Bis-indole-derived NR4A1 antagonists inhibit colon tumor and splenic growth and T-cell exhaustion.

There is evidence that the orphan nuclear receptor 4A1 (NR4A1, Nur77) is overexpressed in exhausted CD8 + T cells and regulates PD-L1 in tumors. This study investigated the effects of potent bis-indole-derived NR4A1 antagonists on reversing T-cell exhaustion and downregulating PD-L1 in colon tumors/cells. NR4A1 antagonists inhibited colon tumor growth and downregulated expression of PD-L1 in mouse colon MC-38-derived tumors and cells.

Piperlongumine is a ligand for the orphan nuclear receptor 4A1 (NR4A1).

Piperlongumine and derivatives are being developed as anticancer agents which act primarily as inducers of reactive oxygen species (ROS) in cancer cell lines. Many of the anticancer activities of piperlongumine resemble those observed for bis-indole derived compounds that bind the orphan nuclear receptor 4A1 (NR4A1) and act as inverse receptor agonists to inhibit NR4A1-regulated pro-oncogenic pathways and genes. In this study we show that like other NR4A1 inverse agonists piperlongumine inhibited RKO, SW480 and HCT116 colon cancer cell growth migration and invasion and induced apoptosis.

Flavonoids Quercetin and Kaempferol Are NR4A1 Antagonists and Suppress Endometriosis in Female Mice.

Nuclear receptor 4A1 (NR4A1) plays an important role in endometriosis progression; levels of NR4A1 in endometriotic lesions are higher than in normal endometrium, and substituted bis-indole analogs (NR4A1) antagonists suppress endometriosis progression in mice with endometriosis. In addition, the flavonoids kaempferol and quercetin are natural products that directly bind NR4A1 and significantly repress the intrinsic NR4A1-dependent transcriptional activity in human endometriotic epithelial and stromal cells and Ishikawa endometrial cancer cells. NR4A1 knockdown and inhibition of NR4A1 by kaempferol and quercetin suppressed proliferation of human endometriotic epithelial cells and Ishikawa cells by inhibiting epidermal growth factor receptor/c-Myc/survivin-mediated growth-promoting and survival pathways, The mammalian target of rapamycin (mTOR) signaling and αSMA/CTGF/COL1A1/FN-mediated fibrosis signaling but increasing Thioredoxin domain Containing 5/SESN2-mediated oxidative/estrogen receptors stress signaling.

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Resveratrol is a polyphenolic phytochemical found in fruits, nuts and vegetables that contributes to the remarkable dietary effects of polyphenolic as inhibitors aging and multiple aging related diseases. In addition, resveratrol has been extensively investigated as an inhibitor of inflammatory diseases including cancer, however, the underlying mechanisms of these chemotherapeutic effects of resveratrol are not completely understood. In cancer cells resveratrol inhibits cell growth, survival, migration and invasion, and many of the effects of resveratrol resemble those observed for bis-indole derived (CDIM) compounds that bind the pro-oncogenic nuclear receptor 4A1 (NR4A1, Nur77) and act as receptor antagonists.

Flavonoids kaempferol and quercetin are nuclear receptor 4A1 (NR4A1, Nur77) ligands and inhibit rhabdomyosarcoma cell and tumor growth.

Background: Flavonoids exhibit both chemopreventive and chemotherapeutic activity for multiple tumor types, however, their mechanisms of action are not well defined. Based on some of their functional and gene modifying activities as anticancer agents, we hypothesized that kaempferol and quercetin were nuclear receptor 4A1 (NR4A1, Nur77) ligands and confirmed that both compounds directly bound NR4A1 with K values of 3.1 and 0.

Transcription factors specificity protein and nuclear receptor 4A1 in pancreatic cancer.

Specificity protein (Sp) transcription factors (TFs) Sp1, Sp3 and Sp4, and the orphan nuclear receptor 4A1 (NR4A1) are highly expressed in pancreatic tumors and Sp1 is a negative prognostic factor for pancreatic cancer patient survival. Results of knockdown and overexpression of Sp1, Sp3 and Sp4 in pancreatic and other cancer lines show that these TFs are individually pro-oncogenic factors and loss of one Sp TF is not compensated by other members. NR4A1 is also a pro-oncogenic factor and both NR4A1 and Sp TFs exhibit similar functions in pancreatic cancer cells and regulate cell growth, survival, migration and invasion.

Nuclear receptor 4A1 (NR4A1) antagonists target paraspeckle component 1 (PSPC1) in cancer cells.

Paraspeckles compound 1 (PSPC1) is a multifunctional protein that plays an important role in cancer cells, where PSPC1 is a master regulator of pro-oncogenic responses that includes activation of TGFβ (TGFβ1), TGFβ-dependent EMT, and metastasis. The pro-oncogenic activities of PSPC1 closely resembled those observed for the orphan nuclear receptor 4A1 (NR4A1, Nur77) and knockdown of NR4A1 decreased expression of PSPC1 in MDA-MB-231 breast, H1299 lung, and SNU449 liver cancer cells. Similar results were observed in these same cell lines after treatment with bisindole-derived (CDIMs) NR4A1 antagonists.

Synthetic curcumin analog: inhibiting the invasion, angiogenesis, and metastasis in human laryngeal carcinoma cells via NF-kB pathway.

Background: Laryngeal carcinoma, the most common among head and neck squamous cell carcinoma (HNSCC), induces 1% of all cancer deaths. Curcumin the active constituent of turmeric, is shown to be effective in the treatment of various cancers. In the present study, we explored the mechanistic role of bis-demethoxy curcumin analog (BDMC-A) as a chemotherapeutic agent.

Orphan nuclear receptor 4A1 (NR4A1) and novel ligands.

The nuclear receptor (NR) superfamily of transcription factors encodes expression of 48 human genes that are important for maintaining cellular homeostasis and in pathophysiology, and this has been observed for all sub-families including orphan receptors for which endogenous ligands have not yet been identified. The orphan NR4A1 (Nur77 and TR3) and other members of this sub-family (NR4A2 and NR4A3) are immediate early genes induced by diverse stressors, and these receptors play an important role in the immune function and are up-regulated in some inflammatory diseases including solid tumors. Although endogenous ligands for NR4A have not been identified, several different classes of compounds have been characterized as NR4A1 ligands that bind the receptor.

NR4A1 Ligands as Potent Inhibitors of Breast Cancer Cell and Tumor Growth.

Nuclear receptor 4A1 (NR4A1, Nur77, TR3) is more highly expressed in breast and solid tumors compared to non-tumor tissues and is a pro-oncogenic factor in solid tumor-derived cancers. NR4A1 regulates cancer cell growth, survival, migration, and invasion, and bis-indole-derived compounds (CDIMs) that bind NR4A1 act as antagonists and inhibit tumor growth. Preliminary structure-binding studies identified 1,1-bis(3'-indolyl)-1-(3,5-disubstitutedphenyl)methane analogs as NR4A1 ligands with low K values; we further investigated the anticancer activity of the four most active analogs (K's ≤ 3.

Flavonoids: structure-function and mechanisms of action and opportunities for drug development.

Flavonoids are polyphenolic phytochemicals produced in fruits, nuts and vegetables and dietary consumption of these structurally diverse compounds is associated with multiple health benefits including increased lifespan, decreased cardiovascular problems and low rates of metabolic diseases. Preclinical studies with individual flavonoids demonstrate that these compounds exhibit anti-inflammatory and anticancer activities and they enhance the immune system. Their effectiveness in both chemoprevention and chemotherapy is associated with their targeting of multiple genes/pathways including nuclear receptors, the aryl hydrocarbon receptor (AhR), kinases, receptor tyrosine kinases and G protein-coupled receptors.

The Histone Methyltransferase Gene G9A Is Regulated by Nuclear Receptor 4A1 in Alveolar Rhabdomyosarcoma Cells.

The histone methyltransferase G9A (EHMT2) gene catalyzes methylation of histone 3 lysine 9 (H3K9), and this gene silencing activity contributes to the tumor promoter-like activity of G9A in several tumor types including alveolar rhabdomyosarcoma (ARMS). Previous studies show the orphan nuclear receptor 4A1 (NR4A1, Nur77) is overexpressed in rhabdomyosarcoma and exhibits pro-oncogenic activity. In this study, we show that knockdown of NR4A1 in ARMS cells decreased expression of G9A mRNA and protein.

Bis-indole derived nuclear receptor 4A1 (NR4A1) antagonists inhibit TGFβ-induced invasion of embryonal rhabdomyosarcoma cells.

Transforming growth factor β (TGFβ) enhances invasion of breast and lung cancer cells through phosphorylation-dependent nuclear export of the nuclear receptor 4A1 (NR4A1, Nur77). This response is inhibited by the NR4A1 antagonist 1,1-bis(3'-indoly)-1-(p-hydroxyphenyl) methane (CDIM8) and we hypothesized that similar effects would be observed in Rhabdomyosarcoma (RMS) cells. Although some kinase inhibitors block TGFβ-induced invasion of embryonal RMS (ERMS) cells, the mechanism differs from breast and lung cancer cells since NR4A1 is extranuclear in ERMS cells.

Bis-Indole-Derived Nuclear Receptor 4A1 (NR4A1, Nur77) Ligands as Inhibitors of Endometriosis.

Endometriosis is an inflammatory disease that primarily affects women during their reproductive years, and since current hormonal therapies are of concern, new hormone-independent treatment regimens are needed. The orphan nuclear receptor 4A1 (NR4A1, Nur77) is expressed in patient-derived (stromal) endometriotic cells and also epithelial cell lines, and we observed that knockdown of NR4A1 in patient-derived ectopic endometrium-isolated ovarian endometrioma (ESECT)-7 and ESECT-40 cells decreased cell proliferation and induced apoptosis. Moreover, the treatment of these cells with bis-indole derived NR4A1 ligands 1,1-bis(3'-indolyl)-1-(p-hydroxyphenyl)methane (DIM-C-pPhOH) and its buttressed 3-chloro-5-methoxy analog (DIM-C-pPhOH-3-Cl-5-OCH3) inhibited cell growth and induced apoptosis and related genes.

A Bis-Indole-Derived NR4A1 Antagonist Induces PD-L1 Degradation and Enhances Antitumor Immunity.

PD-L1 is expressed in tumor cells and its interaction with PD-1 plays an important role in evading immune surveillance; this can be overcome using PD-L1 or PD-1 immunotherapy antibodies. This study reports a novel approach for targeting PD-L1. In human breast cancer cell lines and 4T1 mouse mammary tumor cells, PD-L1 expression was regulated by the nuclear receptor NR4A1/Sp1 complex bound to the proximal germinal center (GC)-rich region of the PD-L1 gene promoter.

Nuclear receptor 4A2 (NR4A2) is a druggable target for glioblastomas.

Introduction: The orphan nuclear receptor 4A2 (NR4A2) has been extensively characterized in subcellular regions of the brain and is necessary for the function of dopaminergic neurons. The NR4A2 ligand, 1,1-bis (3-indoly1)-1-(p-chlorophenyl)methane (DIM-C-pPhCl) inhibits markers of neuroinflammation and degeneration in mouse models and in this study we investigated expression and function of NR4A2 in glioblastoma (GBM).

Methods: Established and patient-derived cell lines were used as models and the expression and functions of NR4A2 were determined by western blots and NR4A2 gene silencing by antisense oligonucleotides respectively.

Inhibition of NR4A1 Promotes ROS Accumulation and IL24-Dependent Growth Arrest in Rhabdomyosarcoma.

Nuclear receptor 4A1 (NR4A1, Nur77) is overexpressed in rhabdomyosarcoma (RMS), and inactivation of NR4A1 (siNR4A1) or treatment with the NR4A1 antagonist 1,1-bis(3'-indoly)-1-(p-hydroxy-phenyl)methane (DIM-C-pPhOH) has antiproliferative and proapoptotic effects on RMS cells. However, the mechanism by which NR4A1 inhibition exerts these effects is poorly defined. Here, we report that NR4A1 silencing or inhibition resulted in accumulation of reactive oxygen species (ROS) and ROS-dependent induction of the tumor suppressor-like cytokine IL24 in RMS cells.

Aryl Hydrocarbon Receptor (AhR) Ligands as Selective AhR Modulators: Genomic Studies.

The aryl hydrocarbon receptor (AhR) binds structurally diverse ligands that vary from the environmental toxicant 2,3,7,8-tetrachlorodibenzo--dioxin (TCDD) to AhR- active pharmaceuticals and health-promoting phytochemicals. There are remarkable differences in the toxicity of TCDD and related halogenated aromatics (HAs) vs. health promoting AhR ligands, and genomic analysis shows that even among the toxic HAs, there are differences in their regulation of genes and pathways.

Potent inhibition of breast cancer by bis-indole-derived nuclear receptor 4A1 (NR4A1) antagonists.

Background: Nuclear receptor 4A1 (NR4A1) is overexpressed in mammary tumors, and the methylene-substituted bis-indole derivative 1,1-bis(3'-indolyl)-1-(p-hydroxyphenyl)methane (DIM-C-pPhOH) acts as an NR4A1 antagonist (inverse agonist) and inhibits NR4A1-regulated pro-oncogenic pathways/genes in breast and other cancer cells.

Methods: Buttressed analogs of DIM-C-pPhOH were synthesized by condensation of the substituted p-hydroxybenzaldehydes with indole. Breast cancer cell growth, survival, and migration assays were carried out by cell counting, Annexin V staining, and Boyden chamber assays, respectively.

Genetic Polymorphisms in the Open Reading Frame of the CCR5 gene From HIV-1 Seronegative and Seropositive Individuals From National Capital Regions of India.

C-C chemokine receptor type 5 (CCR5) serves as a co-receptor for Human immunodeficiency virus (HIV), enabling the virus to enter human CD4 T cells and macrophages. In the absence of CCR5, HIV strains that require CCR5 (R5 or M-tropic HIV) fail to successfully initiate infection. Various natural mutations of the CCR5 gene have been reported to interfere with the HIV-CCR5 interaction, which influences the rate of AIDS progression.

Nuclear receptor 4A1 (NR4A1) antagonists induce ROS-dependent inhibition of mTOR signaling in endometrial cancer.

Objectives: NR4A1 is overexpressed in many solid tumors, and the objectives of this study were to investigate the expression and functional role of this receptor in endometrial cancer cells and demonstrate that NR4A1 antagonist inhibit mTOR.

Methods: Ishikawa and Hec-1B endometrial cells were used as models to investigate the parallel effects of NR4A1 knockdown by RNA interference (siNR4A1) and treatment with bis-indole-derived NR4A1 ligands (antagonists) on cell growth and survival by determining cell numbers and effects on Annexin V staining. Western blot analysis of whole cell lysates was used to determine effects of these treatments on expression of growth promoting, survival and apoptotic genes and mTOR signaling.

Gelatin-Based Matrices as a Tunable Platform To Study in Vitro and in Vivo 3D Cell Invasion.

Hydrogels have been used as synthetic mimics of 3D extracellular matrices (ECM) and their physical properties like stiffness, degradability, and porosity have been known to influence the behavior of encapsulated cells. However, to understand the role of individual properties, the influence of biophysical cues should be decoupled from biochemical ones. In this study, we have used hydrogels as a tunable model matrix to develop a 3D cell culture platform for studying cell invasion.

Reactive Oxygen Species (ROS)-Inducing Triterpenoid Inhibits Rhabdomyosarcoma Cell and Tumor Growth through Targeting Sp Transcription Factors.

Methyl 2-trifluoromethyl-3,11-dioxo-18β-olean-1,12-dien-3-oate (CFDODA-Me) is derived synthetically from glycyrrhetinic acid, a major component of licorice, and this compound induced reactive oxygen species (ROS) in RD and Rh30 rhabdomyosarcoma (RMS) cells. CFDODA-Me also inhibited growth and invasion and induced apoptosis in RMS cells, and these responses were attenuated after cotreatment with the antioxidant glutathione, demonstrating the effective anticancer activity of ROS in RMS. CFDODA-Me also downregulated expression of specificity protein (Sp) transcription factors Sp1, Sp3, and Sp4 and prooncogenic Sp-regulated genes including PAX3-FOXO1 (in Rh30 cells).

Interleukin-24 (IL24) Is Suppressed by PAX3-FOXO1 and Is a Novel Therapy for Rhabdomyosarcoma.

Alveolar rhabdomyosarcoma (ARMS) patients have a poor prognosis, and this is primarily due to overexpression of the oncogenic fusion protein PAX3-FOXO1. Results of RNA-sequencing studies show that PAX3-FOXO1 represses expression of interleukin-24 (IL24), and these two genes are inversely expressed in patient tumors. PAX3-FOXO1 also regulates histone deacetylase 5 (HDAC5) in ARMS cells, and results of RNA interference studies confirmed that PAX3-FOXO1-mediated repression of IL24 is HDAC5-dependent.

TGFβ-Induced Lung Cancer Cell Migration Is NR4A1-Dependent.

TGFβ induces migration of lung cancer cells (A549, H460, and H1299), dependent on activation of c-Jun N-terminal kinase (JNK1), and is inhibited by the JNK1 inhibitor SP600125. Moreover, TGFβ-induced migration of the cells is also blocked by the nuclear export inhibitor leptomycin B (LMB) and the orphan nuclear receptor 4A1 (NR4A1) ligand 1,1-bis(3'-indolyl)-1-(-hydroxyphenyl)methane (CDIM8), which retains NR4A1 in the nucleus. Subsequent analysis showed that the TGFβ/TGFβ receptor/PKA/MKK4 and -7/JNK pathway cascade phosphorylates and induces nuclear export of NR4A1, which in turn forms an active complex with Axin2, Arkadia (RNF111), and RNF12 (RLIM) to induce proteasome-dependent degradation of SMAD7 and enhance lung cancer cell migration.