Methyl 2-Cyano-3,12-dioxooleana-1,9-dien-28-oate Decreases Specificity Protein Transcription Factors and Inhibits Pancreatic Tumor Growth: Role of MicroRNA-27a

Abstract

The anticancer agent 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO) and its methyl ester (CDDO-Me) typically induce a broad spectrum of growth-inhibitory, proapoptotic, and antiangiogenic responses. Treatment of Panc1, Panc28, and L3.6pL pancreatic cancer cells with low micromolar concentrations of CDDO or CDDO-Me resulted in growth inhibition, induction of apoptosis, and down-regulation of cyclin D1, survivin, vascular endothelial growth factor (VEGF), and its receptor (VEGFR2). RNA interference studies indicate that these repressed genes are regulated by specificity protein (Sp) transcription factors Sp1, Sp3, and Sp4, and Western blot analysis of lysates from pancreatic cancer cells treated with CDDO and CDDO-Me shows for the first time that both compounds decreased the expression of Sp1, Sp3, and Sp4. Moreover, CDDO-Me (7.5 mg/kg/day) also inhibited pancreatic human L3.6pL tumor growth and down-regulated Sp1, Sp3, and Sp4 in tumors using an orthotopic pancreatic cancer model. CDDO-Me also induced reactive oxygen species (ROS) and decreased mitochondrial membrane potential (MMP) in Panc1 and L3.6pL cells, and cotreatment with antioxidants (glutathione and dithiothreitol) blocked the formation of ROS, reversed the loss of MMP, and inhibited down-regulation of Sp1, Sp3, and Sp4. Repression of Sp and Sp-dependent genes by CDDO-Me was due to the down-regulation of microRNA-27a and induction of zinc finger and BTB domain containing 10 (ZBTB10), an Sp repressor, and these responses were also reversed by antioxidants. Thus, the anticancer activity of CDDO-Me is due, in part, to activation of ROS, which in turn targets the microRNA-27a:ZBTB10-Sp transcription factor axis. This results in decreased expression of Spregulated genes, growth inhibition, induction of apoptosis, and antiangiogenic responses.Extracts of plants and microorganisms and individual natural products have been extensively used as traditional medicines for the treatment of several diseases, including cancer.Individual natural products including aspirin, morphine, quinine, statins, penicillins, taxanes, and many other compounds are widely used pharmaceutical agents and serve as templates for the synthesis of more potent analogs (Koehn and Carter, 2005). Triterpenoids are derived from cyclization of oxidosqualene, and different cyclization pathways coupled with postcyclization modifications can give several thousand possible analogs, including oleanolic acid, which contains a pentacyclic oleanane skeleton and a C28 carboxyl group. Oleanolic acid has been used by Sporn, Honda, and their collaborators as a template for extensive structure-activity ABBREVIATIONS: CDDO, 2-cyano-3,12-dioxoleana-1,9-dien-28-oic acid; CDDO-IM, imidazole ester of 2-cyano-3,12-dioxoleana-1,9-dien-28-oic acid; CDDO-Me, methyl ester of 2-cyano-3,12-dioxoleana-1,9-dien-28-oic acid; CDODA, 2-cyano-3,11-dioxo-18␤-olean-1,12-dien-30-oic acid; CDODA-Me, methyl ester of 2-cyano-3,11-dioxo-18␤-olean-1,12-dien-30-oic acid; GSH, glutathione; MMP, mitochondrial membrane pote...

Benzyl Isothiocyanate (BITC) Induces Reactive Oxygen Species-dependent Repression of STAT3 Protein by Down-regulation of Specificity Proteins in Pancreatic Cancer

“…We recently reported that many of the responses/ genes affected by PEITC in pancreatic cancer cells were due to induction of ROS and ROS-dependent down-regulation of Sp1, Sp3, Sp4, and pro-oncogenic Sp-regulated genes (30). These results complement studies showing that hydrogen peroxide and t-butylhydroperoxide also down-regulate Sp proteins (22,23,27).BITC is structurally related to PEITC, and both compounds exhibit comparable anticancer activities and modulate expression of some common genes/pathways consistent with their effects on cancer cells (6 -14). It was also reported that BITC inhibits signal transducer and activator of transcription 3 (STAT3) phosphorylation and down-regulates STAT3 protein (6), and another study by the same group showed that BITC also induces ROS in pancreatic and other cancer cell lines (31-33).…”

Mechanism of Metformin-dependent Inhibition of Mammalian Target of Rapamycin (mTOR) and Ras Activity in Pancreatic Cancer

“…Moreover, knockdown of Sp1 in pancreatic cancer cells decreases growth and invasion and induces apoptosis, confirming the pro-oncogenic functions of this factor (49). These results suggest that drugs such as metformin and other agents (31)(32)(33)(34)(35)(36)39) that target Sp1, Sp3, and Sp4 represent a class of new mechanism-based drugs that can be used in combination therapies for treating this deadly disease.…”

“…This involved down-regulation of specificity protein (Sp) transcription factors Sp1, Sp3, and Sp4 and pro-oncogenic Sp-regulated genes such as bcl2, fatty acid synthase (FAS), survivin, vascular endothelial growth factor (VEGF), and VEGF receptor 1 (VEGFR1) (31). The anticancer activities of metformin are also similar to * This work was supported, in whole or in part, by National Institutes of Health that observed after knockdown of Sp1 or all three Sp proteins by RNA interference in cancer cells, and this includes growth inhibition, induction of apoptosis, reversal of epithelial to mesenchymal transition, and decreased migration/invasion (32)(33)(34)(35)(36). Metformin also inhibits NFB and decreases cyclin D1 and ErbB2 in cancer cell lines (13,20,27,28), and these gene products are also decreased after Sp1, Sp3, and Sp4 silencing by RNAi or by other drugs that down-regulate Sp proteins (32)(33)(34)(35)(36).…”

Targeted inhibition of tumor proliferation, survival, and metastasis by pentacyclic triterpenoids: Potential role in prevention and therapy of cancer

“…In another study where transgenic LSL-Kras(G12D/+); LSL-Trp53(R127H/+); Pdx-1-Cre (KPC) mouse model of pancreatic cancer were fed with diet containing CDDO-Me or CDDO-ethyl amide, the rexinoid LG100268 significantly increased the survival of mice by 3-4 weeks [209] and prevented lung cancer development when fed with these terpenoids for 8 weeks [15]. CDDO and CDDO-Me fed for 20 week inhibited the progression of the preneoplastic lesions (low-grade PIN and high-grade-PIN) to adenocarcinoma in the dorsolateral prostate (DLP) and ventral prostate (VP) lobes of TRAMP mice [210,211] prevented breast cancer development in BRCA1-mutated mice [212]; pretreatment with oral CDDO-Me improved survival following lethal-dose LPS challenge in mice by modulating the in vivo immune response to LPS [213]; CDDO-Me inhibited orthotopic pancreatic human L3.6pL tumor growth and down-regulated Sp1, Sp3, and Sp4 in tumors [214]. However, these synthetic terpenoids had very poor oral absorption and low bioavailability.…”

“…However, these synthetic terpenoids had very poor oral absorption and low bioavailability. To improve its pharmacokinetic profile, a novel CDDO anhydride was synthesized with the same potency as CDDO-Me, but with greater bioavailability [214]. Angiogenesis, particularly 'inflammatory angiogenesis', is a common target of many chemopreventive molecules, which most likely suppress angiogenesis in pre-malignant tumors.…”