The Rexinoids LG100268 and LG101506 Inhibit Inflammation and Suppress Lung Carcinogenesis in A/J Mice.

LG101506 was originally synthesized to overcome some of the undesirable side effects of rexinoids. We compared the anticarcinogenic action of LG101506 and LG100268 and for the first time showed that both drugs are useful for prevention of lung cancer in A/J mice. These molecules markedly reduced tumor number, tumor size, and total tumor burden, when chronically administered to A/J mice that had been initiated with the mutagenic carcinogen, vinyl carbamate. Moreover, LG100268 synergized with the histone deacetylase inhibitor, vorinostat, for prevention of experimental lung cancer and enhanced the effect of carboplatin/paclitaxel for treatment of experimental lung cancer. Both rexinoids diminished the percentage of high-grade, highly malignant adenocarcinomas found at autopsy. In cell culture studies, the rexinoids exhibited potent anti-inflammatory properties at nanoMolar concentrations. These drugs suppressed the ability of lipopolysaccharide to stimulate the synthesis and secretion of nitric oxide and inflammatory cytokines and chemokines, such as IL6, IL1ß, CXCL2, and CSF3, in macrophage-like RAW264.7 cells. The present results suggest that LG100268, LG101506, or a related rexinoid may have useful clinical applications in the field of oncology.

Dimethyl fumarate and the oleanane triterpenoids, CDDO-imidazolide and CDDO-methyl ester, both activate the Nrf2 pathway but have opposite effects in the A/J model of lung carcinogenesis.

Lung cancer accounts for the highest number of cancer-related deaths in the USA, highlighting the need for better prevention and therapy. Activation of the Nrf2 pathway detoxifies harmful insults and reduces oxidative stress, thus preventing carcinogenesis in various preclinical models. However, constitutive activation of the Nrf2 pathway has been detected in numerous cancers, which confers a survival advantage to tumor cells and a poor prognosis. In our study, we compared the effects of two clinically relevant classes of Nrf2 activators, dimethyl fumarate (DMF) and the synthetic oleanane triterpenoids, CDDO-imidazolide (CDDO-Im) and CDDO-methyl ester (CDDO-Me) in RAW 264.7 mouse macrophage-like cells, in VC1 lung cancer cells and in the A/J model of lung cancer. Although the triterpenoids and DMF both activated the Nrf2 pathway, CDDO-Im and CDDO-Me were markedly more potent than DMF. All of these drugs reduced the production of reactive oxygen species and inhibited nitric oxide production in RAW264.7 cells, but the triterpenoids were 100 times more potent than DMF in these assays. Microarray analysis revealed that only 52 of 99 Nrf2-target genes were induced by all three compounds, and each drug regulated a unique subset of Nrf2 genes. These drugs also altered the expression of other genes important in lung cancer independent of Nrf2. Although all three compounds enhanced the phosphorylation of CREB, only DMF increased the phosphorylation of Akt. CDDO-Me, at either 12.5 or 50mg/kg of diet, was the most effective drug in our lung cancer mouse model. Specifically, CDDO-Me significantly reduced the average tumor number, size and burden compared with the control group (P < 0.05). Additionally, 52% of the tumors in the control group were high-grade tumors compared with only 14% in the CDDO-Me group. Though less potent, CDDO-Im had similar activity as CDDO-Me. In contrast, 61-63% of the tumors in the DMF groups (400-1200mg/kg diet) were high-grade tumors compared with 52% for the controls (P < 0.05). Additionally, DMF significantly increased the average number of tumors compared with the controls (P < 0.05). Thus, in contrast to the triterpenoids, which effectively reduced pathogenesis in A/J mice, DMF enhanced the severity of lung carcinogenesis in these mice. Collectively, these results suggest that although CDDO-Im, CDDO-Me and DMF all activate the Nrf2 pathway, they target distinct genes and signaling pathways, resulting in opposite effects for the prevention of experimental lung cancer.

The PARP inhibitors, veliparib and olaparib, are effective chemopreventive agents for delaying mammary tumor development in BRCA1-deficient mice.

Poly-ADP ribose polymerase (PARP) inhibitors are effective for the treatment of BRCA-deficient tumors. Women with these mutations have an increased risk of developing breast cancer and would benefit from effective chemoprevention. This study examines whether the PARP inhibitors, veliparib and olaparib, delay mammary gland tumor development in a BRCA1-deficient (BRCA1(Co/Co);MMTV-Cre;p53(+/-)) mouse model. In dose de-escalation studies, mice were fed with control, veliparib (100 mg/kg diet), or olaparib (200, 100, 50, or 25 mg/kg diet) continuously for up to 43 weeks. For intermittent dosing studies, mice cycled through olaparib (200 mg/kg diet) for 2 weeks followed by a 4-week rest period on control diet. To examine biomarkers, mice were fed with olaparib using the intermittent dosing regimen and mammary glands were evaluated by immunohistochemistry. In mice treated with veliparib or olaparib (200 mg/kg diet), the average age of the first detectable tumor was delayed by 2.4 and 6.5 weeks, respectively, compared with controls. Olaparib also increased the average lifespan of mice by 7 weeks. In dose de-escalation studies, lower concentrations of olaparib delayed tumor development but were less effective than the highest dose. When fed intermittently, olaparib delayed the onset of the first palpable tumor by 5.7 weeks and significantly reduced proliferation and induced apoptosis in hyperplastic mammary glands. In summary, veliparib and olaparib are effective for delaying tumor development and extending the lifespan of BRCA1-deficient mice, and intermittent dosing with olaparib was as effective as continuous dosing. These results suggest that the use of PARP inhibitors is a promising chemopreventive option.

The combination of the histone deacetylase inhibitor vorinostat and synthetic triterpenoids reduces tumorigenesis in mouse models of cancer.

Novel drugs and drug combinations are needed for the chemoprevention and treatment of cancer. We show that the histone deacetylase inhibitor vorinostat [suberoylanilide hydroxamic acid (SAHA)] and the methyl ester or ethyl amide derivatives of the synthetic triterpenoid 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid (CDDO-Me and CDDO-Ea, respectively) cooperated to inhibit the de novo synthesis of nitric oxide in RAW 264.7 macrophage-like cells and in primary mouse peritoneal macrophages. Additionally, SAHA enhanced the ability of synthetic triterpenoids to delay formation of estrogen receptor-negative mammary tumors in MMTV-polyoma middle T (PyMT) mice. CDDO-Me (50 mg/kg diet) and SAHA (250 mg/kg diet) each significantly delayed the initial development of tumors by 4 (P < 0.001) and 2 (P < 0.05) weeks, respectively, compared with the control group in the time required to reach 50% tumor incidence. CDDO-Ea (400 mg/kg diet), as a single agent, did not delay tumor development. The combination of either triterpenoid with SAHA was significantly more potent than the individual drugs for delaying tumor development, with a 7 week (P < 0.001) delay before 50% tumor incidence was reached. SAHA, alone and in combination with CDDO-Me, also significantly (P < 0.05) inhibited the infiltration of tumor-associated macrophages into the mammary glands of PyMT mice and levels of the chemokine macrophage colony-stimulating factor in primary PyMT tumor cells. In addition, SAHA and the synthetic triterpenoids cooperated to suppress secreted levels of the pro-angiogenic factor matrix metalloproteinase-9. Similar results were observed in mouse models of pancreatic and lung cancer. At concentrations that were anti-inflammatory, SAHA had no effect on histone acetylation. These studies suggest that both SAHA and triterpenoids effectively delay tumorigenesis, thereby demonstrating a promising, novel drug combination for chemoprevention.

The synthetic triterpenoid CDDO-methyl ester delays estrogen receptor-negative mammary carcinogenesis in polyoma middle T mice.

Novel drugs are needed for the prevention and treatment of breast cancer. Synthetic triterpenoids are a promising new class of compounds with activity in a variety of preclinical cancer models. We tested activity of the methyl ester derivative of the synthetic triterpenoid, 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid (CDDO-Me), in a relevant model of estrogen receptor-negative breast cancer, the polyoma-middle T (PyMT), in which the oncoprotein drives carcinogenesis. The developing tumors recapitulate key features of the human disease. Mice were fed CDDO-Me (50 mg/kg diet), starting at 4 weeks of age. CDDO-Me significantly increased the age of mice at onset of first tumor (P < 0.001) by an average of 4.3 weeks and overall survival (P < 0.001) by 5.2 weeks. The drug also inhibited the infiltration of tumor-associated macrophages into mammary glands of PyMT mice at 12 weeks of age and reduced levels of the chemokines CXCL12 and CCL2 in primary PyMT mammary tumor cells. Treatment with this multifunctional drug also inhibited secretion of matrix metalloproteinase-9 in primary tumor cells from PyMT mice and decreased proliferation of these cells by inhibiting cyclin D1 and decreasing phosphorylation of epidermal growth factor receptor and STAT3.

CDDO-methyl ester delays breast cancer development in BRCA1-mutated mice.

The breast cancer-associated gene 1 (BRCA1) is the most frequently mutated tumor suppressor gene in familial breast cancers. Mutations in BRCA1 also predispose to other types of cancers, pointing to a fundamental role of this pathway in tumor suppression and emphasizing the need for effective chemoprevention in these high-risk patients. Because the methyl ester of the synthetic triterpenoid 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid (CDDO-Me) is a potent chemopreventive agent, we tested its efficacy in a highly relevant mouse model of BRCA1-mutated breast cancer. Beginning at 12 weeks of age, Brca1(Co/Co); MMTV-Cre;p53(+/-) mice were fed powdered control diet or diet containing CDDO-Me (50 mg/kg diet). CDDO-Me significantly (P < 0.05) delayed tumor development in the Brca1-mutated mice by an average of 5.2 weeks. We also observed that levels of ErbB2, p-ErbB2, and cyclin D1 increased in a time-dependent manner in the mammary glands in Brca1-deficient mice, and CDDO-Me inhibited the constitutive phosphorylation of ErbB2 in tumor tissues from these mice. In BRCA1-deficient cell lines, the triterpenoids directly interacted with ErbB2, decreased constitutive phosphorylation of ErbB2, inhibited proliferation, and induced G(0)-G(1) arrest. These results suggest that CDDO-Me has the potential to prevent BRCA1-mutated breast cancer.

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 30days, 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.

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-Kras(G12D/+);LSL-Trp53(R127H/+);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.

Triterpenoids CDDO-methyl ester or CDDO-ethyl amide and rexinoids LG100268 or NRX194204 for prevention and treatment of lung cancer in mice.

We tested members of two noncytotoxic classes of drugs, synthetic oleanane triterpenoids and rexinoids, both as individual agents and in combination, for the prevention and treatment of carcinogenesis in a highly relevant animal model of lung cancer. Lung adenocarcinomas were induced in A/J mice by injection of the carcinogen vinyl carbamate. Mice were fed drugs in diet, beginning 1 week after the carcinogen challenge for prevention or 8 weeks later for treatment. The number, size, and severity of tumors in the lungs were then evaluated. In the prevention studies, the triterpenoids CDDO-ethyl amide and CDDO-methyl ester reduced the average tumor burden (ATB) in the lungs 86% to 92%, respectively, compared with the controls, and the rexinoid LG100268 (268) reduced ATB by 50%. The combination of CDDO-ethyl amide and 268 reduced ATB by 93%. We show for the first time that these drugs also were highly effective for treatment of experimental lung cancer, and all triterpenoid and rexinoid combinations reduced ATB 85% to 87% compared with the control group. The triterpenoids also potently inhibited proliferation of VC1 mouse lung carcinoma cells and directly interacted with key regulatory proteins in these cells. In contrast, the rexinoids had little antiproliferative activity in VC1 cells but were potent inhibitors of the toll-like receptor pathway in macrophage-like cells. Triterpenoids and rexinoids are multifunctional, well-tolerated drugs that target different signaling pathways and are thus highly effective for prevention and treatment of experimental lung cancer.

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, F(2)-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 Parkinson's disease and Huntington's disease.

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.

Prevention and treatment of experimental estrogen receptor-negative mammary carcinogenesis by the synthetic triterpenoid CDDO-methyl Ester and the rexinoid LG100268.

PURPOSE: To test whether the triterpenoid 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid methyl ester (CDDO-Me) and the rexinoid LG100268 (268) prevent the formation of estrogen receptor (ER)-negative mammary tumors or either arrest the growth or cause regression of established tumors in MMTV-neu mice. EXPERIMENTAL DESIGN: For prevention, mice were fed control diet, CDDO-Me (60 mg/kg diet), 268 (20 mg/kg diet), or the combination for 45 weeks. For treatment, mice with established tumors at least 4 mm in diameter were fed control diet, CDDO-Me (100 mg/kg diet), 268 (60 mg/kg diet), or the combination for 4 weeks. RESULTS: CDDO-Me and 268 significantly delayed the development of ER-negative tumors, with a 14- and 24-week delay, respectively, compared with the control group for the time required to reach 50% tumor incidence. The combination of CDDO-Me and 268 was significantly more potent than the individual drugs, as only one tumor was found in the combination group, after 45 weeks on diet, at which time all control animals had tumors. Treating established tumors with CDDO-Me arrested the growth of 86% of the tumors, and 268 induced tumor regression in 85% of tumors. CDDO-Me and 268 target different signaling pathways and cell types. CDDO-Me inhibited constitutive STAT3 phosphorylation and the degradation of IKBalpha in ER-negative breast cancer cells, whereas 268 blocked IKBalpha degradation and the release of interleukin-6 in RAW264.7 macrophage-like cells, inhibited the ability of endothelial cells to organize into networks, and blocked angiogenesis in vivo. CONCLUSIONS: CDDO-Me and 268 are useful as individual drugs to prevent ER-negative mammary tumorigenesis and to treat established tumors. They synergize when used in combination for prevention.

The rexinoid LG100268 and the synthetic triterpenoid CDDO-methyl amide are more potent than erlotinib for prevention of mouse lung carcinogenesis.

Female A/J mice injected with the carcinogen vinyl carbamate develop atypical adenomatous hyperplasias in lungs 4 weeks after injection with the carcinogen. The number and severity of tumors then increase over time, making these mice a useful model for evaluating potential chemopreventive agents. The rexinoid LG100268 (LG268), a selective ligand for the retinoid X receptor, and the methyl amide of the synthetic triterpenoid 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid (CDDO) both significantly reduced the number, size, and severity of the histopathology of lung tumors in female A/J mice when fed in diet for 14 to 20 weeks. The total tumor burden was 85% to 87% lower in mice fed LG268 and CDDO-MA than in controls, and the percentage of high-grade tumors decreased from 59% in the controls to 25% or 30% with CDDO-MA and LG268. Erlotinib, which is used to treat lung cancer patients and is an inhibitor of the epidermal growth factor receptor, was less effective in this model. Immunohistochemical staining of geminin, a marker of cell cycle progression, was higher in lung sections from control mice than in mice treated with LG268. Because rexinoids and triterpenoids signal through different biological pathways, they should be tested in combination for the prevention of lung cancer.

A new rexinoid, NRX194204, prevents carcinogenesis in both the lung and mammary gland.

PURPOSE: We evaluated the anti-inflammatory and growth-inhibitory properties of the novel rexinoid NRX194204 (4204) in vitro and then tested its ability to prevent and/or treat experimental lung and estrogen receptor (ER)-negative breast cancer in vivo. EXPERIMENTAL DESIGN: In cell culture studies, we measured the ability of 4204 to block the effects of lipopolysaccharide and induce apoptosis. For the lung cancer prevention studies, A/J mice were injected with the carcinogen vinyl carbamate and then fed 4204 (30-60 mg/kg diet) for 15 weeks, beginning 1 week after the administration of the carcinogen. For breast cancer prevention studies, mouse mammary tumor virus-neu mice were fed control diet or 4204 (20 mg/kg diet) for 50 weeks; for treatment, tumors at least 32 mm3 in size were allowed to form, and then mice were fed control diet or 4204 (60 mg/kg diet) for 4 weeks. RESULTS: Low nanomolar concentrations of 4204 blocked the ability of lipopolysaccharide and tumor necrosis factor-alpha to induce the release of nitric oxide and interleukin 6 and the degradation of IKBalpha in RAW264.7 macrophage-like cells. In the A/J mouse model of lung cancer, 4204 significantly (P < 0.05) reduced the number and size of tumors on the surface of the lungs and reduced the total tumor volume per slide by 64% to 81% compared with the control group. In mouse mammary tumor virus-neu mice, 4204 not only delayed the development of ER-negative mammary tumors in the prevention studies but also caused marked tumor regression (92%) or growth arrest (8%) in all of the mammary tumors when used therapeutically. CONCLUSIONS: The combined anti-inflammatory and anticarcinogenic actions of 4204 suggest that it is a promising new rexinoid that should be considered for future clinical trials.

Novel semisynthetic analogues of betulinic acid with diverse cytoprotective, antiproliferative, and proapoptotic activities.

Betulinic acid (BA), a pentacyclic triterpene isolated from birch bark and other plants, selectively inhibits the growth of human cancer cell lines. However, the poor potency of BA hinders its clinical development, despite a lack of toxicity in animal studies even at high concentrations. Here, we describe six BA derivatives that are markedly more potent than BA for inhibiting inducible nitric oxide synthase, activating phase 2 cytoprotective enzymes, and inducing apoptosis in cancer cells and in Bax/Bak(-/-) fibroblasts, which lack two key proteins involved in the intrinsic, mitochondrial-dependent apoptotic pathway. Notably, adding a cyano-enone functionality in the A ring of BA enhanced its cytoprotective properties, but replacing the cyano group with a methoxycarbonyl strikingly increased potency in the apoptosis assays. Higher plasma and tissue levels were obtained with the new BA analogues, especially CBA-Im [1-(2-cyano-3-oxolupa-1,20(29)-dien-28-oyl)imidazole], compared with BA itself and at concentrations that were active in vitro. These results suggest that BA is a useful platform for drug development, and the enhanced potency and varied biological activities of CBA-Im make it a promising candidate for further chemoprevention or chemotherapeutic studies.

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.

The combination of the rexinoid, LG100268, and a selective estrogen receptor modulator, either arzoxifene or acolbifene, synergizes in the prevention and treatment of mammary tumors in an estrogen receptor-negative model of breast cancer.

PURPOSE: We tested whether a selective estrogen receptor modulator (SERM) and a rexinoid are active for prevention and treatment in the mouse mammary tumor virus-neu mouse model of estrogen receptor-negative breast cancer. EXPERIMENTAL DESIGN: For prevention, mice were fed a powdered control diet, the SERM arzoxifene (Arz, 20 mg/kg diet), the rexinoid LG100268 (268, 30 mg/kg diet), or the combination for 60 weeks. In a second prevention study, mice were fed Arz (6 mg/kg diet), 268 (30 mg/kg diet), the combination of Arz and 268, the SERM acolbifene (Acol, 3 mg/kg diet), or the combination of Acol and 268 for 52 weeks. For the treatment studies, mice with tumors were fed combinations of a SERM and 268 for 4 weeks. RESULTS: The rexinoid 268 and the SERMs Arz and Acol, as individual drugs, delayed the development of estrogen receptor-negative tumors. Moreover, the combination of a SERM and 268 was strikingly synergistic, as no tumors developed in any mouse fed the combination of 268 and a SERM. Moreover, this drug combination also induced significant tumor regression when used therapeutically. These drugs did not inhibit transgene expression in vitro or in vivo, and the combination of Arz and 268 inhibited proliferation and induced apoptosis in the tumors. CONCLUSION: The combination of a rexinoid and SERM should be considered for future clinical trials.

Design, synthesis, and anti-inflammatory activity both in vitro and in vivo of new betulinic acid analogues having an enone functionality in ring A.

Fifteen new betulinic acid analogues were designed, synthesized, and tested for anti-inflammatory activity. Many of these analogues effectively suppress nitric oxide (NO) production in RAW cells stimulated with interferon-gamma. Analogue 10 is highly and orally active in vivo for induction of the anti-inflammatory and cytoprotective enzyme, heme oxygenase-1.

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.

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.