CDDO drives transcriptomic and epigenetic reprogramming in response to TPA-induced JB6 cell neoplastic transformation [Methyl-seq]
ABSTRACT: 12-O-tetradecanoylphorbol-13-acetate (TPA) promotes skin carcinogenesis. CDDO is a potential antioxidative and antiinflammatory agent to prevent the TPA-induced skin cell transformation at nanomolar scale. We characterized the transcriptome, CpG methylome, and pathway network of JB6 cells treated with TPA and TPA + CDDO using RNA sequencing, methyl sequencing, and QIAGEN Ingenuity Pathway Analysis. Overall design: Samples of JB6 cells were used for extraction of DNA and RNA; DNA was enriched by Agilent SureSelect Kit then bisulfite converted methyl-seq was performed. RNA-seq was performed with routine strategy. This dataset contains data for the Methyl-seq only; Check the other SubSeries under this SuperSeries for RNA-seq data.
Project description:12-O-tetradecanoylphorbol-13-acetate (TPA) promotes skin carcinogenesis. CDDO is a potential antioxidative and antiinflammatory agent to prevent the TPA-induced skin cell transformation at nanomolar scale. We characterized the transcriptome, CpG methylome, and pathway network of JB6 cells treated with TPA and TPA + CDDO using RNA sequencing, methyl sequencing, and QIAGEN Ingenuity Pathway Analysis. Overall design: Samples of JB6 cells were used for extraction of DNA and RNA; DNA was enriched by Agilent SureSelect Kit then bisulfite converted methyl-seq was performed. RNA-seq was performed with routine strategy. This dataset contains data for the RNA-seq only; Check the other SubSeries under this SuperSeries for Methyl-seq data.
Project description:Epigenetic mechanisms play an important role in the early stages of carcinogenesis. Moringa isothiocyanate (MIC-1) is a major bioactive component derived from Moringa oleifera that has considerable antioxidant and anti-inflammatory effects. However, how MIC-1 influences epigenomic alterations in TPA-mediated JB6 cell carcinogenic transformation has not been evaluated. In this study, DNA and RNA isolated from TPA-induced JB6 cells in the presence or absence of MIC-1 were subjected to DNA Methyl-seq and RNA-seq to identify differentially methylated regions (DMRs) and differentially expressed genes (DEGs), respectively. When JB6 cells were challenged with TPA alone, there was a significant alteration of DEGs and DMRs; importantly, MIC-1 treatment reversed the patterns of some of the DEGs and DMRs. Transcriptome and CpG methylome profiling was performed in Ingenuity® Pathway Analysis (IPA) software to analyze the altered signaling pathways. Several anti-inflammatory responses, antioxidative stress-related pathways, and anticancer-related pathways were identified to be affected by MIC-1. These pathways included NF-kB, IL-1, LPS/IL-1-mediated inhibition of RXR function, Nrf2-mediated oxidative stress response, p53, and PTEN signaling pathways. Examination of correlations between transcriptomic and CpG methylome profiles yielded a small subset of genes, including the cancer-related genes Tmpt, Tubb3, and Muc2; the GTPases Gchfr and Igtp; and the cell cycle-related gene Cdc7. Taken together, our results show the potential contributions of epigenomic changes in DNA CpG methylation to gene expression to molecular pathways active in TPA-induced JB6 cells and demonstrate that MIC-1 can reverse these changes, supporting the potential preventive/treatment effects of MIC-1 against skin carcinogenesis.
Project description:Sepsis induced lethality is characterized by amplified host innate immune response. Nrf2, a bZIP transcription factor, regulates a battery of cellular antioxidative genes and maintains cellular redox homeostasis. This study demonstrates that increasing Nrf2 activity by a potent small molecule activator, CDDO-Im (1-[2-cyano-3-,12-dioxooleana-1,9(11)-dien-28-oyl]imidazole), protects from deregulation of lipopolysaccharide (LPS) induced innate immune response. In response to LPS stimuli, nrf2-deficient (nrf2 -/-) peritoneal neutrophils showed increased NADPH oxidase-dependent ROS generation, proinflammatory cytokines (Tnf-alpha and Il-6) and chemokines (Mip2 and Mcp-1) relative to wild-type (nrf2 +/+) cells. Pretreatment of peritoneal neutrophils with CDDO-Im induced antioxidative genes (Ho-1, Gclc, Gclm, and Nqo1) and attenuated LPS induced ROS generation as well as expression of proinflammatory cytokines exclusively in nrf2 +/+ neutrophils but not in nrf2 -/- cells. In corroboration with in vitro studies, pretreatment with CDDO-Im induced Nrf2-dependent antioxidative genes, attenuated LPS induced proinflammatory cytokine expression, and decreased mortality specifically in the nrf2 +/+ mice. In conclusion, the results suggest that Nrf2 is associated with oxidative regulation of LPS induced innate immune response in neutrophils. Activation of Nrf2-dependent compensatory antioxidative pathways by CDDO-Im protects from LPS induced inflammatory response and mortality.
Project description:Ionizing radiation produces reactive oxygen species (ROS) leading to cellular DNA damage. Therefore, patients undergoing radiation therapy or first responders in radiological accident scenarios could both benefit from the identification of specifically acting pharmacological radiomitigators. The synthetic triterpenoid bardoxolone-methyl (CDDO-Me) has previously been shown to exert antioxidant, anti-inflammatory and anticancer activities in several cell lines, in part by enhancing the DNA damage response. In our study, we examined the effect of nanomolar concentrations of CDDO-Me in human peripheral blood mononuclear cells (PBMC). We observed increased cellular levels of the antioxidative enzymes heme oxygenase-1 (HO-1), NAD(P)H dehydrogenase (quinone1) and mitochondrial superoxide dismutase 2 by immunoblotting. Surprisingly, we found increased intracellular ROS-levels using imaging flow-cytometry. However, the radiation-induced DNA double-strand break (DSB) formation using the ?-H2AX?+?53BP1 DSB focus assay and the cytokinesis-block micronucleus assay both revealed, that nanomolar CDDO-Me pre-treatment of PBMC for 2 h or 6 h ahead of X irradiation with 2 Gy did neither significantly affect ?-H2AX?+?53BP1 DSB foci formation nor the frequency of micronuclei. CDDO-Me treatment also failed to alter the nuclear division index and the frequency of IR-induced PBMC apoptosis as investigated by Annexin V-labeled live-cell imaging. Our results indicate that pharmacologically increased cellular concentrations of antioxidative enzymes might not necessarily exert radiomitigating short-term effects in IR-exposed PBMC. However, the increase of antioxidative enzymes could also be a result of a defensive cellular mechanism towards elevated ROS levels.
Project description:Triterpenoids are natural compounds synthesized by plants through cyclization of squalene, known for their weak anti-inflammatory activity. 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid (CDDO), and its C28 modified derivative, methyl-ester (CDDO-Me, also known as bardoxolone methyl), are two synthetic derivatives of oleanolic acid, synthesized more than 20 years ago, in an attempt to enhance the anti-inflammatory behavior of the natural compound. These molecules have been extensively investigated for their strong ability to exert antiproliferative, antiangiogenic, and antimetastatic activities, and to induce apoptosis and differentiation in cancer cells. Here, we discuss the chemical properties of natural triterpenoids, the pathways of synthesis and the biological effects of CDDO and its derivative CDDO-Me. At nanomolar doses, CDDO and CDDO-Me have been shown to protect cells and tissues from oxidative stress by increasing the transcriptional activity of the nuclear factor (erythroid-derived 2)-like 2 (Nrf2). At doses higher than 100 nM, CDDO and CDDO-Me are able to modulate the differentiation of a variety of cell types, both tumor cell lines or primary culture cell, while at micromolar doses these compounds exert an anticancer effect in multiple manners; by inducing extrinsic or intrinsic apoptotic pathways, or autophagic cell death, by inhibiting telomerase activity, by disrupting mitochondrial functions through Lon protease inhibition, and by blocking the deubiquitylating enzyme USP7. CDDO-Me demonstrated its efficacy as anticancer drugs in different mouse models, and versus several types of cancer. Several clinical trials have been started in humans for evaluating CDDO-Me efficacy as anticancer and anti-inflammatory drug; despite promising results, significant increase in heart failure events represented an obstacle for the clinical use of CDDO-Me.
Project description:Patients with cirrhosis have an increased risk of developing liver cancer and a higher rate of mortality. Cirrhosis currently has no known cure, and patients may benefit from new agents aimed at alleviating their complications and slowing down the rate of disease progression. Therefore, the effects of the orally bioavailable synthetic triterpenoid 2-cyano-3,12-dioxooleana- 1,9(11)-dien-28-oate-ethyl amide (CDDO-EA, RTA 405), which has potent antioxidative and antiinflammatory properties, was evaluated in a chronic carbon tetrachloride (CCl(4))-induced model of liver cirrhosis and hepatocellular carcinoma (HCC). Mice were injected with CCl(4) (to induce fibrosis and cirrhosis) or placebo biweekly for 12 weeks followed by CDDO-EA in the diet for 18 weeks with continued biweekly injections of CCl(4). Chronic CCl(4) administration resulted in cirrhosis, ascites, and HCC formation, associated with increased serum transforming growth factor-?1, hepatic hydroxyproline content, and increased serum bilirubin. CDDO-EA, whose administration commenced after establishment of liver fibrosis, decreased liver fibrosis progression, serum bilirubin, ascites, and HCC formation and markedly increased overall survival. CDDO-EA also attenuated -TNF? (tumor necrosis factor-?), ?-SMA (alpha smooth muscle actin), augmented -IL-10 levels, and improved histologic and serologic markers of fibrosis.CDDO-EA mitigates the progression of liver fibrosis induced by chronic CCl(4) administration, which is associated with the induction of antifibrogenic genes and suppression of profibrogenic genes.
Project description:The aim of present study was to explore whether 2-cyano-3, 12-dioxooleana-1, 9-dien-28-oic acid (CDDO)-ethylamide (CDDO-EA) attenuates cerebral ischemic injury and its possible mechanisms using a middle cerebral artery occlusion (MCAO) model in C57BL/6 mice. Our results showed that intraperitoneal injection (i.p.) of CDDO-EA (2 and 4 mg/kg) augmented NFE2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) expression in ischemic cortex after MCAO. Moreover, CDDO-EA (2 mg/kg, i.p.) significantly enhanced Nrf2 nuclear accumulation, associated with increased cytosolic HO-1 expression, reduced neurological deficit and infarct volume as well as neural apoptosis, and shifted polarization of microglia/macrophages toward an antiinflammatory M2 phenotype in ischemic cortex after MCAO. Using an in vitro model, we confirmed that CDDO-EA (100 ?g/mL) increased HO-1 expression and primed microglial polarization toward M2 phenotype under inflammatory stimulation in BV2 microglial cells. These findings suggest that a novel Nrf2 activator CDDO-EA confers neuroprotection against ischemic injury.
Project description:Pulmonary Fibrosis (PF) is a devastating progressive disease in which normal lung structure and function is compromised by scarring. Lung fibrosis can be caused by thoracic radiation, injury from chemotherapy and systemic diseases such as rheumatoid arthritis that involve inflammatory responses. CDDO-Me (Methyl 2-cyano-3,12-dioxooleana-1,9(11)dien-28-oate, Bardoxolone methyl) is a novel triterpenoid with anti-fibrotic and anti-inflammatory properties as shown by our in vitro studies. Based on this evidence, we hypothesized that CDDO-Me would reduce lung inflammation, fibrosis and lung function impairment in a bleomycin model of lung injury and fibrosis. To test this hypothesis, mice received bleomycin via oropharyngeal aspiration (OA) on day zero and CDDO-Me during the inflammatory phase from days -1 to 9 every other day. Bronchoalveolar lavage fluid (BALF) and lung tissue were harvested on day 7 to evaluate inflammation, while fibrosis and lung function were evaluated on day 21. On day 7, CDDO-Me reduced total BALF protein by 50%, alveolar macrophage infiltration by 40%, neutrophil infiltration by 90% (p?0.01), inhibited production of the inflammatory cytokines KC and IL-6 by over 90% (p?0.001), and excess production of the pro-fibrotic cytokine TGF? by 50%. CDDO-Me also inhibited ?-smooth muscle actin and fibronectin mRNA by 50% (p?0.05). On day 21, CDDO-Me treatment reduced histological fibrosis, collagen deposition and ?SMA production. Lung function was significantly improved at day 21 by treatment with CDDO-Me, as demonstrated by respiratory rate and dynamic compliance. These new findings reveal that CDDO-Me exhibits potent anti-fibrotic and anti-inflammatory properties in vivo. CDDO-Me is a potential new class of drugs to arrest inflammation and ameliorate fibrosis in patients who are predisposed to lung injury and fibrosis incited by cancer treatments (e.g. chemotherapy and radiation) and by systemic autoimmune diseases.
Project description:Reactivation of telomerase in cancers provides an attractive target for developing novel agents to selectively destroy tumor cells. Methyl-2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oate (CDDO-Me), a synthetic oleanane triterpenoid, inhibited cell proliferation and induced apoptosis in pancreatic cancer cells at very low concentrations. The antiproliferative and apoptosis-inducing effects of CDDO-Me were associated with the inhibition of human telomerase reverse transcriptase (hTERT) mRNA, hTERT protein and reduction in hTERT telomerase activity. CDDO-Me inhibited multiple transcription factors that regulate hTERT expression positively (Sp1, c-Myc and NF-?B) and negatively (CTCF, E2F-1 and MAD1). CDDO-Me inhibited protein levels of DNA methyl transferases DNMT1 and DNMT3a, which also resulted in hypomethylation of hTERT promoter. In addition, transcriptionally active chromatin markers, such as acetylated histone H3 (Lys 9), acetylated histone H4, di-methyl H3 (Lys 4) and tri-methyl H3 (Lys 9) were all reduced in pancreatic cancer cells treated with CDDO-Me. Chromatin immunoprecipitation analysis showed decreased histone deacetylation and histone demethylation at hTERT promoter. Collectively, these results indicate that down-regulation of telomerase through epigenetic mechanisms plays a critical role in induction of apoptosis in pancreatic cancer cells by CDDO-Me.
Project description:The tumor microenvironment (TME) is an essential contributor to the development and progression of malignancy. Within the TME, tumor associated macrophages (TAMs) mediate angiogenesis, metastasis, and immunosuppression, which inhibits infiltration of tumor-specific cytotoxic CD8+ T cells. In previous work, we demonstrated that the synthetic triterpenoid CDDO-methyl ester (CDDO-Me) converts breast TAMs from a tumor-promoting to a tumor-inhibiting activation state in vitro. We show now that CDDO-Me remodels the breast TME, redirecting TAM activation and T cell tumor infiltration in vivo. We demonstrate that CDDO-Me significantly attenuates IL-10 and VEGF expression but stimulates TNF production, and reduces surface expression of CD206 and CD115, markers of immunosuppressive TAMs. CDDO-Me treatment redirects the TAM transcriptional profile, inducing signaling pathways associated with immune stimulation, and inhibits TAM tumor infiltration, consistent with decreased expression of CCL2. In CDDO-Me-treated mice, both the absolute number and proportion of splenic CD4+ T cells were reduced, while the proportion of CD8+ T cells was significantly increased in both tumors and spleen. Moreover, mice fed CDDO-Me demonstrated significant reductions in numbers of CD4+ Foxp3+ regulatory T cells within tumors. These results demonstrate for the first time that CDDO-Me relieves immunosuppression in the breast TME and unleashes host adaptive anti-tumor immunity.