Charlotte R. Williams

The Synthetic Triterpenoids, CDDO and CDDO-Imidazolide, Are Potent Inducers of Heme Oxygenase-1 and Nrf2/ARE Signaling

The synthetic triterpenoid 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid (CDDO) and its derivative 1-[2-cyano-3-,12-dioxooleana-1,9(11)-dien-28-oyl]imidazole (CDDO-Im) are multifunctional molecules with potent antiproliferative, differentiating, and anti-inflammatory activities. At nanomolar concentrations, these agents rapidly increase the expression of the cytoprotective heme oxygenase-1 (HO-1) enzyme in vitro and in vivo. Transfection studies using a series of reporter constructs show that activation of the human HO-1 promoter by the triterpenoids requires an antioxidant response element (ARE), a cyclic AMP response element, and an E Box sequence. Inactivation of one of these response elements alone partially reduces HO-1 induction, but mutations in all three sequences entirely eliminate promoter activity in response to the triterpenoids. Treatment with CDDO-Im also elevates protein levels of Nrf2, a transcription factor previously shown to bind ARE sequences, and increases expression of a number of antioxidant and detoxification genes regulated by Nrf2. The triterpenoids also reduce the formation of reactive oxygen species in cells challenged with tert-butyl hydroperoxide, but this cytoprotective activity is absent in Nrf2 deficient cells. These studies are the first to investigate the induction of the HO-1 and Nrf2/ARE pathways by CDDO and CDDO-Im, and our results suggest that further in vivo studies are needed to explore the chemopreventive and chemotherapeutic potential of the triterpenoids.

Genetic versus chemoprotective activation of Nrf2 signaling: overlapping yet distinct gene expression profiles between Keap1 knockout and triterpenoid-treated mice

Loss of NF-E2-related factor 2 (Nrf2) signaling increases susceptibility to acute toxicity, inflammation and carcinogenesis in mice due to the inability to mount adaptive responses. In contrast, disruption of Keap1 (a cytoplasmic modifier of Nrf2 turnover) protects against these stresses in mice, although inactivating mutations in Keap1 have been identified recently in some human cancers. Global characterization of Nrf2 activation is important to exploit this pathway for chemoprevention in healthy, yet at-risk individuals and also to elucidate the consequences of hijacking the pathway in Keap1-mutant human cancers. Liver-targeted conditional Keap1-null, Albumin-Cre:Keap1((flox/-)) (CKO) mice provide a model of genetic activation of Nrf2 signaling. By coupling global gene expression analysis of CKO mice with analysis of pharmacologic activation using the synthetic oleanane triterpenoid 1-[2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oyl]imidazole (CDDO-Im), we are able to gain insight into pathways affected by Nrf2 activation. CDDO-Im is an extremely potent activator of Nrf2 signaling. CKO mice were used to identify genes modulated by genetic activation of Nrf2 signaling. The CKO response was compared with hepatic global gene expression changes in wild-type mice treated with CDDO-Im at a maximal Nrf2 activating dose. The results show that genetic and pharmacologic activation of Nrf2 signaling modulates pathways beyond detoxication and cytoprotection, with the largest cluster of genes associated with lipid metabolism. Genetic activation of Nrf2 results in much larger numbers of detoxication and lipid metabolism gene changes. Additionally, analysis of pharmacologic activation suggests that Nrf2 is the primary mediator of CDDO-Im activity, though other cell-signaling targets are also modulated following an oral dose of 30 micromol/kg.

Neuroprotective Effects of the Triterpenoid, CDDO Methyl Amide, a Potent Inducer of Nrf2-Mediated Transcription

The NF-E2-related factor-2 (Nrf2)/antioxidant response element (ARE) signaling pathway regulates phase 2 detoxification genes, including a variety of antioxidative enzymes. We tested neuroprotective effects of the synthetic triterpenoid CDDO-MA, a potent activator of the Nrf2/ARE signaling. CDDO-MA treatment of neuroblastoma SH-SY5Y cells resulted in Nrf2 upregulation and translocation from cytosol to nucleus and subsequent activation of ARE pathway genes. CDDO-MA blocked t-butylhydroperoxide-induced production of reactive oxygen species (ROS) by activation of ARE genes only in wild type, but not Nrf2 knockout mouse embryonic fibroblasts. Oral administration of CDDO-MA resulted in significant protection against MPTP-induced nigrostriatal dopaminergic neurodegeneration, pathological alpha-synuclein accumulation and oxidative damage in mice. Additionally, CDDO-MA treatment in rats produced significant rescue against striatal lesions caused by the neurotoxin 3-NP, and associated increases in the oxidative damage markers malondialdehyde, F2-Isoprostanes, 8-hydroxy-2-deoxyguanosine, 3-nitrotyrosine, and impaired glutathione homeostasis. Our results indicate that the CDDO-MA renders its neuroprotective effects through its potent activation of the Nrf2/ARE pathway, and suggest that triterpenoids may be beneficial for the treatment of neurodegenerative diseases like Parkinsons disease and Huntingtons disease.

The synthetic triterpenoids CDDO-methyl ester and CDDO-ethyl amide prevent lung cancer induced by vinyl carbamate in A/J mice.

We report the first use of new synthetic triterpenoids to prevent lung cancer in experimental animals. Female A/J mice were treated with the mutagenic carcinogen vinyl carbamate, which induces adenocarcinoma of the lung in all animals within 16 weeks. If mice were fed either the methyl ester or the ethyl amide derivative of the synthetic triterpenoid 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid (CDDO-ME and CDDO-EA, respectively), beginning 1 week after dosing with carcinogen, the number, size, and severity of lung carcinomas were markedly reduced. The mechanisms of action of CDDO-ME and CDDO-EA that are germane to these in vivo findings are the following results shown here in cell culture: (a) suppression of the ability of IFN-gamma to induce de novo formation of nitric oxide synthase in a macrophage-like cell line RAW264.7, (b) induction of heme oxygenase-1 in these RAW cells, and (c) suppression of phosphorylation of the transcription factor signal transducers and activators of transcription 3 as well as induction of apoptosis in human lung cancer cell lines.

Triterpenoids CDDO-ethyl amide and CDDO-trifluoroethyl amide improve the behavioral phenotype and brain pathology in a transgenic mouse model of Huntington's disease

Oxidative stress is a prominent feature of Huntingtons disease (HD) due to mitochondrial dysfunction and the ensuing overproduction of reactive oxygen species (ROS). This phenomenon ultimately contributes to cognitive and motor impairment, as well as brain pathology, especially in the striatum. Targeting the transcription of the endogenous antioxidant machinery could be a promising therapeutic approach. The NF-E2-related factor-2 (Nrf2)/antioxidant response element (ARE) signaling pathway is an important pathway involved in antioxidant and anti-inflammatory responses. Synthetic triterpenoids, which are derived from 2-Cyano-3,12-Dioxooleana-1,9-Dien-28-Oic acid (CDDO) activate the Nrf2/ARE pathway and reduce oxidative stress in animal models of neurodegenerative diseases. We investigated the effects of CDDO-ethyl amide (CDDO-EA) and CDDO-trifluoroethyl amide (CDDO-TFEA) in N171-82Q mice, a transgenic mouse model of HD. CDDO-EA or CDDO-TFEA were administered in the diet at various concentrations, starting at 30days of age. CDDO-EA and CDDO-TFEA upregulated Nrf2/ARE induced genes in the brain and peripheral tissues, reduced oxidative stress, improved motor impairment and increased longevity. They also rescued striatal atrophy in the brain and vacuolation in the brown adipose tissue. Therefore compounds targeting the Nrf2/ARE pathway show great promise for the treatment of HD.

Targeting Nrf2-Mediated Gene Transcription by Extremely Potent Synthetic Triterpenoids Attenuate Dopaminergic Neurotoxicity in the MPTP Mouse Model of Parkinson's Disease

UNLABELLED Although the etiology of Parkinsons disease (PD) remains unclear, ample empirical evidence suggests that oxidative stress is a major player in the development of PD and in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) neurotoxicity. Nuclear factor E2-related factor 2 (Nrf2) is a redox-sensitive transcription factor that upregulates a battery of antioxidant response element (ARE)-driven antioxidative and cytoprotective genes that defend against oxidative stress. AIMS We evaluated whether the strategy of activation of Nrf2 and its downstream network of cytoprotective genes with small molecule synthetic triterpenoids (TP) attenuate MPTP-induced PD in mice. RESULTS We show that synthetic TP are thus far the most potent and direct activators of the Nrf2 pathway using a novel Neh2-luciferase reporter. They upregulate several cytoprotective genes, including those involved in glutathione biosynthesis in vitro. Oral administration of TP that were structurally modified to penetrate the brain-induced messenger RNA and protein levels for a battery of Nrf2-dependent cytoprotective genes reduced MPTP-induced oxidative stress and inflammation, and ameliorated dopaminergic neurotoxicity in mice. The neuroprotective effect of these TP against MPTP neurotoxicity was dependent on Nrf2, since treatment with TP in Nrf2 knockout mice failed to block against MPTP neurotoxicity and induce Nrf2-dependent cytoprotective genes. INNOVATION Extremely potent synthetic TP that are direct activators of the Nrf2 pathway block dopaminergic neurodegeneration in the MPTP mouse model of PD. CONCLUSION Our results indicate that activation of Nrf2/antioxidant response element (ARE) signaling by synthetic TP is directly associated with their neuroprotective effects against MPTP neurotoxicity and suggest that targeting the Nrf2/ARE pathway is a promising approach for therapeutic intervention in PD.

The Synthetic Triterpenoid 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-1 and 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.

Triterpenoid CDDO‐methylamide improves memory and decreases amyloid plaques in a transgenic mouse model of Alzheimer’s disease

Oxidative stress is one of the earliest events in the pathogenesis of Alzheimer’s disease (AD) and can markedly exacerbate amyloid pathology. Modulation of antioxidant and anti‐inflammatory pathways represents an important approach for AD therapy. Synthetic triterpenoids have been found to facilitate antioxidant response and reduce inflammation in several models. We investigated the effect of the triterpenoid, 2‐Cyano‐3,12‐Dioxooleana‐1,9‐Dien‐28‐Oic acid‐MethylAmide (CDDO‐MA) in Tg19959 mice, which carry the human amyloid precursor protein with two mutations. These mice develop memory impairments and amyloid plaques as early as 2–3 months of age. CDDO‐MA was provided with chow (800 mg/kg) from 1 to 4 months of age. CDDO‐MA significantly improved spatial memory retention and reduced plaque burden, Aβ42 levels, microgliosis, and oxidative stress in Tg19959 mice.

A novel acetylenic tricyclic bis-(cyano enone) potently induces phase 2 cytoprotective pathways and blocks liver carcinogenesis induced by aflatoxin

A novel acetylenic tricyclic bis-(cyano enone), TBE-31, is a lead compound in a series of tricyclic compounds with enone functionalities in rings A and C. Nanomolar concentrations of this potent multifunctional molecule suppress the induction of the inflammatory protein, inducible nitric oxide synthase, activate phase 2 cytoprotective enzymes in vitro and in vivo, block cell proliferation, and induce differentiation and apoptosis of leukemia cells. Oral administration of TBE-31 also significantly reduces formation of aflatoxin-DNA adducts and decreases size and number of aflatoxin-induced preneoplastic hepatic lesions in rats by >90%. Because of the two cyano enones in rings A and C, TBE-31 may directly interact with DTT and protein targets such as Keap1 that contain reactive cysteine residues. The above findings suggest that TBE-31 should also be tested for chemoprevention and chemotherapy in relevant models of cancer and against other chronic, degenerative diseases in which inflammation and oxidative stress contribute to disease pathogenesis.

Synthetic Triterpenoids Prolong Survival in a Transgenic Mouse Model of Pancreatic Cancer

Pancreatic cancer is the fourth leading cause of cancer-related deaths in the United States and is nearly always fatal. Whereas early detection offers the most promising approach for reducing the mortality of this disease, there is still a need to develop effective drugs for the prevention and treatment of pancreatic cancer. We tested two promising classes of noncytotoxic drugs, synthetic oleanane triterpenoids and rexinoids, for the prevention of carcinogenesis in the highly relevant LSL-KrasG12D/+;LSL-Trp53R127H/+;Pdx-1-Cre (KPC) mouse model of pancreatic cancer. KPC transgenic mice closely recapitulate the genetic mutations, clinical symptoms, and histopathology found in human pancreatic cancer. Beginning at 4 weeks of age, mice were fed powdered control diet or a diet containing the triterpenoids CDDO-methyl ester (CDDO-Me) or CDDO-ethyl amide, the rexinoid LG100268 (LG268), or the combination, until the mice displayed overt symptoms of pancreatic cancer. CDDO-Me, LG268, the combination of CDDO-Me and LG268, and the combination of CDDO-ethyl amide and LG268, all significantly (P < 0.05) increased survival in the KPC mice by 3 to 4 weeks. Recent studies have shown that gemcitabine, the current standard of care for human pancreatic cancer, does not extend survival in KPC mice. In cell lines developed from the KPC mice, the triterpenoids directly interact with both signal transducer and activator of transcription 3 and IκB kinase (IKK) to decrease constitutive interleukin-6 secretion, inhibit constitutive signal transducer and activator of transcription 3 phosphorylation, and block the degradation of IκBα when challenged with tumor necrosis factor α. These results suggest that oleanane triterpenoids and rexinoids have the potential to prevent pancreatic cancer. Cancer Prev Res; 3(11); 1427–34. ©2010 AACR.