Retinoic Acid Receptors Alpha Antagonist Research Articles

Investigation of selective retinoic acid receptor alpha antagonist ER-50891 and related analogs for male contraception.

Retinoic acid receptor alpha (RARα) antagonist ER-50891 and 15 analogs were prepared and tested in vitro for potency and selectivity at RARα, RARβ, and RARγ using transactivation assays. Minor modifications to the parent molecule such as the introduction of a C4 tolyl group in place of the C4 phenyl group on the quinoline moiety slightly increased the RARα selectivity but larger substituents significantly decreased the potency. Replacement of the pyrrole moiety of ER-50891 with triazole, amides, or a double bond produced inactive compounds. ER-50891 was found to be stable in male mouse liver microsomes and was tested in male mice to assess its effects on spermatogenesis. Characteristic, albeit modest and transient, effects on spermatogenesis were observed.

Myeloid differentiation and retinoblastoma phosphorylation changes in HL-60 cells induced by retinoic acid receptor- and retinoid X receptor- selective retinoic acid analogs

The ability of subtypes of retinoic acid receptors (RARs) and retinoid X receptors (RXRs) singly and in combination to elicit myeloid differentiation, G1/0-specific growth arrest, and retinoblastoma (RB) tumor suppressor protein dephosphorylation was determined in the human myeloblastic leukemia cell line HL-60 using subtype-selective retinoic acid (RA) analogs. RA analogs that selectively bind only to RARs (Am580 and/or TTNPB) or to RXRs (Ro 25-6603, SR11237, and/or SR11234) did not elicit the above-mentioned three cellular responses. In contrast, simultaneous treatment with both an RAR-selective ligand (Am580 or TTNPB) and an RXR-selective ligand (Ro 25-6603, SR11237, or SR11234) induced all three cellular processes. An RAR alpha-selective ligand used with an RXR-selective ligand generated the same responses as did all-trans RA or 9-cis RA, which affect both families of receptors, suggesting an important role for RAR alpha among RAR subtypes in eliciting cellular response. Consistent with this finding, the RAR alpha antagonist, Ro 41-5253, reduced the level of the cellular responses elicited by treatment with an RAR alpha-selective ligand plus RXR-selective ligand. The coupling of the shift of RB to its hypophosphorylated form with G1/0 arrest and differentiation in response to ligands is consistent with a possible role of RB as a downstream target or effector of RAR alpha and RXR in combination.

Practical synthesis of a chromene analog for use as a retinoic acid receptor alpha antagonist lead compound

Retinoic acid receptor alpha (RARα) selective compounds may guide the design of drugs that can be used in conjunction with hormonal adjuvant therapy in the treatment of breast cancer. Herein we report a modified synthesis of a known RARα antagonist, 2-fluoro-4-[[[8-bromo-2,2-dimethyl-4-(4-methylphenyl)chroman-6-yl]carbonyl]amino]benzoic acid and a synthesis of its unknown, desfluoro analog, 4-[[[8-bromo-2,2-dimethyl-4-(4-methylphenyl)chroman-6-yl]carbonyl]amino]benzoic acid. The modified route allows for facile reaction workups, increased yields, lower cost and incorporates a green alternative step. Structure–activity relationship studies determined through functional cell-based assays, demonstrated antagonism to RARα for both compounds. Molecular modeling within the RARα binding pocket was used to compare binding interactions of the desfluoro analog to a known RAR antagonist.

Retinoic acid signaling biomarkers after treatment with retinoic acid and retinoic acid receptor alpha antagonist (Ro 41-5253) in canine testis: An in vitro organ culture study

Retinoic acid (RA) is an essential component for development and maintenance of the male genital tract and for spermatogenesis. Aldehyde dehydrogenase (ALDH)1, cytochrome P450 (CYP)26b1, RA receptor (RAR)α, cellular RA-binding protein (CRAB)II, and stimulated by RA gene (STRA)8 are involved in synthesis, metabolism signaling pathways, and as downstream effectors of RA. The objective was to elucidate the effects of exogenous RA and a RARα antagonist on gene expression of ALDH1, CYP26b1, RARα, cellular RA-binding protein II, and STRA8 in an in vitro organ culture model of canine testis. Testicular tissues from medium-sized mixed breed dogs (N = 5; age 8 ± 0.17 mo) were subjected to exogenous all trans-RA (final concentrations of 1, 2, and 10 μM, and DMSO as control) for 24 h. Similarly, testicular tissues were treated with Ro 41-5253 (RARα antagonist), at 1, 10, and 50 μM final concentrations (DMSO as control) for 24 h. Exogenous RA or the RARα antagonist decreased (P < 0.05) mRNA abundance of ALDH1 in a dose-dependent manner compared with control. The CRABII mRNA abundance was greater after RA treatment compared with control (P < 0.01), but only 50 μM Ro 41-5253 effectively decreased CRABII mRNA abundance compared with control (P < 0.01). Although RA did not affect RARα mRNA abundance, the RARα antagonist treatment lowered RARα mRNA abundance compared with control (P < 0.05). Abundance of CYP26b1and STRA8 mRNA were greater (P < 0.05) after RA treatment, but lower (P < 0.05) after RARα antagonist treatment compared with control. In conclusion, exogenous RA decreased mRNA abundance of ALDH1 and increased mRNA abundance of RA signaling molecules and its downstream effectors (CYP26b1, CRABII, and STRA8), whereas treatment with a RARα antagonist effectively decreased RARα and RA metabolism molecules and its downstream effectors in canine testis. Perhaps pharmacological intervention via the RA pathway would enable canine male contraception or treatment of testicular pathology.

Expression of the Cancer Stem Cell Marker ALDH1A1 in Primary Breast Cancer: A Mechanism for Chemotherapy Resistance.

Abstract Background: Aldehyde dehydrogenase 1 (ALDH1) has been identified as a marker of breast cancer stem cells, and ALDH1 expression in primary breast tumors has been associated with resistance to chemotherapy and poor prognosis. ALDH1 is an enzyme that can detoxify reactive aldehyde compounds; however the mechanism of ALDH1 chemotherapy resistance and its promotion of the breast cancer stem cell phenotype is unclear. Materials and Methods: Illumina cDNA array was performed on primary breast tumors that were snap-frozen, and RNA was isolated from dense, viable tumor regions. Samples were normalized by rank-invariant method and gene expression was analyzed. Immunohistochemistry was performed for ALDH1A1 on initial and post-neoadjuvant tumor samples from patients who have undergone six cycles of chemotherapy. Clinical data including age, tumor pathology, lymph node status, and response to neoadjuvant therapy were available for analysis. Human breast cancer cell lines (MCF-7 and MDA-MB-468) were treated with increasing doses of retinoic acid (RA) with and without the retinoic acid receptor alpha antagonist, Ro 41-5253. Effects on cell proliferation and signal transduction pathways were assessed. Results: The expression of ALDH1A1 and/or ALDH1A3 isoform mRNA was found to be present in primary human breast tumors. The majority of these cases were noted as triple negative or Her-2 positive tumors. Evaluation of RA receptor expression revealed repressed RAR beta levels in all tumors, whereas RAR alpha expression was retained. Observations of patient tumor samples (pre and post neoadjuvant chemotherapy treatment) demonstrated high ALDH1A1 expression in tumor and stromal cell compartments. The expression was predominant in partial and non-responders to the neoadjuvant therapy. Interestingly, the tumor from a partial responder was converted to extremely high expresser of ALDH1A1 post-neoadjuvant therapy indicating a positive survival selection. Since ALDH1A1 can convert retinaldehyde to retinoic acid, breast cancer cell lines were treated with all-trans RA and responded with a significant decrease in cell proliferation. This effect was partially attenuated by pre-treatment with the RAR alpha antagonist, Ro 41-5253. Importantly, RA treatment of MCF-7 cells repressed MAPK signaling, which was also attenuated by pre-treatment with Ro 41-5253. Discussion: The observations from this study suggest that the expression of the breast cancer stem cell marker, ALDH1A1 on tumor cells or in tumor-associated stromal cells can provide a mechanism for survival of cancer during chemotherapy treatment. The conversion of therapeutic oxidant compounds to retinoic acid by ALDH1 may lead to increased RAR alpha signaling and suppression of proliferation. The suppression of proliferation correlates well with chemotherapy resistance in these neoadjuvant cases. This data demonstrates a possible mechanism for ALDH1 promotion of tumor cell survival under chemotherapy and suggests that treatment with RAR alpha antagonists may promote tumor cell susceptibility to current treatment modalities and improve the therapeutic window for primary, metastatic and chemo-refractory breast cancer. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 1135.

Effect of all-trans-retinoic acid on the differentiation, maturation and functions of dendritic cells derived from cord blood monocytes

We investigated the effects of all-trans-retinoic acid on dendritic cells derived from human cord blood monocytes to clarify how vitamin A affects immune function in children. Monocytes were separated from 18 cord blood samples, and dendritic cells were differentiated by culture. The percentage of dendritic cells was markedly lower in all-trans-retinoic acid treated cells than in untreated cells. After exposure to tumour necrosis factor-alpha for 3 days, all-trans-retinoic acid treated dendritic cells showed a reduced capacity to activate alloreactive T cells compared to untreated cells. In addition, all-trans-retinoic acid-treated dendritic cells could drive T cells towards T-helper cell type 2 responses with decreased secretion of interleukin-12, interferon-gamma, and increased production of interleukin-10 and interleukin-4. However, when Ro 41-5253, a selective retinoic acid receptor alpha antagonist, was add to culture, all the above actions were reversed. Thus, all-trans-retinoic acid may act at the first step of the immune response by inhibiting the differentiation of dendritic cells, maturation and induction of the T-helper cell type-2 response. The actions of all-trans-retinoic acid on dendritic cells were mediated through retinoic acid receptor alpha.

The effect of all‐trans retinoic acid on myeloid dendritic cell adhesion

Myeloid dendritic cells (DC) are specialized antigen presenting cells. Activation of DC by antigen triggers migration to secondary lymphoid tissues where DC activate naive T cells. Vitamin A has long been known for its role in immune function. We investigated the effect of all-trans retinoic acid (atRA), a bioactive metabolite of vitamin A, on DC adhesion. Mouse bone marrow cells cultured with granulocyte-macrophage colony-stimulating factor in the presence of a retinoic acid receptor-alpha antagonist (AGN) demonstrated increased adherence compared to cell cultures rescued with atRA treatment for the final 48 hours of culture. When floating cells, predominantly granulocytes, are removed on day 6 and 8 of the 10-day culture period, there is no difference in percentage of adherent cells between the AGN and atRA rescued treatments, indicating that atRA and granulocytes act together to cause loss of DC adherence. AtRA rescue treatment is found to result in decreased surface expression of the cellular adhesion molecule CD11a. Adherent cells and floating DC treated with atRA produce more matrix metalloproteinase (MMP) mRNA and protein and lower levels of MMP inhibitor than adherent cells cultured with AGN. However, there is no difference in RNA expression of CD11a between treatment groups. These data suggest that atRA up-regulates MMPs to release DC for migration, partly through degradation of CD11a. Supported by NIH grant 5R21AI58994-2 to KAH.

Retinoid receptors involved in the effects of retinoic acid on rat testis development.

We have previously shown that retinoic acid (RA) is able to act on the development of Leydig, Sertoli, and germ cells in the testis in culture (Livera et al., Biol Reprod 2000; 62:1303-1314). To identify which receptors mediate these effects, we have now added selective agonists and antagonists of retinoic acid receptors (RARs) or retinoid X receptors (RXRs) in the same organotypic culture system. The RAR alpha agonist mimicked most of the effects of RA on the cultured fetal or neonatal testis, whereas the RAR beta, gamma, and pan RXR agonists did not. The RAR alpha agonist decreased the testosterone production, the number of gonocytes, and the cAMP response to FSH of fetal testis explanted at 14.5 days postconception (dpc). The RAR alpha agonist disorganized the cords of the 14.5-dpc cultured testis and increased the cord diameter in cultured 3-days-postpartum (dpp) testis in the same way as RA. All these RA effects could be reversed by an RAR alpha antagonist and were unchanged by an RAR beta/gamma antagonist. The RAR beta agonist, however, increased Sertoli cell proliferation in the 3-dpp testis in the same way as RA, and this effect was blocked by an RAR beta antagonist. The RAR gamma and the pan RXR agonists had no selective effect. These results suggest that all the effects of RA on development of the fetal and neonatal testis are mediated via RAR alpha, except for its effect on Sertoli cell proliferation, which involves RAR beta.

Effects of retinoids on the production of tumour necrosis factor-alpha and nitric oxide by lipopolysaccharide-stimulated rat Kupffer cells in vitro: evidence for participation of retinoid X receptor signalling pathway.

Kupffer cells play important roles in the development of liver injury by producing cytokines and free radicals. In consequence inhibition of these inflammatory mediators will be one of the targets for treating liver diseases. Retinoids modulate a wide variety of functions of monocytes/macrophages. Cellular effects of retinoids are mediated by two families of nuclear receptors, retinoic acid receptors (RARs) and retinoid X receptors (RXRs). We examined the effects of several kinds of natural and synthetic retinoids on the production of tumour necrosis factor-alpha (TNF-alpha) and nitric oxide (NO) by LPS-stimulated rat Kupffer cells in vitro. Of the various retinoids tested, 9-cis-retinoic acid (9-cis-RA) and Ro 13-6307 which are agonists of both RARs and RXRs, suppressed the production of TNF-alpha and NO in a concentration-dependent fashion, whereas three types of RAR-selective agonists, Ro 13-7410, Ro 40-6055 and Ro 19-0645 did not show any effect. Furthermore, the RAR alpha antagonist, Ro 41-5253, did not prevent the effects induced by 9-cis-RA. The results suggest that these effects of 9-cis RA and Ro 13-6307 were induced by the RXRs-dependent signalling pathway.

Enhancement of HL-60 Differentiation by a New Class of Retinoids with Selective Activity on Retinoid X Receptor

Cellular responsiveness to retinoic acid and its metabolites is conferred through two distinct families of receptors: the retinoic acid receptors (RARs) and the retinoid X receptors (RXRs). Herein, we report on the identification and characterization of several conformationally restricted retinoids, which selectively bind and activate RX receptors. Under the influence of retinoids, HL-60 myelocytic leukemia cells differentiate into granulocytes. This effect is mediated by RAR alpha, as has been demonstrated through the use of a selective RAR alpha antagonist (Apfel, C., Bauer, F., Crettaz, M., Forni, L., Kamber, M., Kaufmann, F., LeMotte, P., Pirson, W., and Klaus, M. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 7129-7133). Here, we show that conformationally restricted RXR-specific retinoids, at doses that are per se inactive, are able to potentiate by up to one order of magnitude the pro-differentiating effects of all-trans retinoic acid and an RAR alpha-selective synthetic retinoid. We also present evidence that these RXR-selective ligands are able to bind to a DNA RXR.RAR heterodimer complex. This finding demonstrates that agonists for RARs and RXRs can synergistically promote HL-60 differentiation, which could be mediated through a heterodimer of these receptors.

Different agonist- and antagonist-induced conformational changes in retinoic acid receptors analyzed by protease mapping.

The pleiotropic effects of retinoic acid on cell differentiation and proliferation are mediated by two subfamilies of nuclear receptors, the retinoic acid receptors (RARs) and the retinoid X receptors (RXRs). Recently the synthetic retinoid Ro 41-5253 was identified as a selective RAR alpha antagonist. As demonstrated by gel retardation assays, Ro 41-5253 and two related new RAR alpha antagonists do not influence RAR alpha/RXR alpha heterodimerization and DNA binding. In a limited trypsin digestion assay, complexation of RAR alpha with retinoic acid or several other agonistic retinoids altered the degradation of the receptor such that a 30-kDa proteolytic fragment became resistant to proteolysis. This suggests a ligand-induced conformational change, which may be necessary for the interaction of the DNA-bound RAR alpha/RXR alpha heterodimer with other transcription factors. Our results demonstrate that antagonists compete with agonists for binding to RAR alpha and may induce a different structural alteration, suggested by the tryptic resistance of a shorter 25-kDa protein fragment in the digestion assay. This RAR alpha conformation seems to allow RAR alpha/RXR alpha binding to DNA but not the subsequent transactivation of target genes. Protease mapping with C-terminally truncated receptors revealed that the proposed conformational changes mainly occur in the DE regions of RAR alpha. Complexation of RAR beta, RAR gamma, and RXR alpha, as well as the vitamin D3 receptor, with their natural ligands resulted in a similar resistance of fragments to proteolytic digestion. This could mean that ligand-induced conformational changes are a general feature in the hormonal activation of vitamin D3 and retinoid receptors.

Different agonist- and antagonist-induced conformational changes in retinoic acid receptors analyzed by protease mapping

The pleiotropic effects of retinoic acid on cell differentiation and proliferation are mediated by two subfamilies of nuclear receptors, the retinoic acid receptors (RARs) and the retinoid X receptors (RXRs). Recently the synthetic retinoid Ro 41-5253 was identified as a selective RAR alpha antagonist. As demonstrated by gel retardation assays, Ro 41-5253 and two related new RAR alpha antagonists do not influence RAR alpha/RXR alpha heterodimerization and DNA binding. In a limited trypsin digestion assay, complexation of RAR alpha with retinoic acid or several other agonistic retinoids altered the degradation of the receptor such that a 30-kDa proteolytic fragment became resistant to proteolysis. This suggests a ligand-induced conformational change, which may be necessary for the interaction of the DNA-bound RAR alpha/RXR alpha heterodimer with other transcription factors. Our results demonstrate that antagonists compete with agonists for binding to RAR alpha and may induce a different structural alteration, suggested by the tryptic resistance of a shorter 25-kDa protein fragment in the digestion assay. This RAR alpha conformation seems to allow RAR alpha/RXR alpha binding to DNA but not the subsequent transactivation of target genes. Protease mapping with C-terminally truncated receptors revealed that the proposed conformational changes mainly occur in the DE regions of RAR alpha. Complexation of RAR beta, RAR gamma, and RXR alpha, as well as the vitamin D3 receptor, with their natural ligands resulted in a similar resistance of fragments to proteolytic digestion. This could mean that ligand-induced conformational changes are a general feature in the hormonal activation of vitamin D3 and retinoid receptors.

A retinoic acid receptor alpha antagonist selectively counteracts retinoic acid effects.

Retinoic acid (RA) exerts its pleiotropic effects on cell growth and differentiation through the activation of a family of transcription factors-the RA receptors (RARs). Three subtypes of these receptors exist, RAR alpha, RAR beta, and RAR gamma. The receptors are differentially expressed in different cell types and stages of development, suggesting that they may regulate different sets of genes. We have identified a synthetic retinoid with the characteristics of a selective RAR alpha antagonist. This antagonist counteracts RA effects on HL-60 cell differentiation and on B-lymphocyte polyclonal activation. Beyond its potential practical relevance, this and other specific antagonists will be useful to dissect the RAR system and to assign to one given receptor each of the many RA-regulated functions.