trans-3,4,5 -Trihydroxystibene Inhibits Hypoxia-Inducible Factor 1 and Vascular Endothelial Growth Factor Expression in Human Ovarian Cancer Cells

trans-3,4,5 -Trihydroxystibene (resveratrol) is a natural product commonly found in the human diet and has been shown recently to have anticancer effects on various human cancer cells. However, the molecular basis for its anticancer action remains to be elucidated. In this study, we investigated the effect of resveratrol on hypoxia-inducible factor 1 (HIF-1 ) and vascular endothelial growth factor (VEGF) expression in human ovarian cancer cells A2780/CP70 and OVCAR-3. We found that although resveratrol did not affect HIF-1 mRNA levels, it did dramatically inhibit both basal-level and growth factor-induced HIF-1 protein expression in the cells. Resveratrol also greatly inhibited VEGF expression. Mechanistically, we demonstrated that resveratrol inhibited HIF-1 and VEGF expression through multiple mechanisms. First, resveratrol inhibited AKT and mitogen-activated protein kinase activation, which played a partial role in the down-regulation of HIF-1 expression. Second, resveratrol inhibited insulin-like growth factor 1induced HIF-1 expression through the inhibition of protein translational regulators, including Mr 70,000 ribosomal protein S6 kinase 1, S6 ribosomal protein, eukaryotic initiation factor 4E-binding protein 1, and eukaryotic initiation factor 4E. Finally, we showed that resveratrol substantially induced HIF-1 protein degradation through the proteasome pathway. Our data suggested that resveratrol may inhibit human ovarian cancer progression and angiogenesis by inhibiting HIF-1 and VEGF expression and thus provide a novel potential mechanism for the anticancer action of resveratrol. INTRODUCTION trans-3,4,5 -Trihydroxystibene (resveratrol), a polyphenolic, was identified originally as a phytoalexin produced by plants in response to injury, UV irradiation, and insect or fungal attack (1). Resveratrol is present in more than 70 plant species and is especially abundant in food products such as grapes, peanuts, and mulberries (2). During the past few years, resveratrol has attracted considerable attention as one of the most promising cancer chemopreventive agents. Resveratrol was shown to affect diverse cellular events associated with each step of carcinogenesis, i.e., tumor initiation, promotion, and progression (2). At the molecular level, these effects corresponded with the inhibition of free radical formation, cyclooxygenase, and cytochrome P450 activity, as well as the inhibition of protein kinase C activity (3, 4). Additionally, resveratrol inhibited cell proliferation by decreasing DNA synthesis through its inhibitory effects on ribonucleotide reductase, DNA polymerase, and ornithine decarboxylase activities (3, 4). Resveratrol induces apoptosis in various malignant cells through multiple mechanisms, such as up-regulation of CD95L expression, enhancement of p53 expression and activity, induction of B-cell CLL/lymphoma 2-associated X protein expression, suppression of B-cell CLL/ lymphoma 2 expression, and inhibition of nuclear factor B activity (3, 4). Therefore, resveratrol possesses therapeutic potential based on its suppression of tumor cell growth by inducing cell cycle arrest and apoptosis. For example, in a rat ascetic hepatoma model, i.p. administration of resveratrol caused apoptosis in the tumor cell population and significantly decreased tumor cell numbers (5). Despite these findings, however, the molecular mechanisms by which resveratrol exerts its anticancer effects remain largely unknown. Ovarian cancer represents the fourth leading cause of cancer-related death for women and is the most common cause of death from gynecologic cancer in the Western world (6). The overall 5-year survival rate of ovarian cancer is 50% and about 30% for advanced stage disease (7). The symptoms of the disease are observed only after it has spread to the surfaces of the peritoneal cavity. At this stage, it is impossible to remove all apparent lesions by surgical operations, and this accounts for the high rate of cancer recurrence after surgery. Consequently, the majority of ovarian cancer patients require chemotherapy. However, the major challenge in ovarian cancer treatment is the broad resistance to available chemotherapeutic drugs (6). The combination of cisplatin and paclitaxel as a chemotherapy regimen has improved the survival rate of ovarian cancer patients (8), but in the majority of cases, the cancer ultimately progresses, and the ovarian cancer patient dies from chemotherapy-refractory cancer (6). It has been well established that solid tumor growth is angiogenesis dependent (9). Advanced solid tumors have a characteristic property of intratumoral hypoxia, which is caused Received 11/14/03; revised 4/27/04; accepted 5/6/04. Grant support: NIH Grant RR16440 and American Cancer Society Research Scholar Grant 04-076-01-TBE (B. H. Jiang). 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. Requests for reprints: Bing-Hua Jiang, Mary Babb Randolph Cancer Center, Department of Microbiology, Immunology and Cell Biology, West Virginia University, Morgantown, WV 26506-9300. Fax: (304) 293-4667; E-mail: [email protected]. 5253 Vol. 10, 5253–5263, August 1, 2004 Clinical Cancer Research Cancer Research. on October 23, 2017. © 2004 American Association for clincancerres.aacrjournals.org Downloaded from by the structural and functional abnormalities of the tumor microvasculature, rapid expansion of tumor mass, and tumorassociated anemia (10). Hypoxia condition is a strong stimulus for angiogenesis, and this is predominately accomplished by hypoxia-inducible factor 1 (HIF-1)-mediated up-regulation of vascular endothelial growth factor (VEGF) expression (11–13). VEGF, also known as the vascular permeability factor, is a potent and endothelial cell-specific mitogen that plays a crucial role during the process of tumor angiogenesis. VEGF expression is elevated in many human cancers, including ovarian carcinoma (14, 15). HIF-1 is a heterodimeric transcriptional factor composed of HIF-1 and HIF-1 subunits (16). HIF-1 binds to the hypoxia-responsive element in the promoter region of the VEGF gene and up-regulates VEGF expression (16). HIF-1 -deficient cells have reduced VEGF production under hypoxia (17, 18). VEGF expression levels in vivo are also much lower in HIF-1 null tumors (17). HIF-1 expression increases dramatically under hypoxia. However, under normoxic conditions, HIF-1 protein is expressed at a very low level due to rapid degradation via the ubiqitin-proteasomal pathway. Certain oncogenic proteins and growth factors have been shown to up-regulate HIF-1 expression in normoxic cells (19–23). HIF-1 was also shown to be elevated in various human tumors, including ovarian cancer (24). The effect of VEGF on vascular permeability has been implicated in the pathogenesis of ovarian cysts and malignant ascites (25, 26). In addition, increased levels of VEGF expression and the microvessel density in ovarian cancer directly correlate with poor prognosis (27, 28). Therefore, an anti-angiogenic therapy that targets the HIF-1 /VEGF system would be a rational strategy for the treatment of ovarian cancer. In this study, we have demonstrated for the first time that resveratrol has a strong inhibitory effect on HIF-1 and VEGF expression in human ovarian cancer cells. Our data showed that resveratrol did not affect HIF-1 mRNA levels; rather, it interfered with the protein translational machinery and promoted HIF-1 protein degradation. These unique actions of resveratrol provide important clues to the molecular basis for its anticancer effects. MATERIALS AND METHODS Cell Culture and Reagents. A2780/CP70 and OVCAR-3 human ovarian cancer cells were cultured in RPMI 1640 (Life Technologies, Inc., Grand Island, NY), supplemented with 10% fetal bovine serum, 50 nM insulin (Sigma), 100 units/ml penicillin, and 100 g/ml streptomycin. The cells were maintained at 37°C and 5% CO2 in a humid environment. Resveratrol was purchased from ICN Biomedicals, Inc. (Aurora, OH). Cycloheximide was obtained from EMD Biosciences, Inc. (San Diego, CA). Recombinant human insulin and insulin-like growth factor 1 (IGF-1) were obtained from Sigma. LY294002, rapamycin, and PD98059 were purchased from Calbiochem (La Jolla, CA). Monoclonal HIF-1 antibody was obtained from BD Transduction Laboratories (Lexington, KY). Antibodies specific for phosphorylated (Thr-202/Tyr-204) or total p44/p42 mitogenactivated protein kinase (MAPK) were from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies against phosphorylated (Ser-473) or total AKT, phosphorylated (Thr-421/Ser-424) or total Mr 70,000 ribosomal protein S6 kinase 1 (p70S6K1), phosphorylated (Ser-235/236) S6 ribosomal protein, phosphorylated (Ser-65) eukaryotic initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1), and phosphorylated (Ser-209) eIF4E were obtained from Cell Signaling Technology (Beverly, MA). Antiglyceraldehyde-3-phosphate dehydrogenase (GAPDH) monoclonal antibody was from R&D Systems (Minneapolis, MN). Treatment of the Cells with Resveratrol. Exponentially growing cells (about 80% confluence) were treated with resveratrol at 12.5, 25, 37.5, 50, 75, 100, and 150 M for 6 h in complete medium. For time-dependent studies, cells were treated with 50 M resveratrol from 0 to 24 h. The control cells were incubated with the highest amount of solvent (DMSO) used for dissolving corresponding doses of resveratrol in the dose-dependent studies. For experiments in which cells received growth factor stimulation, cells were starved in serum-free and insulin-free medium overnight and then pretreated with resveratrol for 30 min, followed by incubation with growth factors for 6 h. Western Blotting. Cells were washed with ice-cold PBS [140 mM NaCl, 3 mM KCl, 6 mM Na2HPO4, and 1 mM KH2PO4 (pH 7.4)], scrapped, and pelleted by centrifugation. Whole-cell extracts were prepared using modified radioimmune precipitation buffer [100 mM Tris, 5 mM EDTA, 1% Triton X-100, 1% deoxycholate acid, 0.1% SDS, 2 mM phenylmethylsulfonyl fluoride, 1 mM sodium orthovanadate, 2 mM DTT, 20 g/ml leupeptin, and 20 g/ml pepstatin (pH 7.4)]. Protein concentrations of the lysates were assayed using a protein assay reagent (Bio-Rad). Aliquots (50 g) of protein samples were fractionated by 8% SDS-PAGE, transferred to a nitrocellulose membrane (Schleicher & Schuell Biosciences, Keene, NH), and subjected to immunoblotting analysis. Monoclonal HIF-1 antibody was used at a dilution of 1:3,000 in blocking buffer [1 Tris-buffered saline plus Tween 20: 20 mM Tris (pH 7.4), 137 mM NaCl, and 0.1% Tween 20] containing 5% nonfat dry milk. Anti-GAPDH antibody was used at a dilution of 1:10,000. All other polyclonal antibodies were diluted at 1:2,000 in 1 Trisbuffered saline plus Tween 20 containing 5% BSA. The blots were blocked in 1 Tris-buffered saline plus Tween 20 containing 5% nonfat dry milk for 2 h at room temperature, followed by incubation with the appropriately diluted primary antibodies overnight at 4°C. Immunoreactivity was visualized with appropriate horseradish peroxidase-conjugated secondary antibodies and enhanced chemiluminescence (Perkin-Elmer Life Sciences, Boston, MA). Northern Blotting. Total cellular RNA was extracted from the cells using Trizol reagent (Invitrogen) according to the manufacturer’s instructions. Aliquots (15 g) of total RNA were fractionated by electrophoresis in 1% agarose gel with 2.2 M formaldehyde, transferred to a Nytron supercharge membrane (Schleicher & Schuell) by capillary transfer with a downward transfer system (Schleicher & Schuell), and cross-linked to the membrane by UV irradiation. The blot was prehybridized for 1 h at 42°C in 10 ml of Ultrahyb buffer (Ambion). Human VEGF, HIF-1 , and -actin cDNA probes were labeled with [ -P]dATP by random priming using the RadPrime DNA labeling system (Invitrogen) and purified with the ProbeQuant G-50 Micro Columns (Amersham Biosciences, Piscataway, NJ). Heat-denatured probes were added to the hybridization buffer to a final concentration of 1 10 cpm/ml, and hybridization was 5254 Resveratrol Inhibits HIF-1 and VEGF Expression Cancer Research. on October 23, 2017. © 2004 American Association for clincancerres.aacrjournals.org Downloaded from continued overnight at 42°C. The membrane was washed twice for 15 min in 2 SSC/0.1% SDS at 42°C, and 2 15 min in 0.1 SSC/0.1% SDS at 60°C. The membrane was wrapped and overlaid with a Kodak Biomax MS film, and an intensifying screen. Autoradiography was performed overnight at 80°C. Enzyme-Linked Immunosorbent Assay. The levels of VEGF protein secreted by the cells in the medium were determined by a VEGF ELISA kit (R&D Systems). In brief, subconfluent cells were changed into fresh medium in the presence of solvent or various concentrations of resveratrol for 12 h, or the cells were cultured in serum-free and insulin-free medium overnight, followed by incubation with IGF-1 in the absence or presence of various concentrations of resveratrol for 12 h. The medium was collected, and VEGF protein concentrations were measured by ELISA according to the manufacturer’s instructions. The results were normalized to the number of cells per plate. The data were presented as mean SD from three replicate experiments. Transient Transfection and Luciferase Reporter Assays. The VEGF promoter reporter was constructed by inserting 47 bp of human VEGF promoter 5 -flanking sequence between 985 and 939, which contains the HIF-1 binding site, into the pGL2-basic luciferase vector (Promega) as described previously (11). The dominant-negative HIF-1 expressing plasmid was described previously (11). The cells were cotransfected with the reporter, pCMV-gal plasmid, and a dominant-negative or wild-type HIF-1 -expressing plasmid using LipofectAMINE reagent (Invitrogen). An empty vector plasmid was used to adjust the equal amounts of plasmids used in each experiment. The cells were cultured overnight after transfection. The cells were then treated with resveratrol for 12 h. Luciferase activity was measured using a luciferase assay reagent (Promega) and normalized to -galactosidase activity. The data were mean SD from three replicate experiments. Cell Viability Assays. Cell viability was assayed by the trypan blue dye exclusion method. A2780/CP70 and OVCAR-3 cells were seeded into 6-well plate at a density of 1 10/well. The cells were treated with 50 or 100 M resveratrol for 12 h and then trypsinized and resuspended. A 1:1 dilution of the cell suspension using 0.4% trypan blue was loaded into the counting chambers of a hemocytometer, and the number of stained cells and the total number of cells were counted. Cell viability was the percentage of unstained cells. The data were mean SD from three replicate experiments. Statistical Analysis. When applicable, the data were analyzed by Student’s t test using SPSS statistical software (SPSS, Inc., Chicago, IL).