The SyntheticTriterpenoid CDDO-Imidazolide Suppresses STAT Phosphorylation and Induces Apoptosis in Myeloma and Lung Cancer Cells

Purpose: Excessive activity of the transcription factors known as signal transducers and activators of transcription (STAT) contributes to the development and progression of malignancy in many organs. It is, therefore, important to develop new drugs to control the STATs, particularly their phosphorylation state, which is required for their transcriptional activity. Experimental Design: Myeloma and lung cancer cells were treated with the new synthetic triterpenoid CDDO-Imidazolide, and STAT phosphorylation and apoptosis were evaluated by immunoblotting and fluorescence-activated cell sorting analysis. Results:We now report that CDDO-Imidazolide, previously shown to be a potent agent for control of inflammation, cell proliferation, and apoptosis, rapidly (within 30-60 minutes) and potently (at nanomolar levels) suppresses either constitutive or interleukin-6-induced STAT3 and STAT5 phosphorylation in human myeloma and lung cancer cells. Furthermore, in these cells, CDDO-Imidazolide also up-regulates critical inhibitors of STATs, such as suppressor of cytokine signaling-1and SH2-containing phosphatase-1 (a tyrosine phosphatase). Moreover, gene array studies reported here show that CDDO-Imidazolide potently regulates the transcription of important genes that are targets of the STATs. Conclusions: Our new data thus show that CDDO-Imidazolide is a potent suppressor of STAT signaling and provide a further mechanistic basis for future clinical use of this agent to control inflammation or cell proliferation. Studies on the role of the signal transducer and activator of transcription (STAT) family of transcription factors in carcinogenesis and on the development of novel inhibitors of the STAT pathway for both prevention and treatment of cancer are evoking increasing interest (1–3). Most notably, it is clear that the STAT transcription factors, particularly STAT3 and STAT5, are frequently overactivated in a wide variety of cancers (2, 4–7), including many hematogenous malignancies (such as leukemia, lymphoma, and multiple myeloma), and in many carcinomas at most major target sites (such as lung, breast, prostate, pancreas, ovary, and head and neck). In this context, it becomes increasingly important to discover small molecules capable of directly inhibiting the activity of both STAT3 and STAT5 (2, 3, 8, 9). STAT3 and STAT5 become active transcription factors upon their phosphorylation by a unique set of Janus-activated kinases (JAK; refs. 2, 10, 11). Their functional activity is regulated not only by these kinases but also by a set of inhibitory proteins (2, 10, 11), which either bind to activated STATs (such as protein inhibitors of activated STATs), bind to the JAKs [such as suppressor of cytokine signaling (SOCS)], or dephosphorylate activated STATs [protein tyrosine phosphatases, such as SH2-containing phosphatases (SHP)]. We now report here for the first time that a new synthetic triterpenoid, CDDO-Imidazolide, is a potent agent (active at nanomolar concentrations) for suppression of the phosphorylated state of either STAT3 or STAT5. These new results are a mechanistic extension of previous studies, in which it was first shown that CDDO-Imidazolide has marked anti-inflammatory and antiproliferative activity, both in cell culture and in vivo (12). The biological activity of CDDO-Imidazolide had originally been identified (13) by its ability to block the ability of IFN-g, a known activator of STATs (14), to stimulate the de novo production of inducible nitric oxide synthase; subsequent studies then showed that CDDO-Imidazolide had marked antiproliferative and proapoptotic activity on a variety of human and murine tumor cells (15–19). The suppression of STAT activity, shown here for the first time, is likely an important mechanism contributing to these activities of CDDO-Imidazolide. Cancer Therapy: Preclinical Authors’ Affiliations: Dartmouth Medical School and Dartmouth College, Hanover, New Hampshire; National Cancer Institute, Bethesda, Maryland; and CaseWestern Reserve University School of Medicine, Cleveland, Ohio Received1/30/06; revised 4/13/06; accepted 4/20/06. Grant support: National Foundation for Cancer Research and NIH grant RO1 CA78814 (M.B. Sporn). 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 with18 U.S.C. Section1734 solely to indicate this fact. Note: K. Liby and N.Voong contributed equally to this work. M.B. Sporn and J.J. Letterio are co-senior authors. Requests for reprints: John J. Letterio, Division of Pediatric Hematology Oncology, Rainbow Babies and Children’s Hospital, 11100 Euclid Avenue, Cleveland OH 44106. Fax: 216-844-5431; E-mail: John.Letterio@uhhs.com. F2006 American Association for Cancer Research. doi:10.1158/1078-0432.CCR-06-0215 www.aacrjournals. org Clin Cancer Res 2006;12(14) July15, 2006 4288 Cancer Research. on December 4, 2021. © 2006 American Association for clincancerres.aacrjournals. org Downloaded from Materials andMethods Synthesis of CDDO-Imidazolide (1-[2-cyano-3,12-dioxooleana-1,9dien-28-oyl] imidazole). A brief report of the synthesis of CDDOImidazolide has been published (13), and the structure of CDDOImidazolide is shown in ref. (12). Full details are as follows: Oxalyl chloride was added dropwise to a solution of CDDO (ref. 20; 3 g, 6.1 mmol) in anhydrous methylene chloride (50 mL) under nitrogen atmosphere at room temperature. The mixture was stirred at room temperature overnight. The reaction mixture was evaporated in vacuo to give a residue, which was dissolved in benzene (20 mL). This solution was evaporated in vacuo to remove excess oxalyl chloride; this procedure was repeated twice. The resultant residue was triturated with a small amount of methylene chloride to give crystals. The methylene chloride was removed in vacuo to give CDDO acid chloride (3.1 g; yield, 100%). For conversion of this acid chloride to CDDO-imidazolide, 400 mg (0.78 mmol), was dissolved in anhydrous benzene (2.7 mL), and imidazole (109 mg, 1.6 mmol, 2.1 equivalents) was added under nitrogen at room temperature. The mixture was vigorously stirred at room temperature under nitrogen overnight. The reaction mixture was diluted with ethyl acetate (30 mL), washed with water (15 mL, twice) and a saturated sodium chloride solution (15 mL, once), dried over MgSO4, and filtered. The filtrate gave a crystalline solid (366 mg), containing about 10% ethyl acetate. To remove the ethyl acetate, the crystalline solid was dissolved in methylene chloride, and this solution was evaporated in vacuo to give CDDO-Imidazolide as an amorphous solid (330 mg; yield, 78%). The purity was 99.7%, as determined by high-performance liquid chromatography (C18 column; eluant = 80% acetonitrile, 20% water). Cell culture. CDDO-Imidazolide was dissolved in DMSO, and controls containing equal concentrations of DMSO (V0.1%) were included in all experiments. The human myeloma cell lines RPMI 8226 and JJN3 were provided by Dr. John Shaughnessy (Myeloma Research Institute, University of Arkansas, Little Rock, AR). The mouse plasmacytoma cell line used in the present study, 1254, was developed in the laboratory of Dr. Michael Potter (National Cancer Institute, Bethesda, MD). The A549 and H358 human lung cancer cell lines were either obtained from the American Type Culture Collection (Manassas, VA) or provided by Dr. William Petty (Dartmouth Medical School), respectively. All cells were maintained in RPMI containing 10% fetal bovine serum (Invitrogen, Carlsbad, CA). For all myeloma experiments, cells were centrifuged over Lympholyte-M (Cedar Lane, Burlington, NC) to remove the dead cells and then kept in media containing 0.2% fetal bovine serum overnight before treatment with CDDO-Imidazolide, recombinant human interleukin-6 (IL-6), or calyculin A (Invi-