A novel dicyanotriterpenoid, 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-onitrile, active at picomolar concentrations for inhibition of nitric oxide production

New oleanane triterpenoids with various substituents at the C-17 position of 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid (CDDO) and methyl 2-carboxy-3,12-dioxooleana-1,9(11)-dien-28-oate were synthesized. Among them, 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-onitrile shows extremely high inhibitory activity (IC(50)=1 pM level) against production of nitric oxide induced by interferon-gamma in mouse macrophages. This potency is about 100 times and 30 times more potent than CDDO and dexamethasone, respectively.     

Synthesis and biological evaluation of amino acid methyl ester conjugates of 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid against the production of nitric oxide (NO)

2-Cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid (CDDO, 2) was condensed with various amino acid methyl esters at the C-28 carboxylic acid. The new amide conjugates were evaluated for their inhibition of nitric oxide (NO) production in RAW264.7 cells stimulated with interferon-γ (IFNγ). Of these new compounds, CDDO conjugates with alanine, valine, and serine are nearly equipotent to CDDO-ethyl amide (4), a triterpenoid with promising biological activity in numerous disease models. Some of these conjugates also induce the in vitro expression of heme oxygenase-1, and inhibit the proliferation of Panc-1343 pancreatic cells.     

Synthetic Triterpenoids Target the Arp2/3 Complex and Inhibit Branched Actin Polymerization

Synthetic triterpenoids are anti-tumor agents that affect numerous cellular functions including apoptosis and growth inhibition. Here, we used mass spectrometric and protein array approaches and uncovered that triterpenoids associate with proteins of the actin cytoskeleton, including actin-related protein 3 (Arp3). Arp3, a subunit of the Arp2/3 complex, is involved in branched actin polymerization and the formation of lamellipodia. 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO)-Im and CDDO-Me were observed to 1) inhibit the localization of Arp3 and actin at the leading edge of cells, 2) abrogate cell polarity, and 3) inhibit Arp2/3-dependent branched actin polymerization. We confirmed our drug effects with siRNA targeting of Arp3 and observed a decrease in Rat2 cell migration. Taken together, our data suggest that synthetic triterpenoids target Arp3 and branched actin polymerization to inhibit cell migration.     

Triterpenoid CDDO-Me blocks the NF-kappaB pathway by direct inhibition of IKKbeta on Cys-179

The novel oleanane triterpenoid 2-cyano-3,12-dioxooleana-1,9,-dien-28-oic acid (CDDO) and the C-28 methyl ester (CDDO-Me) induce apoptosis of human tumor cells by disruption of redox balance and are currently in clinical trials. The present studies show that CDDO and CDDO-Me block tumor necrosis factoralpha-induced targeting of NF-kappaB p65 to the nucleus. CDDO-Me also blocked tumor necrosis factor alpha-induced phosphorylation of IkappaBalpha. In concert with these results, we found that CDDO-Me inhibits IkappaBalpha kinasebeta (IKKbeta) activity in cells. In support of a direct mechanism, CDDO-Me inhibited recombinant IKKbeta activity in vitro. The results also demonstrate that (i) CDDO and CDDO-Me form adducts with IKKbeta, but not IKKbeta with mutation of Cys-179 to Ala, and (ii) CDDO-Me inhibits IKKbeta by a mechanism dependent on oxidation of Cys-179. These findings indicate that CDDO and CDDO-Me directly block IKKbeta activity and thereby the NF-kappaB pathway by interacting with Cys-179 in the IKKbeta activation loop.     

2-Cyano-3,10-dioxooleana-1,9(11)-dien-28-oic acid anhydride. A novel and highly potent anti-inflammatory and cytoprotective agent

2-Cyano-3,10-dioxooleana-1,9(11)-dien-28-oic acid anhydride (CDDO anhydride) has been synthesized, which is the first example of an oleanane triterpenoid anhydride. CDDO anhydride shows potency similar to or higher than the corresponding acid (CDDO) in various in vitro and in vivo assays related to inflammation and carcinogenesis. Notably, preliminary phamacokinetics studies show that CDDO anhydride levels are higher than CDDO levels in mouse tissues and blood. Further evaluation of CDDO anhydride is in progress.     

Triterpenoid CDDO-Im downregulates PML/RARalpha expression in acute promyelocytic leukemia cells

The triterpenoid 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO) induces differentiation and apoptosis of diverse human tumor cells. In the present study, we examined the effects of the CDDO imidazolide imide (CDDO-Im) on the NB4 acute promyelocytic leukemia (APL) cell line and primary APL cells. The results show that CDDO-Im selectively downregulates expression of the PML/retinoic receptor alpha fusion protein by a caspase-dependent mechanism and sensitizes APL cells to the differentiating effects of all-trans retinoic acid (ATRA). CDDO-Im treatment of APL cells was also associated with disruption of redox balance and activation of the extrinsic apoptotic pathway. In concert with these results, CDDO-Im sensitizes APL cells to arsenic trioxide (ATO)-induced apoptosis. Our findings indicate that CDDO-Im may be effective in the treatment of APL by: (i) downregulation of PML/RARalpha; (ii) enhancement of ATRA-induced differentiation; and (iii) sensitization of ATO-induced APL cell death.     

A dicyanotriterpenoid induces cytoprotective enzymes and reduces multiplicity of skin tumors in UV-irradiated mice

Inducible phase 2 enzymes constitute a primary line of cellular defense. The oleanane dicyanotriterpenoid 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-onitrile (TP-225) is a very potent inducer of these systems. Topical application of TP-225 to SKH-1 hairless mice increases the levels of NAD(P)H-quinone acceptor oxidoreductase 1 (NQO1) and heme oxygenase 1 (HO-1) and protects against UV radiation-induced dermal thickening. Daily topical treatments of 10 nmol of TP-225 to the backs of mice that were previously subjected to low-level chronic UVB radiation (30 mJ/cm²/session, twice a week for 17 weeks), led to 50% reduction in multiplicity of skin tumors. In addition, the total tumor burden of squamous cell carcinomas was reduced by 5.5-fold. The identification of new agents for protection against UV radiation-induced skin cancer and understanding of their mechanism(s) of action is especially important in view of the fact that human skin cancers represent a significant source of increasing morbidity and mortality.     

The synthetic triterpenoid 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid-imidazolide alters transforming growth factor beta-dependent signaling and cell migration by affecting the cytoskeleton and the polarity complex

The anti-tumor synthetic triterpenoid 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO)-imidazolide (CDDO-Im) ectopically activates the transforming growth factor beta (TGFbeta)-Smad pathway and extends the duration of signaling by an undefined mechanism. Here we show that CDDO-Imdependent persistence of Smad2 phosphorylation is independent of Smad2 phosphatase activity and correlates with delayed TGFbeta receptor degradation and trafficking. Altered TGFbeta trafficking parallels the dispersal of EEA1-positive endosomes from the perinuclear region of CDDO-Im-treated cells. The effect of CDDO-Im on the EEA1 compartment led to an analysis of the cytoskeleton, and we observed that CDDO-Im alters microtubule dynamics by disrupting the microtubule-capping protein, Clip-170. Interestingly, biotinylated triterpenoid was found to localize to the polarity complex at the leading edge of migrating cells. Furthermore, CDDO-Im disrupted the localization of IQGAP1, PKCzeta, Par6, and TGFbeta receptors from the leading edge of migrating cells and inhibited TGFbeta-dependent cell migration. Thus, the synthetic triterpenoid CDDO-Im interferes with TGFbeta receptor trafficking and turnover and disrupts cell migration by severing the link between members of the polarity complex and the microtubule network.     

Oleanane triterpenoid CDDO-Me inhibits Akt activity without affecting PDK1 kinase or PP2A phosphatase activity in cancer cells

Our previous studies have shown that methyl-2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oate (CDDO-Me), a oleanane synthetic triterpenoid induces apoptosis in prostate cancer cells by inhibiting the Akt/NF-κB/mTOR signaling cascade; however, the mechanism by which CDDO-Me inhibits Akt/NF-κB/mTOR signaling has remained undetermined. Present studies show that Akt plays a critical role in the response of prostate cancer cells to CDDO-Me. Silencing of Akt sensitized PC-3 cells to CDDO-Me, whereas its overexpression rendered them resistant to CDDO-Me. Evaluation of the effect of CDDO-Me on Akt which lies upstream of NF-κB and mTOR showed that CDDO-Me directly inhibits the Akt kinase activity in cell-free kinase activity assay and in vivo without modulating the activity of PDK1, the upstream kinase that phosphorylates and activates Akt. The inhibition of Akt activity resulted in inhibition of phosphorylation/inactivation of proapoptotic procaspase-9, Bad and Foxo3a. Further, inhibition of p-Akt by CDDO-Me was not attributable to an increase in the activity of protein phosphatase 2A (PP2A) or PH domain/leucine-rich repeat protein phosphatase1 (PHLPP1) both of which dephosphorylate p-Akt. These findings show that Akt is a direct target of CDDO-Me in the Akt/NF-κB/mTOR prosurvival signaling axis.     

Neuroprotective effect of Nrf2/ARE activators, CDDO ethylamide and CDDO trifluoroethylamide, in a mouse model of amyotrophic lateral sclerosis

Oxidative damage, neuroinflammation, and mitochondrial dysfunction contribute to the pathogenesis of amyotrophic lateral sclerosis (ALS), and these pathologic processes are tightly regulated by the Nrf2/ARE (NF-E2-related factor 2/antioxidant response element) signaling program. Therefore, modulation of the Nrf2/ARE pathway is an attractive therapeutic target for neurodegenerative diseases such as ALS. We examined two triterpenoids, CDDO (2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid) ethylamide and CDDO trifluoroethylamide (CDDO-TFEA), that potently activate Nrf2/ARE in a cell culture model of ALS and in the G93A SOD1 mouse model of ALS. Treatment of NSC-34 cells stably expressing mutant G93A SOD1 with CDDO-TFEA upregulated Nrf2 expression and resulted in translocation of Nrf2 into the nucleus. Western blot analysis showed an increase in the expression of Nrf2/ARE-regulated proteins. When treatment started at a “presymptomatic age” of 30 days, both of these compounds significantly attenuated weight loss, enhanced motor performance, and extended the survival of G93A SOD1 mice. Treatment started at a “symptomatic age,” as assessed by impaired motor performance, was neuroprotective and slowed disease progression. These findings provide further evidence that compounds that activate the Nrf2/ARE signaling pathway may be useful in the treatment of ALS.     

Protection of HepG2 cells against acrolein toxicity by 2-cyano-3,12-dioxooleana-1,9-dien-28-imidazolide via glutathione-mediated mechanism

Acrolein is an environmental toxicant, mainly found in smoke released from incomplete combustion of organic matter. Several studies showed that exposure to acrolein can lead to liver damage. The mechanisms involved in acrolein-induced hepatocellular toxicity, however, are not completely understood. This study examined the cytotoxic mechanisms of acrolein on HepG2 cells. Acrolein at pathophysiological concentrations was shown to cause apoptotic cell death and an increase in levels of protein carbonyl and thiobarbituric acid reactive acid substances. Acrolein also rapidly depleted intracellular glutathione (GSH), GSH-linked glutathione-S-transferases, and aldose reductase, three critical cellular defenses that detoxify reactive aldehydes. Results further showed that depletion of cellular GSH by acrolein preceded the loss of cell viability. To further determine the role of cellular GSH in acrolein-mediated cytotoxicity, buthionine sulfoximine (BSO) was used to inhibit cellular GSH biosynthesis. It was observed that depletion of cellular GSH by BSO led to a marked potentiation of acrolein-mediated cytotoxicity in HepG2 cells. To further assess the contribution of these events to acrolein-induced cytotoxicity, triterpenoid compound 2-cyano-3,12-dioxooleana-1,9-dien-28-imidazolide (CDDO-Im) was used for induction of GSH. Induction of GSH by CDDO-Im afforded cytoprotection against acrolein toxicity in HepG2 cells. Furthermore, BSO significantly inhibited CDDO-Im-mediated induction in cellular GSH levels and also reversed cytoprotective effects of CDDO-Im in HepG2 cells. These results suggest that GSH is a predominant mechanism underlying acrolein-induced cytotoxicity as well as CDDO-Im-mediated cytoprotection. This study may provide understanding on the molecular action of acrolein which may be important to develop novel strategies for the prevention of acrolein-mediated toxicity.     

The triterpenoid 2-cyano-3,12-dioxooleana-1,9-dien-28-oic-acid methyl ester has potent anti-diabetic effects in diet-induced diabetic mice and Lepr(db/db) mice

The triterpenoid 2-Cyano-3,12-dioxooleana-1,9-dien-28-oic-acid (CDDO) and its methyl ester (CDDO-Me) are undergoing clinical trials in cancer and leukemia therapy. Here we report that CDDO-Me ameliorates diabetes in high fat diet-fed type 2 diabetic mice and in Lepr(db/db) mice. CDDO-Me reduces proinflammatory cytokine expression in these animals. Oral CDDO-Me administration reduces total body fat, plasma triglyceride, and free fatty acid levels. It also improves glucose tolerance and insulin tolerance tests. Its potent glucose-lowering activity results from enhanced insulin action. Hyperinsulinemic-euglycemic clamp reveals an increased glucose infusion rate required to maintain euglycemia and showed a significant increase in muscle-specific insulin-stimulated glucose uptake (71% soleus, 58% gastrocnemius) and peripheral glucose clearance as documented by a 48% increase in glucose disposal rate. CDDO-Me activates AMP-activated protein kinase (AMPK) and via LKB1 activation in muscle and liver in vivo. Treatment of isolated hepatocytes with CDDO-Me directly stimulates AMPK activity and LKB1 phosphorylation and decreases acetyl-coA carboxylase activity; it also down-regulates lipogenic gene expression, suppresses gluconeogenesis, and increases glucose uptake. Inhibition of AMPK phosphorylation using compound C and lentiviral-mediated knockdown of AMPK completely blocks the CDDO-Me-induced effect on hepatocytes as well as C(2)C(12) cells. We conclude that the triterpenoid CDDO-Me has potent anti-diabetic action in diabetic mouse models that is mediated at least in part through AMPK activation. The in vivo anti-diabetogenic effects occur at a dose substantially lower than that used for anti-leukemia therapy. We suggest that CDDO-Me holds promise as a potential anti-diabetic agent.     

Active NF-E2-related Factor (Nrf2) Contributes to Keep Endothelial NO Synthase (eNOS) in the Coupled State: ROLE OF REACTIVE OXYGEN SPECIES (ROS), eNOS, AND HEME OXYGENASE (HO-1) LEVELS*

The aim of our study was to examine in detail the impact of NF-E2-related factor (Nrf2) activation on endothelial cell function with focus on redox homeostasis and the endothelial nitric oxide synthase (eNOS) system. We administered 2-cyano-3,12-dioxooleana-1,9-dien-28-oic imidazolide (CDDO-IM), a known activator of Nrf2, to primary human umbilical vein endothelial cells. Activation of Nrf2 by CDDO-IM increased the amount of bioavailable nitric oxide (NO), a major contributor to vascular homeostasis, in naive and stressed cells. Concomitantly, intracellular reactive oxygen species were dose-and time-dependently reduced. In apparent contrast to elevated NO levels, eNOS protein expression was transiently decreased in an Nrf2-dependent manner. Employing pharmacological inhibitors as well as a small interfering RNA approach, we identified de novo protein synthesis of heme oxygenase 1 (HO-1) and its enzymatic activity as cause for the observed reduction of eNOS. We hypothesize that under redox stress, when the availability of tetrahydrobiopterin, a pivotal stoichiometric cofactor for eNOS, is limited, activation of Nrf2 leads (a) to transient reduction of eNOS protein levels and (b) to an antioxidant defense in human umbilical vein endothelial cells. Both activities ensure that a stoichiometric ratio of eNOS and tetrahydrobiopterin is sustained and that the risk of eNOS uncoupling is reduced. Our study is the first to provide a causal link between Nrf2 activation and eNOS expression and NO levels, respectively.