr-1) independently of p53 in response to curcumin treatment in U-87MG human glioblastoma cells. Egr-1 is a transcription factor that helps regulate differentiation, growth, and apoptosis in many cell types. Egr-1 expression is induced by curcumin through extracellular signal-regulated kinase (ERK) and c-Jun NH2terminal kinase (JNK), but not the p38, mitogen-activated protein kinase (MAPK) pathways, which mediate the transactivation of Elk-1. Transient expression of Egr-1 enhanced curcumin-induced p21 promoter activity, whereas suppression of Egr-1 expression by small interfering RNA abrogated the ability of curcumin to induce p21 promoter activity. In addition, stable knockdown of Egr-1 expression in U-87MG cells suppressed curcumin-induced p21 expression. Our results indicate that ERK and JNK MAPK/Elk1/Egr-1 signal cascade is required for p53-independent transcriptional activation of p21 in response to curcumin in U-87MG human glioblastoma cells. [Cancer Res 2008;68(5):1369–77] Introduction The p21 protein (hereafter, p21) is a well-characterized cell cycle regulator that inhibits the activity of cyclin/cyclindependent kinase (cdk)2 complexes (1). It inhibits cell cycle progression through the transactivation of p53 when cells are exposed to DNA-damaging agents, such as doxorubicin and girradiation (2). In addition to playing a role in DNA damage responses, p21 also plays a crucial role in differentiation, senescence, and apoptosis. Although p21 expression was initially identified as being p53dependent, a variety of transcription factors, including SP1/SP3, Smads, AP2, p150(Sal2), the vertebrate homologue of the Drosophila melanogaster homeotic transcription factor Spalt, sterol regulatory element-binding protein–1a, STATs, ets-related transcription factor E1AF, AP2, CAAT/enhancer binding protein a/h, Ets-1, and hepatocyte nuclear factor-4a, bind to specific cis-acting elements in the p21 promoter to activate transcription of the gene via a p53-independent mechanism (3–12). In addition, the region between 58 and 51 bp from the p21 transcription initiation site contains consensus early growth response-1 (Egr-1) binding sequences (EBS), which play a major role in regulating p21 transcription in response to resveratrol treatment in K562 cells (13) and to tamoxifen in MDA-MB-361 breast cancer cells (14). Egr-1, also known as nerve growth factor I-A, zif268, krox24, and Tis8, is an immediate early-response gene induced by stress, injury, mitogens, and differentiation (15). Egr-1 regulates expression of genes involved in the control of growth and apoptosis by transactivating p21, p53, PTEN, transforming growth factor h1, fibronectin, and Gadd45 (13–17). Significantly reduced Egr-1 expression has been observed during tumor formation in various mammalian cells and tissues (18). On the other hand, ectopic expression of Egr-1 inhibits cell proliferation and soft agar growth of NIH3T3 cells transformed with v-sis , suggesting that Egr-1 functions as a tumor suppressor (19). Curcumin (diferuloylmethane) is a natural compound with potent antioxidant, antiinflammatory, anticarcinogenic, and chemopreventive activities in a variety of cancer cell types (20). Curcumin-induced suppression of cellular proliferation is mediated at least in part by blocking cell cycle progression at G1. This arrest may result from the down-regulation of cyclins D1 (21) and E (22), as well as up-regulation of cell cycle inhibitors such as p21 and p27 (22, 23) in multiple human tumor cell lines. Curcumin-induced down-regulation of cyclin D1 is caused by inhibition of nuclear factor-nB (NF-nB) activity through the suppression of InB kinase (IKK) and activation of Akt (22) because the promoter of cyclin D1 is regulated by NF-nB (24). However, the molecular mechanism underlying the curcumin-induced up-regulation of p21 remains unanswered. In this study, we assessed the mechanism by which curcumin stimulates transcriptional activation of p21 in U-87MG cells. We show for the first time that the induction of p21 by curcumin is mediated by transactivation of Egr-1 independently of p53. We also show that curcumin activates the extracellular signal-regulated kinase (ERK) and c-Jun NH2-terminal kinase (JNK) mitogenactivated protein kinase (MAPK) pathways, which activate the transcription factor Elk-1. This activation leads to up-regulation of Egr-1, which results in the transcriptional activation of the p21 Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). Requests for reprints: Young Han Lee and Soon Young Shin, Department of Biomedical Science and Technology, Research Center for Transcription Control, Institute of Biomedical Science and Technology, Konkuk University, 1 Hwayang-dong, Kwangjin-gu, Seoul 143-701, South Korea. Phone: 82-2-2049-6115; Fax: 82-2-3437-9781; E-mail: [email protected] and [email protected]. I2008 American Association for Cancer Research. doi:10.1158/0008-5472.CAN-07-5222 www.aacrjournals.org 1369 Cancer Res 2008; 68: (5). March 1, 2008 Research Article Research. on June 6, 2017. © 2008 American Association for Cancer cancerres.aacrjournals.org Downloaded from promoter. Our results indicate that Egr-1 is required for p53independent transcriptional activation of p21. Materials and Methods Cell culture and reagents. Human U-87MG glioblastoma cells and rat C6glioma cells were obtained from the American Type Culture Collection and maintained in DMEM supplemented with 10% fetal bovine serum (Hyclone). Curcumin was purchased from Sigma Co. Antibodies against phospho-ERK1/2 MAPK (Thr/Tyr), phospho-JNK1/2 (Thr/Tyr), phospho-p38 MAPK (Thr/Tyr), and phosphor-Elk-1 (Ser) were obtained from Cell Signaling Technology. Antibodies against Egr-1, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and ERK2 were obtained from Santa Cruz Biotechnology. The firefly and Renilla Dual-Glo Luciferase Assay System was purchased from Promega. Construction and mutagenesis of the p21 promoter. The construction of p21-Luc( 150/+38), a p21 promoter fragment that spans nt 150 to +39 and lacks the p53 binding site, and a mutant construct, p21-Luc( 150/+38)mtEgr1, which contains a mutated EBS, has been described elsewhere (14). Plasmids. The luciferase reporter plasmid pGL-2.4, which contains 2.4 kb of the human p21 promoter, was provided by Dr. Jae-Yong Lee (Department of Biochemistry, College of Medicine, Hanlym University, Korea). Full-length [Pegr1-Luc( 780/+1)] and serial deletion mutant reporter constructs of the Egr-1 promoter expressing firefly luciferase were provided by Dr. H. Eibel (Department of Orthopedic Surgery, University of Tubingen Medical Center, Germany) and are described elsewhere (25). The plasmid pRL-null, which encodes Renilla luciferase, was purchased from Promega, whereas the p53 cis-acting reporter plasmid p53-Luc was purchased from Stratagene. Plasmids expressing dominantnegative (dn) JNK1 (pSRa/HA-JNK T183A/Y185F) and kinase-dead (kd) p38 kinase (pCDNA3/flag-p38 T180A/Y182F) were donated by Dr. D. S. Min (Department of Molecular Biology, College of Natural Science, Pusan National University, Korea). Figure 1. Curcumin arrests the cell cycle at G1. A, U-87MG cells were seeded onto 96-well culture plates and treated with various concentrations of curcumin for the indicated lengths of time. Cellular proliferation was measured using Cell Counting kit-8. Points, mean of one experiment performed in triplicate; bars, SD. Similar results were obtained from two other independent experiments. B, U-87MG cells were treated with 20 Amol/L curcumin. After the indicated times (12 h and 24 h), the cells were harvested, fixed with ethanol, and stained with propidium iodide. The DNA content was analyzed using flow cytometry. The percentages of the cell population at each phase of the cell cycle are indicated in each histogram. Similar results were obtained from three independent experiments. C, exponentially growing U-87MG cells were cultured in the absence or presence of 20 Amol/L curcumin for the indicated lengths of time. At the indicated time points, cells were collected and analyzed for protein expression using Western blotting. The same blot was reprobed with anti–a-tubulin antibody as an internal control. Each blot represents three separate experiments. D, U-87MG cells were transfected with 0.5 Ag 5 p53-Luc cis -reporting plasmid containing five repeats of the p53 binding site, along with 50 ng pRL-null vector. After 24 h, the cells were treated with doxorubicin (1 Ag/mL), curcumin (10 or 20 Amol/L), or a combination for an additional 8 h. Firefly luciferase activity was normalized to the Renilla activity. Columns, mean of three independent experiments performed in triplicate; bars, SD. Cancer Research Cancer Res 2008; 68: (5). March 1, 2008 1370 www.aacrjournals.org Research. on June 6, 2017. © 2008 American Association for Cancer cancerres.aacrjournals.org Downloaded from Cell proliferation assay. U-87MG cells were seeded onto 96-well plates (2 10 cells per well) and treated with various concentrations of curcumin for the various lengths of time. Proliferation was measured using a Cell Counting kit-8 (Dojindo Molecular Technologies) with the water-soluble tetrazolium salt WST-8 [2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt] as a substrate. Cell cycle analysis. Cellular DNA content was analyzed by flow cytometry as described previously. Briefly, U-87MG cells were collected after 12 or 24 h of exposure to 20 Amol/L curcumin, fixed in 70% ethanol, washed twice with PBS, and stained with a 50 Ag/mL propidium iodide solution containing 0.1% Triton X-100, 0.1 mmol/L EDTA, and 50 Ag/mL RNase A. Fluorescence was measured and analyzed using a FACSCalibur Flow Cytometer (Becton Dickinson Immunocytometry Systems). Western blot analysis. Cells were lysed in a buffer consisting of 20 mmol/L HEPES (pH 7.2), 1% Triton X-100, 10% glycerol, 150 mmol/L NaCl, 10 Ag/mL leupeptin, and 1 mmol/L phenylmethylsulfonyl fluoride. The protein extracts (20 Ag each) were separated by 10% SDS-PAGE and transferred to nitrocellulose filters. The blots were incubated with the corresponding primary antibodies and developed using an enhanced chemiluminescence detection system (Amersham Pharmacia Biotech). Northern blot analysis. For each sample, 10 Ag total RNA was electrophoresed on a formaldehyde/agarose gel and transferred to a Hybond N nylon membrane (Amersham Pharmacia Biotech). Northern blotting was performed with a [g-P]dCTP-labeled Egr-1 cDNA probe followed by hybridization with a GAPDH cDNA probe. Transient transfection and promoter reporter assay. U-87MG cells were seeded onto 12-well plates and transfected with 0.5 Ag p21 promoter (14) or Egr-1 promoter construct (26) using Lipofectamine 2000 (Invitrogen Life Technologies) according to the manufacturer’s instructions. To monitor the transfection efficiency, a pRL-null plasmid (50 ng) encoding Renilla luciferase was included in all transfections. Where indicated, a mammalian expression vector encoding dn-MEK1, dn-JNK1, or kd-p38 kinase was also included. At 24 h posttransfection, the cells were serum starved by culturing them for 12 h in 0.5% serum and then treated with curcumin. After 6 to 12 h, the levels of firefly and Renilla luciferase activity were measured sequentially from a single sample using the Dual-Glo Luciferase Assay System. Luminescence was measured with a luminometer (Centro LB960;