alitretinoin and 4-(2-(5-6-7-8-tetrahydro-5-5-8-8-tetramethyl-2-naphthalenyl)-1-propenyl)benzoic-acid

Mechanisms regulating CYP4F genes remain under investigation, although characterization of CYP4F regulatory modalities would facilitate the discovery of new drug targets. This present study shows that all-trans- and 9-cis-retinoic acids can inhibit CYP4F11 expression in human keratinocyte-derived HaCaT cells. Transrepression of many genes by retinoic acids is mediated by interactions between retinoid receptors and the activator protein 1 (AP-1) complex. Proinflammatory cytokines tumor necrosis factor alpha (TNF-alpha) and interleukin 1beta, which can activate the AP-1 complex, induce CYP4F11 transcription in HaCaT cells. The c-Jun N-terminal kinase (JNK)-specific inhibitor 1,9-pyrazoloanthrone (SP600125) blocked the induction of CYP4F11 by both cytokines, indicating involvement of the JNK pathway. Furthermore, TNF-alpha failed to induce CYP4F11 transcription when HaCaT cells were preincubated with retinoic acids. Retinoic acids are ligands for the retinoic acid receptors (RARs) and the retinoid X receptors (RXRs). The RXR agonist 6-(1(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydronaphthalen-2-yl)cyclopropyl) nicotinic acid (LG268) greatly induced CYP4F11 transcription, whereas the RAR agonist 4-(2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propenyl)benzoic acid (TTNPB) markedly inhibited CYP4F11 transcription, indicating that down-regulation of CYP4F11 transcription by retinoic acid is mediated by RARs and may also be related to ligand competition for RXRs. Thus, the CYP4F11 gene is positively regulated by multiple signaling pathways in HaCaT keratinocytes, including RXR and JNK signaling pathways.

Topics: Alitretinoin; Anthracenes; Benzoates; Cell Line; Cytochrome P-450 Enzyme System; Cytochrome P450 Family 4; Gene Expression; Gene Expression Regulation; Humans; Interleukin-1alpha; JNK Mitogen-Activated Protein Kinases; Keratinocytes; Nicotinic Acids; Phosphorylation; Protein Kinase Inhibitors; Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; Retinoic Acid Receptor gamma; Retinoid X Receptor alpha; Retinoid X Receptor beta; Retinoids; Signal Transduction; Tetrahydronaphthalenes; Tretinoin; Tumor Necrosis Factor-alpha

We investigated the effects of retinoic acids (RAs) on steroid hormone production and mRNA expression of steroidogenic enzymes in rat placenta in vitro and in vivo. In the rat trophoblast giant cell line Rcho-1, the natural retinoid X receptor (RXR) agonist 9-cis retinoic acid (9cRA) and synthetic RXR agonist LG100268 slightly promoted production of progesterone and androgen, whereas the natural retinoic acid receptor (RAR) agonist all-trans retinoic acid (atRA) and synthetic RAR agonist TTNPB did not. Furthermore, although administration of atRA and 9cRA into the rat uterus at 13.5days postcoitum robustly induced mRNA expression of cellular retinol binding protein II, the gene for which is targeted by RAR and/or RXR, in the placenta, neither RA affected the expression of placental steroidogenic enzymes, and both had little effect on progesterone and androgen levels in the placenta and embryo, suggesting that placental steroidogenesis is not regulated by RAs in rats.

Topics: Alitretinoin; Androgens; Animals; Antineoplastic Agents; Benzoates; Cell Line; Chromatography, Liquid; Female; Nicotinic Acids; Placenta; Pregnancy; Progesterone; Rats; Rats, Wistar; Retinoid X Receptors; Retinoids; Retinol-Binding Proteins, Cellular; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Spectrometry, Mass, Electrospray Ionization; Tandem Mass Spectrometry; Tetrahydronaphthalenes; Tretinoin

Human UDP-glucuronosyltransferase 2B7 (UGT2B7) is one of the major isoforms involved in the glucuronidation of endogenous compounds and xenobiotics. This isoform is the only human UGT shown to glucuronidate retinoids and their oxidized derivatives. In this study, the effects of all-trans retinoic acid (atRA), 9-cis RA, and the RAR agonist TTNPB, on UGT2B7 and UGT2B15 mRNA expression in Caco-2 cells have been examined. Each of these retinoids significantly suppressed UGT2B7 mRNA expression in a concentration-dependent manner with IC50 values of 3.5, 0.3, and 0.2 microM, respectively. However, no inhibition was observed when two other UGTs, UGT2B15 or -1A6, were exposed to atRA, 9-cis RA, or TTNPB, demonstrating that the inhibitory effect of retinoids might be specific for the UGT2B7 isoform. Further, experiments with oxidized atRA derivatives, 4-OH-atRA, 4-oxo-atRA, and 5,6-epoxy-atRA showed that these RA degradation products have no inhibitory effect on UGT2B7 mRNA expression. These data lead us to hypothesize that biologically active forms of RA suppress the expression of UGT2B7 in intestinal cells. This information provides a new pathway by which retinoids may enhance their own toxicity when accumulated in the body at pharmacological concentrations by down-regulating the enzymes involved in their biotransformation into soluble derivatives.

Topics: Alitretinoin; Benzoates; Caco-2 Cells; Cell Survival; Cells, Cultured; Down-Regulation; Glucuronosyltransferase; Humans; Retinoids; RNA, Messenger; Tretinoin

Activation of hepatic stellate cells (HSC) is a central event in the pathogenesis of liver fibrosis during chronic liver injury. We examined the expression of retinoic acid (RAR) and retinoid X receptors (RXR) during HSC activation and evaluated the influence of natural and synthetic retinoic acids (RA) on the phenotype of culture-activated HSC. The expression of the major RAR/RXR subtypes and isoforms was analyzed by Northern hybridization. Presence of functional receptor proteins was established by gel shift analysis. Retinoic acids, RAR, and RXR selective agonists and an RAR antagonist were used to evaluate the effects of retinoid signalling on matrix synthesis by Northern blotting and immunoprecipitation, and on cell proliferation by BrdU incorporation. The 9-cisRA and synthetic RXR agonists reduced HSC proliferation and synthesis of collagen I and fibronectin. All-trans RA and RAR agonists both reduced the synthesis of collagen I, collagen III, and fibronectin, but showed a different effect on cell proliferation. Synthetic RAR agonists did not affect HSC proliferation, indicating that ATRA inhibits cell growth independent of its interaction with RARs. In contrast, RAR specific antagonists enhance HSC proliferation and demonstrate that RARs control proliferation in a negative way. In conclusion, natural RAs and synthetic RAR or RXR specific ligands exert differential effects on activated HSC. Our observations may explain prior divergent results obtained following retinoid administration to cultured stellate cells or to animals subjected to fibrogenic stimuli.

Topics: Alitretinoin; Amino Acid Sequence; Animals; Antineoplastic Agents; Base Sequence; Benzoates; Cell Division; Dimerization; Extracellular Matrix Proteins; Fatty Acids, Unsaturated; Gene Expression; Liver; Liver Cirrhosis; Male; Molecular Sequence Data; Phenotype; Rats; Rats, Wistar; Receptors, Retinoic Acid; Retinoid X Receptors; Retinoids; RNA, Messenger; Tetrahydronaphthalenes; Transcription Factors; Tretinoin

Hepatic insulin-like growth factor binding protein (IGFBP) expression is controlled by diverse factors including thyroid hormone, which enhances IGFBP-4 production in hepatocytes. In the present work, we have investigated whether hepatic IGFBP-4 expression is regulated by retinoic acid (RA), which acts via nuclear receptors belonging to the steroid/thyroid hormone receptor superfamily. Primary cultures of adult rat hepatocytes were incubated with two natural stereoisomers of RA, all-trans RA and 9-cis RA (atRA and 9cRA), and with the synthetic RA receptor (RAR)-selective agonist TTNPB. IGFBP-4 mRNA abundance was measured by Northern blot and protein production was evaluated by Ligand blot on hepatocyte-conditioned culture media. Our results indicate that atRA, 9cRA, and TTNPB increase IGFBP-4 expression by cultured hepatocytes, both at the mRNA and protein level. The RARs play a definite role in this regulation, which is independent from ongoing protein synthesis but dependent on active transcription. AtRA and thyroid hormone act synergistically in increasing hepatic IGFBP-4 expression. Our data establish a role for hormonal factors such as thyronines and retinoids in regulating the hepatic IGF system directly at the IGFBP-4 level.

Topics: Alitretinoin; Animals; Benzoates; Blotting, Northern; Cells, Cultured; Culture Media, Conditioned; Gene Expression; Hepatocytes; Insulin-Like Growth Factor Binding Protein 4; Kinetics; Male; Rats; Rats, Wistar; Receptors, Retinoic Acid; Retinoids; RNA, Messenger; Tretinoin

In a search for retinoic acid (RA) receptor ligands endowed with potent apoptotic activity, a series of novel arotinoids were prepared. Because the stereochemistry of the C9-alkenyl portion of natural 9-cis-RA and the olefinic moiety of the previously synthesized isoxazole retinoid 4 seems to have particular importance for their apoptotic activity, novel retinoid analogues with a restricted or, vice versa, a larger flexibility in this region were designed and prepared. The new compounds were evaluated in vitro for their ability to activate natural retinoid receptors and for their differentiation-inducing activity. Cytotoxic and apoptotic activities were, in addition, evaluated. In general, these analogues showed low cytotoxicity, with the restricted structures being slightly more active than the more flexible ones. As an exception, however, the isoxazole retinoid 15b proved to be particularly able to induce apoptosis at concentrations <5 microM, showing a higher activity than the classical retinoids such as all-trans-RA, 13-cis-RA, and 9-cis-RA and the previously described synthetic retinoid 4. 15b also exhibited a good affinity for the retinoid receptors. Interestingly, another important property of 15b was its ability to induce apoptosis in the HL60R multidrug-resistant (MDR) cell line, at the same concentration as is effective in HL60. Therefore, 15b represents a new retinoid possessing high apoptotic activity in an MDR cell line. The ability of 15b to act on K562 and HL60R cells suggests that this compound may have important implications in the treatment of different leukemias, and its structure could offer an interesting model for the design of new compounds endowed with apoptotic activity on MDR- and retinoid-resistant malignancies.

Topics: Antineoplastic Agents; Apoptosis; Benzoates; Cell Differentiation; Cell Division; Drug Resistance, Multiple; Drug Resistance, Neoplasm; Drug Screening Assays, Antitumor; Humans; Inhibitory Concentration 50; Isoxazoles; Receptors, Retinoic Acid; Retinoids; Tetrahydronaphthalenes; Transcriptional Activation; Tumor Cells, Cultured

Stearoyl-CoA desaturase (SCD) is a regulatory enzyme involved in the synthesis of the monounsaturated fatty acids palmitoleate and oleate. The regulation of SCD is of physiological importance because the ratio of saturated fatty acids to unsaturated fatty acids is thought to modulate membrane fluidity. Differential display analysis of retinal pigment epithelial (ARPE-19) cells identified SCD as a gene regulated by retinoic acid. Two SCD transcripts of 3.9 and 5.2 kilobases in size were found to be expressed in these cells by Northern blot analysis. All-trans-retinoic acid (all-trans-RA) increased SCD mRNA expression in a dose- and time-dependent manner; an approximately 7-fold increase was observed with 1 microm all-trans-RA at 48 h. SCD mRNA expression was also increased by 9-cis-retinoic acid (9-cis-RA) as well as 4-(E-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propenyl)benzoic acid (TTNPB), a retinoic acid receptor (RAR)-specific agonist. AGN194301, a RAR alpha-specific antagonist, suppressed the SCD expression induced by all-trans-RA, TTNPB, and 9-cis-RA. These results indicate the involvement of RAR alpha in the induction of SCD expression by retinoic acid. However, AGN194204, a RXR (retinoid X receptor) pan agonist, also increased SCD mRNA expression. This increase was not blocked by AGN194301, suggesting that an RAR-independent mechanism may also be involved. Thus, SCD expression in retinal pigment epithelial cells is regulated by retinoic acid, and the regulation appears to be mediated through RAR and RXR.

Topics: Alitretinoin; Animals; Antineoplastic Agents; Benzoates; Blotting, Northern; Carcinoma, Hepatocellular; Cell Line; Cells, Cultured; Chlorocebus aethiops; COS Cells; Fatty Acids, Unsaturated; Gene Expression Regulation, Enzymologic; Gingiva; HeLa Cells; Humans; Kinetics; Liver Neoplasms; Pigment Epithelium of Eye; Polymerase Chain Reaction; Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; Retinoid X Receptors; Retinoids; RNA, Messenger; Stearoyl-CoA Desaturase; Tetrahydronaphthalenes; Transcription Factors; Transcription, Genetic; Tretinoin; Tumor Cells, Cultured

Retinoic acid (RA) regulates the transcription of various genes required for several essential functions in vertebrates through binding to two classes of nuclear receptors, the retinoic acid receptors (RAR) and retinoid X receptors (RXR). We investigated nuclear RA binding in tissues from rainbow trout using the radiolabeled all-trans and 9-cis isomers of RA. Specific binding (indicative of receptor binding) of both all-trans- and 9-cis-RA was found in all tissues tested, including the adult trout ovary, testis, gill, liver, kidney, blood, white muscle, and heart. The kinetics and absolute amount of RA binding were dependent on both the tissue and the isomer of RA used. All-trans-RA bound with high affinity (K(d) approximately 1.0-3.9 nM), and low capacity (B(max) approximately 75-484 fmol RA/mg protein), while 9-cis-RA bound with lower affinity (K(d) approximately 7-56 nM), but with a greater capacity (B(max) approximately 214-1076 fmol RA/mg protein). The B(max) results were used to estimate RAR and RXR levels and revealed that the gill possesses primarily RARs while the liver possesses primarily RXRs. The RAR-specific competitor TTNPB was able to effectively displace all-trans-[3H]RA in most tissues, and the RXR-specific competitor AGN 194204 was able to effectively displace 9-cis-[3H]RA. However, TTNPB and AGN 194204 could not displace all of the RA in the kidney and testis, suggesting the existence of another nuclear RA binding protein. Binding of all-trans- and 9-cis-RA was also found in developing trout embryos and fry. Kinetic analysis revealed that RAR levels predominated at the eyed-embryo stage, but decreased 87% by the swim-up fry stage, while RXR levels remained relatively constant over the same time period. These findings suggest that RA and its receptors may play a key role in early trout development. This study has provided a simple and rapid radioligand binding assay that can identify RAR and RXRs in trout tissues.

Topics: Alitretinoin; Aminobenzoates; Animals; Benzoates; Fatty Acids, Unsaturated; Gills; Kidney; Kinetics; Liver; Male; Oncorhynchus mykiss; Receptors, Retinoic Acid; Retinoid X Receptors; Retinoids; Testis; Tetrahydronaphthalenes; Tissue Distribution; Transcription Factors; Tretinoin

In the NB4 model of acute promyelocytic leukemia (APL), ATRA, 9-cis retinoic acid (9-cis RA), the pan-RAR and RARalpha-selective agonists, TTNPB and AM580, induce growth inhibition, granulocytic differentiation and apoptosis. By contrast, two RXR agonists, a RARbeta agonist and an anti-AP1 retinoid have very limited activity, ATRA- and AM580-dependent effects are completely inhibited by RAR antagonistic blockade, while 9-cis RA-induced cell-growth-inhibition and apoptosis are equally inhibited by RAR and RXR antagonists. ATRA, 9-cis RA and AM580 cause upregulation of the mRNAs coding for pro-caspase-1, -7, -8, and -9, which, however, results in increased synthesis of only pro-caspase-1 and -7 proteins. These phenomena are associated with activation of pro-caspase-6, -7 and -8, cytochrome c release from the mitochondria, inversion of Bcl-2/Bax ratio and degradation of PML-RARalpha. Caspase activation is fundamental for retinoid-induced apoptosis, which is suppressed by the caspase-inhibitor z-VAD.

Topics: Alitretinoin; Antineoplastic Agents; Apoptosis; Benzoates; Caspases; Cell Differentiation; Cell Division; Enzyme Activation; Gene Expression; Humans; Leukemia, Promyelocytic, Acute; Receptors, Retinoic Acid; Retinoid X Receptors; Retinoids; Tetrahydronaphthalenes; Transcription Factors; Tretinoin; Tumor Cells, Cultured

Retinoids are important agents which regulate differentiation and proliferation processes in various cell types, including cancer cells. Growth arrest and induction of terminal differentiation demonstrate the tumor-suppressive effects of retinoids on leukemic cells. We studied differentiation, proliferation, and death processes in the cell line of v-myb-transformed monoblasts BM2 and their retinoic acid receptor (RAR) alpha- and retinoid X receptor (RXR) alpha-expressing derivatives after exposure to four different retinoids: all-trans retinoic acid, 9-cis retinoic acid, TTNPB, and LG1000153. The effects of retinoids on the phenotype of BM2, BM2RAR, and BM2RXR cells were correlated with the transcription activation function of the v-Myb oncoprotein of avian myeloblastosis virus. We found that the efficiency of terminal differentiation of BM2RAR and BM2RXR cells induced by retinoids is indirectly proportional to the v-Myb transcription activation activity. In contrast, the effects of liganded retinoid receptors on growth of BM2 cells are more complex. Activated RAR protein induces growth inhibition of BM2 cells by suppression of v-Myb function. However, liganded RXR protein is less efficient in cell cycle arrest and rather decreases cellular viability. This process can occur in the presence of active v-Myb protein. These results suggest that ligand-activated RARalpha protein is primarily engaged in control of proliferation and differentiation of v-myb-transformed monoblasts, while activated RXRalpha protein controls their differentiation and death.

Topics: Alitretinoin; Animals; Benzoates; Cell Count; Cell Differentiation; Cell Division; Cell Survival; Cell Transformation, Viral; Genes, myb; Humans; Ligands; Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; Retinoid X Receptors; Retinoids; Transcription Factors; Transcriptional Activation; Tretinoin; Tumor Cells, Cultured

Retinoids constitute a very promising class of agents for the chemoprevention or treatment of breast cancer. These retinoids exert their biological activity through two distinct classes of retinoic acid (RA) receptors (R), the RAR isotypes (alpha, beta, and gamma) and the three RXR isotypes (alpha, beta, and gamma) and their numerous isoforms which bind as RXR/RAR heterodimers to the polymorphic cis-acting response elements of RA target genes. With respect to these numerous receptor sub-types, the retinoid-induced effects at the biological level include marked modifications with respect to both cell proliferation and cell death (apoptosis), and also in the induction of differentiation processes. The present study aims to characterize the effect which four retinoids (TTNPB, 9-cis-RA, LGD 1069, 4-HPR) with distinct RAR/RXR binding properties induced on various in vitro and in vivo mouse and human breast cancer models. The experiments with the retinoids were carried out in comparison with the anti-estrogen tamoxifen and the anti-progestagen RU-486 compounds. The results show that the 6 compounds under study were markedly more efficient in terms of growth inhibition in the human T-47D cell line when maintained under anchorage-independent culture conditions than when maintained under anchorage-dependent ones. While RU-486 exhibited a weak statistically significant (p < 0.05) influence on the growth of the T-47D stem cells, tamoxifen had a marked inhibitory influence on the growth of these cells. Of the four retinoids, 4-HPR was the least effective since the lowest doses tested (1 and 0.1 nM) exhibited no statistically (p > 0.05) significant influence on the growth of the stem cells. The most efficient retinoid was TTNPB. It was only at the highest dose (10 microM) that tamoxifen and RU-486 showed a weak inhibitory influence on the growth of the T-47D non-stem cells while all 4 retinoids exerted a significant inhibitory influence on the growth of these non-stem cells, with 4-HPR being the most efficient (P < 0.001) at the highest dose, but ineffective (P > 0.05) at the lowest. Tamoxifen and TTNPB were tested in vivo on hormone-sensitive (HS) and hormone-insensitive (HI) strains of the MXT murine mammary carcinoma. While TTNPB appeared to be equally efficient in terms of growth inhibition in both MXT-HS and MXT-HI models, tamoxifen had only a marginal inhibitory influence on the growth of the MXT-HI strain but did inhibit growth in the case of the MXT-HS one. TT

Topics: Alitretinoin; Animals; Anticarcinogenic Agents; Apoptosis; Benzoates; Bexarotene; Breast Neoplasms; Cell Division; Disease Models, Animal; Female; Fenretinide; Hormone Antagonists; Humans; Mammary Neoplasms, Animal; Mice; Mice, Inbred C57BL; Mifepristone; Retinoids; Tamoxifen; Tetrahydronaphthalenes; Tretinoin; Tumor Cells, Cultured

Retinoids and vitamin D (VD) cooperate to induce the differentiation and inhibit the proliferation of human myelomonocytic leukemia cells. Two classes of retinoids receptors, the RARs and RXRs, respectively, can mediate these effects. RXR forms heterodimers with a variety of nuclear receptors, including RAR and the VD receptor. We have previously found that VD treatment increases RXR alpha levels in myelomonocytic leukemia cells. By immunoanalysis, we observed in the present work that the RAR alpha protein is expressed in proliferating U937, HL-60 and THP-1 human leukemia cells and that VD treatment induces alterations of its electrophoretic pattern, although with large differences between cell lines. In the three cell lines, 9-cis RA, an agonist of both RARs and RXRs, cooperated with VD more efficiently than all-trans RA and RAR-specific synthetic ligands, thus suggesting an involvement of both RAR and RXR pathways in cell differentiation. Using U937 cells as a model, we delineated the relative contributions of RAR and RXR by assessing the effects of receptor-selective synthetic retinoids. The synergy between VD and all-trans RA or RAR-specific agonists (TTNPB and Ro 40-6055) was abrogated by a RAR alpha-specific antagonist (Ro 41-5253), confirming an involvement of RAR alpha. However, the cooperation between VD and 9-cis RA, although reduced, was not suppressed by the antagonist, suggesting also an involvement of the RXR pathway. The role of RXR as a ligand-activated receptor was confirmed using RXR-specific agonists (CD2608 and LGD1069), which also proved able to cooperate with VD. Finally, while each synthetic agonist alone was significantly less potent than 9-cis RA, combinations of the RAR and RXR selective agonists TTNPB and LGD1069 appeared to be as effective as the pan agonist 9-cis-RA. These results confirm that various retinoids can cooperate with VD and demonstrate that, at a whole cell level, optimal effects require the activation of both RAR and RXR receptors.

Topics: Alitretinoin; Animals; Benzoates; Bexarotene; Cell Differentiation; Chromans; COS Cells; HL-60 Cells; Humans; Leukemia, Monocytic, Acute; Lymphoma, Large B-Cell, Diffuse; Molecular Structure; Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; Retinoid X Receptors; Retinoids; Signal Transduction; Tetrahydronaphthalenes; Transcription Factors; Transfection; Tretinoin; Tumor Cells, Cultured; Vitamin D

Retinoic acid receptor (RAR) alpha has been shown to play a role in retinoid-induced growth inhibition of human breast cancer cell lines that express the estrogen receptor (ER). The dogma in the field has been that ER-positive breast cancer cell lines respond to retinoid treatment because they express RAR alpha, whereas ER-negative breast cancer cell lines are refractory to retinoid treatment and have been thought to express little or no RAR alpha. We set out to test several ER-negative breast cancer cell lines for expression of RAR alpha protein and responsiveness to retinoids in growth inhibition assays. Of six ER-negative breast cancer cell lines that were tested, one (SK-BR-3) had high levels of RAR alpha protein as measured by ligand-binding immunoprecipitation (approximately 55 fmol/mg protein) and also displayed sensitivity to growth inhibition by retinoids (9-cis-retinoic acid; EC50, approximately 3 nM). These cells were more sensitive than an ER-positive cell line, T-47D, which expressed approximately 35 fmol RAR alpha/mg total protein (9-cis retinoic acid; EC50, approximately 50-100 nM). Another ER-negative cell line, Hs578T, also expressed RAR alpha (approximately 23 fmol/mg) and was sensitive to retinoid-induced growth inhibition, albeit to a lesser extent than SK-BR-3 or T-47D cells. In contrast, the other ER-negative cell lines tested expressed low (<10 fmol/mg) or no detectable levels of RAR alpha protein and also did not respond to retinoids in growth inhibition assays. A RAR alpha agonist displayed 100 times greater potency than a RARgamma agonist in growth inhibition of both T-47D and SK-BR-3 cells, suggesting RAR alpha involvement in the process. Furthermore, a RAR alpha antagonist completely abolished the growth inhibition induced by RAR agonists, implying that the activity of the agonists is exerted solely through RAR alpha, not RARgamma, which is also expressed in both cell lines. Additionally, although retinoid X receptor (RXR) compounds are weakly active in growth inhibition of the RAR alpha-positive cell lines, they markedly increased the growth-inhibitory activity of RAR ligands. RXR compounds also potentiated the action of the antiestrogen 4-hydroxytamoxifen to inhibit the growth of T-47D cells. These findings have clinical ramifications in that patients with ER-negative tumors that are RAR alpha positive may be candidates for retinoid therapy. Additionally, combinations of RXR ligands with RAR ligands (especially RAR alpha agon

Topics: Alitretinoin; Aminobenzoates; Antineoplastic Agents; Benzoates; Bexarotene; Breast Neoplasms; Cell Division; Chromans; Female; Humans; Nicotinic Acids; Nuclear Proteins; Receptors, Estrogen; Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; Retinoid X Receptors; Retinoids; Tetrahydronaphthalenes; Transcription Factors; Tretinoin; Tumor Cells, Cultured

We showed previously that retinoids stimulate apolipoprotein A-I (apoA-I) synthesis in cultured cynomolgus hepatocytes only after a 24-h lag phase. Here we report on the biochemical background of the slow response, the requirement for high retinoic acid concentrations, and the involvement of different retinoid receptors. The time course of the effect of 10 microM all-trans retinoic acid (at-RA) on apoA-I mRNA levels and protein secretion were comparable, i.e., minor increases were observed after a 24-h incubation and mRNA levels were increased 2.2- and 3.5-fold after 48 h and 72 h, respectively. In contrast, apoA-I gene transcription was already increased (2.6-fold) after a 4-h incubation with 10 microM at-RA. At-RA disappeared rapidly from the cultures: after 2 h of incubation 40% of the added amount was left and after 24 h only 2%. RAR beta mRNA and gene expression were increased after incubation with 10 microM at-RA, whereas RAR alpha and RXR alpha mRNA levels and expression remained unchanged. No transcriptional activity and mRNA for other retinoid receptors were detectable. Both RAR-selective (TTNPB) and RXR-selective (3-methyl-TTNEB) agonists induced apoA-I synthesis at 1 and 10 microM. These results show that i) the slow increase in apoA-I secretion is caused by a slow increase of its mRNA level; ii) the apoA-I gene transcription in cynomolgus hepatocytes is induced rapidly by retinoids; iii) the added at-RA disappeared rapidly from the cultures, explaining the necessity for high initial concentrations; iv) RLR alpha and/or RAR beta and RXR alpha are involved in the activation of apoA-I expression by retinoids.

Topics: Alitretinoin; Animals; Apolipoprotein A-I; Benzoates; Bexarotene; Blotting, Northern; Cells, Cultured; Female; Gene Expression Regulation; Liver; Macaca fascicularis; Male; Receptors, Retinoic Acid; Retinoid X Receptors; Retinoids; RNA, Messenger; Tetrahydronaphthalenes; Time Factors; Transcription Factors; Transcriptional Activation; Tretinoin

Human bronchial epithelial (HBE) cells undergo squamous differentiation in response to a variety of conditions in tissue culture, and retinoid treatment has been shown to reverse this process. Retinoids mediate their effects through the retinoic acid and retinoid X nuclear receptors (RAR and RXR, respectively), which form RAR-RXR heterodimers, RXR homodimers, and heterodimers of RXR and certain orphan receptors. These receptor dimers bind to distinct response elements, activating separate pathways. In this study, we investigated the roles of RAR and RXR signaling pathways in the inhibition of HBE squamous differentiation. After induction of squamous differentiation by confluent growth, HBE cells were treated with retinoids that selectively activate RARs (E-4-[2-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-2-naphthaienyl)-1- propenyl] benzoic acid), RXRs (4-[1-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-2-naphthyl)ethenyl]benzoic acid), or both RARs and RXRs (9-c/s retinoic acid). These retinoids inhibited the mRNA expression of the squamous differentiation markers transglutaminase type I, involucrin, keratin 5, and keratin 13, suggesting that inhibition of HBE squamous differentiation could be mediated by activation of either RAR or RXR signaling pathways. We examined the role of AP-1 as a potential effector of these retinoid pathways. AP-1 transcriptional activity was reduced markedly by these retinoids, and a concomitant, but proportionally smaller, reduction in AP-1 DNA binding was observed. Furthermore, treatment of squamous HBE cells with the retinoid SR11238, which inhibited AP-1 without activating retinoid receptor transcriptional properties, reduced the expression of transglutaminase type I and involucrin. These findings support the hypothesis that, in HBE cells, RAR and RXR signaling pathways inhibit AP-1 transcriptional activity, and this contributes to retinoid-induced reversal of HBE squamous differentiation.

Topics: Alitretinoin; Benzoates; Bexarotene; Blotting, Northern; Bronchi; Cell Differentiation; Epithelium; Humans; Isomerism; Luciferases; Receptors, Retinoic Acid; Retinoid X Receptors; Retinoids; Signal Transduction; Tetrahydronaphthalenes; Transcription Factor AP-1; Transcription Factors; Transfection; Tretinoin

Retinoids elicit biological responses by activating a series of nuclear receptors. Six retinoid receptors belonging to two families are currently known: retinoic acid receptors (RAR alpha,beta,and gamma) and retinoid X receptors (RXR alpha,beta,and gamma). Stilbene retinoid analogs of retinoic acid (RA), such as (E)-4-[2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)prope n-1- yl]benzoic acid (TTNPB, 1) and (E)-4-[2-(5,6,7,8-tetrahydro-3,5,5,8,8-pentamethyl-2-naphthalenyl)pro pen-1- yl]benzoic acid (3-methyl-TTNPB, 2), display differential RAR and RXR activities, depending on the substituent at C3 of the naphthalene ring. We report here structural modifications of the benzoate moiety of 2 that result in analogs with greater RXR selectivity as well as those with pan-agonist (activate both RAR and RXR receptors) activities, analyze the structural features that impart receptor selectivity, and describe a stereoselective method for the synthesis of these analogs. The biological activities associated with the RAR and RXR receptors were examined by testing representative examples with different receptor activation profiles for their ability to induce tissue transglutaminase (Tgase) activity in a human promyelocytic leukemia cell line (HL-60 cdm-1) and to inhibit tumor-promoter-induced ornithine decarboxylase (ODC) activity in hairless mouse skin. These results suggest that RAR agonists and RXR agonists may have different therapeutic applications. Finally, we show that RXR agonists are significantly reduced in teratogenic potency relative to RAR agonists and may therefore have significant advantages in clinical practice.

Topics: Abnormalities, Drug-Induced; Animals; Antineoplastic Agents; Benzoates; Female; Gene Expression; Humans; Mice; Mice, Hairless; Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; Retinoid X Receptors; Retinoids; Stereoisomerism; Stilbenes; Structure-Activity Relationship; Substrate Specificity; Transcription Factors; Transcription, Genetic; Transcriptional Activation; Transfection

Retinoids induce myeloblastic leukemia (HL-60) cells to differentiate into granulocytes, which subsequently die by apoptosis. Retinoid action is mediated through at least two classes of nuclear receptors: retinoic acid receptors, which bind both all-trans retinoic acid and 9-cis retinoic acid, and retinoid X receptors, which bind only 9-cis retinoic acid. Using receptor-selective synthetic retinoids and HL-60 cell sublines with different retinoid responsiveness, we have investigated the contribution that each class of receptors makes to the processes of cellular differentiation and death. Our results demonstrate that ligand activation of retinoic acid receptors is sufficient to induce differentiation, whereas ligand activation of retinoid X receptors is essential for the induction of apoptosis in HL-60 cell lines.

Topics: Apoptosis; Benzoates; Binding, Competitive; Cell Differentiation; Cell Division; Dose-Response Relationship, Drug; Hematopoietic Stem Cells; Humans; Leukemia; Receptors, Retinoic Acid; Retinoid X Receptors; Retinoids; Signal Transduction; Structure-Activity Relationship; Tetrahydronaphthalenes; Transcription Factors; Tretinoin; Tumor Cells, Cultured

all-trans-Retinoic acid is known to bind to the retinoic acid receptors (RARs) resulting in an increase in their transcriptional activity. In contrast, recently identified 9-cis-retinoic acid (9-cis-RA), which is an additional endogenous RA isomer, is capable of binding to both RARs and retinoid X receptors (RXRs). These distinct properties have raised questions as to the biological role governed by these two retinoic acid isomers and the set of target genes that they regulate. Herein, we report the synthesis of high specific activity [3H]-9-cis-RA and its application to study the ligand-binding properties of the various retinoid receptor subtypes. We examined the binding properties of RARs and RXRs for a series of synthetic retinoids and compared the ligand-binding properties of these arotinoid analogs with their ability to regulate gene expression via the retinoid receptors in a cotransfection assay. The utilization of the [3H]-9-cis-RA competitive binding assay and the cotransfection assay has made it possible to rapidly identify important structural features of retinoids leading to increased selectivity for either the RAR or RXR receptor subtypes.

Topics: Animals; Baculoviridae; Benzoates; Binding, Competitive; Methylation; Molecular Structure; Moths; Receptors, Cytoplasmic and Nuclear; Receptors, Retinoic Acid; Recombinant Proteins; Retinoid X Receptors; Retinoids; Structure-Activity Relationship; Transcription Factors; Transfection; Tretinoin; Tritium