Nrf2 expression in pancreatic stellate cells promotes progression of cancer

It was previously identified that systemic Nrf2-deletion attenuates pancreatic cancer progression in a mutant K-ras/p53-expressing mouse model (KPC mouse). In this study, the type of cell that is responsible for the retarded cancer progression was elucidated. Human pancreatic cancers were first examined, and elevated expression of NRF2-target gene products in a-smooth muscle actin-positive cells was found, suggesting that pancreatic stellate cells (PSCs) are involved in this process. Closer examination of primary cultured PSCs from Nrf2-deleted mice revealed that the cells were less proliferative and retained a lower migration capacity. The conditioned medium of Nrf2-deleted PSCs exhibited reduced growth-stimulating effects in pancreatic cancer cells. KPC mouse-derived pancreatic cancer cells co-injected with wild-type PSCs developed significantly larger subcutaneous tumors in immunodeficient mice than those co-injected with Nrf2-deleted PSCs. These results demonstrate that Nrf2 actively contributes to the function of PSCs to sustain KPC cancer progression, thus, suggesting that Nrf2 inhibition in PSCs may be therapeutically important in pancreatic cancer.

Brusatol Sensitizes Endometrial Hyperplasia and Cancer to Progestin by Suppressing Nrf2-Tet1-AKR1C1-Mediated Progestin Metabolism

BackgroundProgestin resistance is the main obstacle for the conservative therapy to maintain fertility in women with endometrial hyperplasia and cancer. Up to 30% of endometrial cancers fail to respond to progestin, a rate that has not significantly changed due to the lack of a detailed understanding of progestin resistance.MethodsCell Counting Kit-8 (CCK-8) assays were used to detect the synergistic effects of brusatol in combination with progestin. Using commercial kits, the conversion of progestin to 20α-dihydroxyprogesterone following btusatol treatment or AKR1C1 silence was investigated. The correlation betwwen AKR1C1 expression profile and prgestin response was further analyzed in paired endometrial hyperplasia and cancer samples from the same individuals before and after progestin therapy. The effects of brusatol-mediated reversal of progestin resistance was explored in both mouse xenograft and human organoid models. DNA dot blot, HMeDIP, and dural-luciferase reporter assays were performed to uncover the mechanism through which brusatol inhibits AKR1C1 and sensitizes endometrial hyperplasia and cancer to progestin.ResultsBrusatol sensitizes endometrial cancer cell to progestin by downregulating the expression of Nrf2 and its target AKR1C1. Increased AKR1C1 facilitated production of 20-α-dihydroxyprogeserone and was associated with declined progesterone. Suppression of AKR1C1 by brusatol resulted in decreased progesterone catabolism and maintained potent progesterone to inhibit endometrial cancer growth. Aberrant overexpression of AKR1C1 was found in paired endometrial hyperplasia and cancer samples from the same individuals with progestin resistance, whereas attenuated or loss of AKR1C1 was observed in post-treatment samples with well progestin response as compared with paired pre-treatment tissues. Tet1 was identified as a novel Nrf2 target gene. It in turn upregulated AKR1C1 expression by increasing hydroxymethylation levels in its promoter regions. ConclusionsWe found that Nrf2-Tet1-AKR1C1 axis plays an essential role in progestin resistance, and brusatol sensitizes endometrial hyperplasia and cancer to progestin by suppressing Nrf2-Tet1-AKR1C1-mediated progestin metabolism. Our findings suggest that AKR1C1 expression pattern may serve as an important biomarker of progestin resistance in endometrial hyperplasia and cancer .

Heme-Mediated Activation of the Nrf2/HO-1 Axis Attenuates Calcification of Valve Interstitial Cells

Calcific aortic valve stenosis (CAVS) is a heart disease characterized by the progressive fibro-calcific remodeling of the aortic valves, an actively regulated process with the involvement of the reactive oxygen species-mediated differentiation of valvular interstitial cells (VICs) into osteoblast-like cells. Nuclear factor erythroid 2-related factor 2 (Nrf2) regulates the expression of a variety of antioxidant genes, and plays a protective role in valve calcification. Heme oxygenase-1 (HO-1), an Nrf2-target gene, is upregulated in human calcified aortic valves. Therefore, we investigated the effect of Nrf2/HO-1 axis in VIC calcification. We induced osteogenic differentiation of human VICs with elevated phosphate and calcium-containing osteogenic medium (OM) in the presence of heme. Heme inhibited Ca deposition and OM-induced increase in alkaline phosphatase and osteocalcin (OCN) expression. Heme induced Nrf2 and HO-1 expression in VICs. Heme lost its anti-calcification potential when we blocked transcriptional activity Nrf2 or enzyme activity of HO-1. The heme catabolism products bilirubin, carbon monoxide, and iron, and also ferritin inhibited OM-induced Ca deposition and OCN expression in VICs. This study suggests that heme-mediated activation of the Nrf2/HO-1 pathway inhibits the calcification of VICs. The anti-calcification effect of heme is attributed to the end products of HO-1-catalyzed heme degradation and ferritin.

Docosahexanoic acid signals through the Nrf2–Nqo1 pathway to maintain redox balance and promote neurite outgrowth

Evidence suggests that n-3 polyunsaturated fatty acids (PUFA) may act as activators of the Nrf2 antioxidant pathway. The antioxidant response in-turn promotes neuronal differentiation and neurite outgrowth. Nrf2 has recently been suggested to be a cell intrinsic mediator of DHA signaling. In the current study, we assessed whether DHA mediated axodendritic development was dependent on activation of the Nrf2-pathway and whether Nrf2 protected from agrochemical-induced neuritic retraction. Expression profiling of the DHA-enriched Fat-1 mouse brain relative to WT showed a significant enrichment of genes associated with neuronal development and neuronal projection and genes associated with the Nrf2-transcriptional pathway. Moreover, we found that primary cortical neurons treated with DHA showed a dose dependent increase in Nrf2 transcriptional activity and Nrf2-target gene expression. DHA-mediated activation of Nrf2 promoted neurite outgrowth and inhibited oxidative stress induced neuritic retraction evoked by exposure to agrochemicals. Finally, we provide evidence that this effect is largely dependent on induction of the Nrf2-target gene NAD(P)H: (quinone acceptor) oxidoreductase 1 (NQO1), and that silencing of either Nrf2 or Nqo1 blocks the effects of DHA on the axodendritic compartment. Collectively, these data support a role for the Nrf2-NQO1 pathway in DHA-mediated axodendritic development and protection from agrochemical exposure.

Comparation of Anti-Inflammatory and Antioxidantactivities of Curcumin, Tetrahydrocurcuminand Octahydrocurcuminin LPS-Stimulated RAW264.7 Macrophages

Curcumin (CUR) possesses pronounced anti-inflammatory and antioxidant activities. Generally, the clinical application of CUR is restricted due to its apparent unstability and poor absorption, and the biological activities of CUR may be closely associated with its metabolites. Tetrahydrocurcumin (THC) and octahydrocurcumin (OHC) are two major hydrogenated metabolites of CUR with appreciable biological potentials. Here, we comparatively explored the anti-inflammatory and antioxidant activities of CUR, THC, and OHC in lipopolysaccharide- (LPS-) induced RAW264.7 macrophages. The results revealed that CUR, THC, and OHC dose-dependently inhibited the generation of NO and MCP-1 as well as the gene expression of MCP-1 and iNOS. Additionally, CUR, THC, and OHC significantly inhibited NF-κB activation and p38MAPK and ERK phosphorylation, while substantially upregulated the Nrf2 target gene expression (HO-1, NQO-1, GCLC, and GCLM). Nevertheless, zinc protoporphyrin (ZnPP), a typical HO-1 inhibitor, significantly reversed the alleviative effect of CUR, THC, and OHC on LPS-stimulated ROS generation. These results demonstrated that CUR, THC, and OHC exerted beneficial effect on LPS-stimulated inflammatory and oxidative responses, at least partially, through inhibiting the NF-κB and MAPKs pathways and activating Nrf2-regulated antioxidant gene expression. Particularly, THC and OHC might exert superior antioxidant and anti-inflammatory activities to CUR in LPS-stimulated RAW264.7 cells, which can be further explored to be a promising novel effective agent for inflammatory treatment.

Allyl Isothiocyanate Protects Acetaminophen-Induced Liver Injury via NRF2 Activation by Decreasing Spontaneous Degradation in Hepatocyte

Acetaminophen (APAP) is one of the most frequently prescribed analgesic and anti-pyretic drugs. However, APAP-induced hepatotoxicity is a major cause of acute liver failure globally. While the therapeutic dose is safe, an overdose of APAP produces an excess of the toxic metabolite N-acetyl-p-benzoquinone imine (NAPQI), subsequently resulting in hepatotoxicity. Allyl isothiocyanate (AITC), a bioactive molecule in cruciferous plants, is reported to exert various biological effects, including anti-inflammatory, anti-cancer, and anti-microbial effects. Notably, AITC is known for activating nuclear factor erythroid 2-related factor 2 (NRF2), but there is limited evidence supporting the beneficial effects on hepatocytes and liver, where AITC is mainly metabolized. We applied a mouse model in the current study to investigate whether AITC protects the liver against APAP-induced injury, wherein we observed the protective effects of AITC. Furthermore, NRF2 nuclear translocation and the increase of target genes by AITC treatment were confirmed by in vitro experiments. APAP-induced cell damage was attenuated by AITC via an NRF2-dependent manner, and rapid NRF2 activation by AITC was attributed to the elevation of NRF2 stability by decreasing its spontaneous degradation. Moreover, liver tissues from our mouse experiment revealed that AITC increases the expression of heme oxygenase-1 (HO-1), an NRF2 target gene, confirming the potential of AITC as a hepatoprotective agent that induces NRF2 activation. Taken together, our results indicate the potential of AITC as a natural-product-derived NRF2 activator targeting the liver.

The Integrative Role of Sulforaphane in Preventing Inflammation, Oxidative Stress and Fatigue: A Review of a Potential Protective Phytochemical

Cruciferous vegetables hold a myriad of bioactive molecules that are renowned for possessing unique medicinal benefits. Sulforaphane (SFN) is one of the potential nutraceuticals contained within cruciferous vegetables that is useful for improving health and diseased conditions. The objective of this review is to discuss the mechanistic role for SFN in preventing oxidative stress, fatigue, and inflammation. Direct and indirect research evidence is reported to identify the nontoxic dose of SFN for human trials, and effectiveness of SFN to attenuate inflammation and/or oxidative stress. SFN treatment modulates redox balance via activating redox regulator nuclear factor E2 factor-related factor (Nrf2). SFN may play a crucial role in altering the Keap1/Nrf2/ARE pathway (an intricate response to many stimuli or stress), which induces Nrf2 target gene activation to reduce oxidative stress. In addition, SFN reduces inflammation by suppressing centrally involved inflammatory regulator nuclear factor-kappa B (NF-κB), which in turn downregulates the expression of proinflammatory cytokines and mediators. Exercise may induce a significant range of fatigue, inflammation, oxidative stress, and/or organ damage due to producing excessive reactive oxygen species (ROS) and inflammatory cytokines. SFN may play an effective role in preventing such damage via inducing phase 2 enzymes, activating the Nrf2/ARE signaling pathway or suppressing nuclear translocation of NF-κB. In this review, we summarize the integrative role of SFN in preventing fatigue, inflammation, and oxidative stress, and briefly introduce the history of cruciferous vegetables and the bioavailability and pharmacokinetics of SFN reported in previous research. To date, very limited research has been conducted on SFN’s effectiveness in improving exercise endurance or performance. Therefore, more research needs to be carried out to determine the effectiveness of SFN in the field of exercise and lifestyle factors.

TET Dioxygenase Inhibition As a Therapeutic Strategy in TET2 Mutant Myeloid Neoplasia

TET2 is one of the most commonly mutated genes in myeloid neoplasia. Somatic TET2 mutations (TET2MT) cause complete or partial loss of enzymatic activity. TET2 (along with TET1/3) are Fe2+ and αKG-dependent DNA-dioxygenases that catalyze the oxidation of 5mC→5hmC→5fC→5caC. Ultimately, 5hmC generated by TET2-dioxygenase passively prevents maintenance methylation due to DNA methyltransferase's inability to recognize 5hmC. Alternatively, demethylation may also be a result of base excision repair of fC and caC. TET2MT can serve as therapeutic targets because they are often initiating lesions and present in a large fraction of patients. In this study, a comprehensive analysis of the configurations of TET2MT in myeloid neoplasia including MDS (n=1809) and AML (n=808), showed a remarkable exclusivity with 2-HG producing neomorphic IDH1/2MT (Fig.A). TET2 expression in 97 healthy and 909 MDS/MPN or AML patients from two independent studies showed that IDH1/2MT cases have significantly higher TET2 expression and were also mutually exclusive in cases with lower TET2 expression. Doxycycline inducible expression of IDH1MT led to profound growth inhibition of both a natural TET2MT cell line SIG-M5 and engineered TET2-/- K562, while the effect to parental K562 was mild (Fig.B-E). These observations suggest that mutual exclusivity of TET2MT and IDH1/2MT is due to synthetic lethality of TET2MT cell caused by 2-HG production, rather than redundancy of the consequences of IDH1/2MT and TET2MT. In TET2MT cell 2-HG further inhibit the residual TET-activity (TET1/3) and may cause synthetic lethality to cells with affected TET2 function. SIG-M5 cells expresses significant amount of TET3 while negligible levels of TET1. The reliance on relative compensation through residual TET3 activity has been confirmed in cells by inducible TET3 knockdown. We hypothesized that transient suppression of the residual DNA dioxygenase activity with inhibitors may selectively eliminate TET2-deficient clones. The known TET inhibitors 2-HG, N-oxalylglycine (NOG) and dimethyl methyl fumarate (DMF) lack specificity, pharmacologic properties and potency. Based on the results of in silico docking simulations, we designed and synthesized 16 aKG derivatives. Among them, TETi76 showed best inhibition effect in both TET activity and cell growth of TET2 low expressing cell. TETi76 binds to the α-KG co-factor site of TET2 that principally involves H1801, H1381 and S1898. These amino acids are conserved in all three TET enzymes. To test the in vitro efficacy and specificity of TETi, we used several human myeloid cell lines that harbor loss of function TET2 mutations or constitutively express low TET2 levels as well as bone marrow derived from Tet2+/+, Tet2+/- and Tet2-/- mice (Fig.F-G). Results showed that cells with low 5hmC level were more sensitive to TETi76 treatment. Specificity of TETi76 was further confirmed by RNAseq analyses of TETi76 treated K562, TET2-/- K562 and parental control cells. Moreover, TETi treatment did not appear to affect the function of α-KG-dependent histone dioxygenases. Mechanistically, treatment of SIG-M5 cells with TETi76 induced early and late stages of apoptotic cell death, a finding further confirmed by PARP1 and caspase-3 cleavage. RNAseq analyses of SIGM5 cells after treatment with TETi demonstrated a significant down-regulation of genes involved in transcription and peptide elongation, consistent with the consequences of TET inhibition. Interestingly, we also observed significant up-modulation of oxidative stress response pathway genes consistent with the inhibition of dioxygenases. In particular, TETi76 treatment induces 8-fold increase of oxidative stress sensor NQO1 a NRF2 target gene. To further probe the effects of TETi76 on TET2 deficient cells, Tet2MT/Tet2WT BM cells were co-cultured at fixed ratios to mimic the evolving Tet2MT clones. TETi76 effectively eliminated otherwise dominating Tet2MT cells (Fig.H). To determine the in vivo effects of TETi e.g., on elimination of Tet2MT clones, we performed bone marrow competitive reconstitution assays in PEP mice. TETi treatment selectively restricted the proliferative advantage of Tet2MT HSC compared to vehicle control where, as expected, TET2 mutant clones took over the WT cells. In clinical applications, TET inhibitors may constitute a new class of agents to be used in a targeted fashion in TET2 mutant neoplasia. Figure. Disclosures Meggendorfer: MLL Munich Leukemia Laboratory: Employment. Abazeed:Bayer AG: Honoraria, Other: Travel Support, Research Funding; Siemens: Research Funding. Sekeres:Millenium: Membership on an entity's Board of Directors or advisory committees; Syros: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Maciejewski:Novartis: Consultancy; Alexion: Consultancy.