Retinoic Acid Analogs Research Articles

Trifarotene alleviates skin photoaging injury by inhibition of JNK/c-Jun/MMPs.

Long-term exposure to ultraviolet (UV) radiation induces skin photoaging, which manifests as oxidative stress, inflammation, and collagen degradation. Multiple approaches (topical or systemic retinoids, antioxidants, alpha-hydroxy acids, laser, surgery) are used in the treatment of photoaged skin, and the use of topical retinoids is currently a primary clinical treatment. Previous studies revealed that retinoic acid promotes keratinocyte proliferation and reduces melanin deposition and matrix metalloproteinase (MMP) secretion; it also causes potential allergic and inflammatory damage to the skin. This study aimed to investigate the therapeutic effects and mechanisms of trifarotene, a functional retinoic acid analog, on UV-irradiated photoaging ICR and BALB/c nude mice and UVB photodamaged human epidermal keratinocyte (HaCaT) cells by examining indicators such as collagen, oxidoreductase, and inflammatory factor presence through histochemical staining, Western blot, and ELISA. Results suggested that trifarotene significantly reduced UV-induced photoaging in mouse skin tissue, potentially by reducing oxidative stress damage and inflammatory factor release, and inhibiting melanin deposition and collagen degradation by downregulating MMP expression. Concentrations of malondialdehyde, tyrosinase, interleukin-6, interleukin- 12, and tumor necrosis factor-alpha in photoaged skin decreased, while SOD content in photodamaged HaCaT cells significantly increased. Trifarotene (3.3 μmol L-1) inhibited phosphorylated JNK and c-Jun expression both independently and collaboratively with the JNK activator anisomycin, demonstrating that trifarotene mitigates UV-induced collagen degradation and apoptosis through inhibition of the JNK/c-Jun/MMPs signaling pathway.

A retinoid analogue, TTNPB, promotes clonal expansion of human pluripotent stem cells by upregulating CLDN2 and HoxA1

Enzymatic dissociation of human pluripotent stem cells (hPSCs) into single cells during routine passage leads to massive cell death. Although the Rho-associated protein kinase inhibitor, Y-27632 can enhance hPSC survival and proliferation at high seeding density, dissociated single cells undergo apoptosis at clonal density. This presents a major hurdle when deriving genetically modified hPSC lines since transfection and genome editing efficiencies are not satisfactory. As a result, colonies tend to contain heterogeneous mixtures of both modified and unmodified cells, making it difficult to isolate the desired clone buried within the colony. In this study, we report improved clonal expansion of hPSCs using a retinoic acid analogue, TTNPB. When combined with Y-27632, TTNPB synergistically increased hPSC cloning efficiency by more than 2 orders of magnitude (0.2% to 20%), whereas TTNPB itself increased more than double cell number expansion compared to Y-27632. Furthermore, TTNPB-treated cells showed two times higher aggregate formation and cell proliferation compared to Y-27632 in suspension culture. TTNPB-treated cells displayed a normal karyotype, pluripotency and were able to stochastically differentiate into all three germ layers both in vitro and in vivo. TTNBP acts, in part, by promoting cellular adhesion and self-renewal through the upregulation of Claudin 2 and HoxA1. By promoting clonal expansion, TTNPB provides a new approach for isolating and expanding pure hPSCs for future cell therapy applications.

Strategies for developing retinoic acid receptor alpha-selective antagonists as novel agents for male contraception

Reported here are the synthesis and in vitro evaluation of a series of 26 retinoic acid analogs based on dihydronaphthalene and chromene scaffolds using a transactivation assay. Chromene amide analog 21 was the most potent and selective retinoic acid receptor α antagonist identified from this series. In vitro evaluation indicated that 21 has favorable physicochemical properties and a favorable pharmacokinetic PK profile in vivo with significant oral bioavailability, metabolic stability, and testes exposure. Compound 21 was evaluated for its effects on spermatogenesis and disruption of fertility in a mouse model. Oral administration of compound 21 at low doses showed reproducibly characteristic albeit modest effects on spermatogenesis, but no effects on fertility were observed in mating studies. The inhibition of spermatogenesis could not be enhanced by raising the dose and lengthening the duration of dosing. Thus, 21 may not be a good candidate to pursue further for effects on male fertility.

Differential responsiveness of spermatogonia to retinoic acid dictates precocious differentiation but not meiotic entry during steady-state spermatogenesis†.

The foundation of mammalian spermatogenesis is provided by undifferentiated spermatogonia, which comprise of spermatogonial stem cells (SSCs) and transit-amplifying progenitors that differentiate in response to retinoic acid (RA) and are committed to enter meiosis. Our laboratory recently reported that the foundational populations of SSCs, undifferentiated progenitors, and differentiating spermatogonia are formed in the neonatal testis in part based on their differential responsiveness to RA. Here, we expand on those findings to define the extent to which RA responsiveness during steady-state spermatogenesis in the adult testis regulates the spermatogonial fate. Our results reveal that both progenitor and differentiating spermatogonia throughout the testis are capable of responding to exogenous RA, but their resulting fates were quite distinct-undifferentiated progenitors precociously differentiated and proceeded into meiosis on a normal timeline, while differentiating spermatogonia were unable to hasten their entry into meiosis. This reveals that the spermatogonia responding to RA must still complete the 8.6day differentiation program prior to their entry into meiosis. Addition of exogenous RA enriched testes with preleptotene and pachytene spermatocytes one and two seminiferous cycles later, respectively, supporting recent clinical studies reporting increased sperm production and enhanced fertility in subfertile men on long-term RA analog treatment. Collectively, our results reveal that a well-buffered system exists within mammalian testes to regulate spermatogonial RA exposure, that exposed undifferentiated progenitors can precociously differentiate, but must complete a normal-length differentiation program prior to entering meiosis, and that daily RA treatments increased the numbers of advanced germ cells by directing undifferentiated progenitors to continuously differentiate.

Pretreatment of human retinal pigment epithelial cells with sterculic acid forestalls fenretinide-induced apoptosis

The ratio of saturated to monounsaturated fatty acids, thought to play a critical role in many cellular functions, is regulated by stearoyl-CoA desaturase (SCD), a rate-limiting enzyme in the biosynthesis of monounsaturated fatty acids. Previously, we observed a decrease in both SCD protein and enzymatic activity in apoptosis induced by fenretinide, a synthetic analog of retinoic acid, in the human retinal pigment epithelial (RPE) cell line ARPE-19. Here, we investigated the effect of pretreating ARPE-19 with sterculic acid, a cyclopropenoic fatty acid inhibitor of SCD, on preventing fenretinide-induced apoptosis, given the role of SCD in cell proliferation and apoptosis. We show that sterculic acid pretreatment prevents the effects of fenretinide-induced apoptosis shown by changes in cell morphology, viability, and caspase-3 activation. Analysis of endoplasmic reticulum (ER)-associated proteins shows that sterculic acid pretreatment reduced the fenretinide-induced upregulation of heme oxygenase-1, ATF3 and GADD153 expression that are in response to reactive oxygen species (ROS) generation. Sterculic acid is as effective as allopurinol in inhibition of xanthine oxidase (XDH), and this may play a role in reducing the potential role of XDH in fenretinide-induced ROS generation. Sterculic acid pretreatment also results in a reduction in SOD2 mRNA expression. Dihydroceramide accumulation, compared to ceramide, and ROS generation indicate that a ceramide-independent pathway mediates fenretinide-induced apoptosis, and ROS mediation is borne out by activation of the NF-κBp50 and NF-κBp65 downstream signaling cascade. Its prevention by sterculic acid pretreatment further indicates the latter’s antioxidant/anti-inflammatory effect. Taken together, our results suggest that sterculic acid pretreatment can mitigate ROS-mediated fenretinide-induced apoptosis. Thus, sterculic acid may serve as a potential antioxidant and therapeutic agent. These effects may be independent of its effects on SCD activity.

Inspired by vitamin A for anti‐ageing: Searching for plant‐derived functional retinoid analogues

BackgroundCosmetic treatments that inspire one's appearance to resemble their younger portrait often utilize ingredients that confer acute effects, particularly hydration by creating hydrophobic barriers or transient elevation of barrier water content. But superior therapies successfully promote morphogenesis of the dermal‐epidermal junction, inspiring extracellular matrix (ECM) formation. This can be achieved by agonism of the very well‐known retinoid nuclear receptors using the endogenous ligand all‐trans retinoic acid (tRA), tRA precursors or plant‐based functional analogues, with reduced side effects.Aims, Materials and MethodsWhile there are already many promising cosmetic ingredients available from the world's flora, higher potency is favoured, so increasing known candidates is a worth undertaking. Functional analogues of retinoic acid can be identified by culturing fibroblasts with lipophilic candidates from the plant kingdom and assessing gene‐arrays. Modern approaches to validating these findings include the coculturing of fibroblasts with keratinocytes as a measure to predict the potential effects of crosstalk.Results and DiscussionIn this regard, the most promising plant‐derived candidates are of terpene or meroterpene origin, including derivatives of squalene and phytol. Surprisingly pimaric or abietic acids and labdane diterpenes are also noteworthy agonists of the retinoic acid receptor, stimulating collagen expression in dermal fibroblasts.ConclusionThere are numerous derivatives of these terpenes available from the world's flora and research conducted thus far encourages further screening of these chemical candidates.

Chemical Biology Studies Using Vitamin A Analogs

"Retinoid" is the general term for vitamin A derivatives and chemical compounds that act like vitamin A. Vitamin A are composed of four isoprene units and are named according to their terminal functional group, such as retinol (OH, 1), retinal (CHO, 2), and retinoic acid (CO2H, 3). Vitamin A usually refers to retinol. In the past few decades, major advances in research on vitamin A have improved our understanding of its fundamental roles and physiological significance in living cells. In this review, three types of chemical biology studies using vitamin A analogs are described: (1) conformational studies of the chromophore in retinal proteins (rhodopsin, phoborhodopsin, and retinochrome), especially the conformation around the cyclohexene ring; (2) structure-activity relationship studies of retinoic acid analogs to create new signaling molecules for activating nuclear receptors; and (3) development of a new channelrhodopsin with an absorption maximum at longer wavelength to overcome the various demerits of channelrhodopsins used in optogenetics, as well as the stereoselective synthesis of retinoid isomers and their analogs using a diene-tricarbonyliron complex or a palladium-catalyzed cross-coupling reaction between vinyl triflates and stannyl olefins.

CFTR Correctors and Antioxidants Partially Normalize Lipid Imbalance but not Abnormal Basal Inflammatory Cytokine Profile in CF Bronchial Epithelial Cells.

A deficiency in cystic fibrosis transmembrane conductance regulator (CFTR) function in CF leads to chronic lung disease. CF is associated with abnormalities in fatty acids, ceramides, and cholesterol, their relationship with CF lung pathology is not completely understood. Therefore, we examined the impact of CFTR deficiency on lipid metabolism and pro-inflammatory signaling in airway epithelium using mass spectrometric, protein array. We observed a striking imbalance in fatty acid and ceramide metabolism, associated with chronic oxidative stress under basal conditions in CF mouse lung and well-differentiated bronchial epithelial cell cultures of CFTR knock out pig and CF patients. Cell-autonomous features of all three CF models included high ratios of ω-6- to ω-3-polyunsaturated fatty acids and of long- to very long-chain ceramide species (LCC/VLCC), reduced levels of total ceramides and ceramide precursors. In addition to the retinoic acid analog fenretinide, the anti-oxidants glutathione (GSH) and deferoxamine partially corrected the lipid profile indicating that oxidative stress may promote the lipid abnormalities. CFTR-targeted modulators reduced the lipid imbalance and oxidative stress, confirming the CFTR dependence of lipid ratios. However, despite functional correction of CF cells up to 60% of non-CF in Ussing chamber experiments, a 72-h triple compound treatment (elexacaftor/tezacaftor/ivacaftor surrogate) did not completely normalize lipid imbalance or oxidative stress.Protein array analysis revealed differential expression and shedding of cytokines and growth factors from CF epithelial cells compared to non-CF cells, consistent with sterile inflammation and tissue remodeling under basal conditions, including enhanced secretion of the neutrophil activator CXCL5, and the T-cell activator CCL17. However, treatment with antioxidants or CFTR modulators that mimic the approved combination therapies, ivacaftor/lumacaftor and ivacaftor/tezacaftor/elexacaftor, did not effectively suppress the inflammatory phenotype.We propose that CFTR deficiency causes oxidative stress in CF airway epithelium, affecting multiple bioactive lipid metabolic pathways, which likely play a role in CF lung disease progression. A combination of anti-oxidant, anti-inflammatory and CFTR targeted therapeutics may be required for full correction of the CF phenotype.

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.

Myeloid Differentiation and Retinoblastoma Phosphorylation Changes in HL-60 Cells Induced by Retinoic Acid Receptor- and Retinoid X Receptor-Selective Retinoic Acid Analogs

Myeloid Differentiation and Retinoblastoma Phosphorylation Changes in HL-60 Cells Induced by Retinoic Acid Receptor- and Retinoid X Receptor-Selective Retinoic Acid Analogs

Identification and characterization of human PEIG-1/GPRC5A as a 12-O-tetradecanoyl phorbol-13-acetate (TPA) and PKC-induced gene

Homo sapiens orphan G protein-coupling receptor PEIG-1 was first cloned and characterized by applying differential display to T84 colonic carcinoma cells incubated in the presence of phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) (GenBank AF506289.1). Later, Lotan's laboratory found the same gene product in response to retinoic acid analogues, naming it with the symbol RAIG1. Now the official HGNC symbol is GPRC5A. Here, we report the extension of its original cDNA fragment towards the 5' and 3' end. In addition, we show that TPA (100 ng/ml, 162 nM) strongly stimulated GPRC5A mRNA in T84 colonic carcinoma cells, with maximal expression at 4 h and 100 ng/ml (162 nM). Western blots showed several bands between 35 and 50 kDa, responding to TPA stimulation. Confocal microscopy confirmed its TPA upregulation and the location in the plasma membrane. The PKC inhibitor Gö 6983 (10 μM), and the Ca2+ chelator BAPTA-AM (150 μM), strongly inhibited its TPA induced upregulation. The PKA inhibitor H-89 (10 μM), and the MEK1/2 inhibitor U0126 (10 μM), also produced a significant reduction in the TPA response (~50%). The SGK1 inhibitor GSK650394 stimulated GPRC5A basal levels at low doses and inhibit its TPA-induced expression at concentrations ≥10 μM. The IL-1β autocrine loop and downstream signalling did not affect its expression. In conclusion, RAIG1/RAI3/GPRC5A corresponds to the originally reported PEIG-1/TIG1; the inhibition observed in the presence of Gö 6983, BAPTA and U0126, suggests that its TPA-induced upregulation is mediated through a PKC/Ca2+ →MEK1/2 signalling axis. PKA and SGK1 kinases are also involved in its TPA-induced upregulation.

Fenretinide reduces angiogenesis by downregulating CDH5, FOXM1 and eNOS genes and suppressing microRNA-10b.

Angiogenesis is a new vessel formation process that plays a role in various physiological and pathological conditions. This process is controlled by the balance between pro-angiogenic and anti-angiogenic mediators in the organism. Angiogenesis is needed for the growth and metastasis of solid tumors. Therefore, the anti-angiogenic treatment approach is seen as an interesting option in cancers. Fenretinide, a synthetic retinoic acid analog, is an effective agent on angiogenesis. In this study, we aimed to investigate the effects of the fenretinide on some miRNAs involving in angiogenesis process and on the expression of CDH5, FOXM1 and eNOS genes upregulated in angiogenesis. In addition, it was shown the effects of this agent on cell proliferation, cell migration and capillary-like tube formation. In our study, the data were analyzed using Kruskal-Wallis and Dunn's test. Fenretinide applied to the cells for 24 and 48h periods reduced cell proliferation (P < 0.001) and cell migration, and suppressed tube formation (P < 0.001) as a dose dependent manner. Endothelial cells were cultured in growth-inducing media containing a variety of growth factors such as VEGF, FGF, IGF and EGF. As a result of simultaneous PCR analysis, we found that angiogenesis-promoting miR-10b was effectively suppressed (P < 0.001) and interestingly angiogenesis-modulating miR-126 was slightly increased (P < 0.05), but other miRNAs, including miR-31, miR-21, miR-101, miR-340, miR-29c, miR-206 and miR-146a were not affected. Besides, a significant decrease was observed in the levels of some angiogenesis-inducing genes, CDH5 (P < 0.001), FOXM1 (P < 0.001) and eNOS (P < 0.01 and P < 0.001) in endothelial cells treated with fenretinide. Our results have shown that fenretinide exhibited anti-angiogenic activity through the down-regulation of CDH5, FOXM1 and eNOS genes, and suppression of miR-10b.

Fluorescent Retinoic Acid Analogues as Probes for Biochemical and Intracellular Characterization of Retinoid Signaling Pathways.

Retinoids, such as all- trans-retinoic acid (ATRA), are endogenous signaling molecules derived from vitamin A that influence a variety of cellular processes through mediation of transcription events in the cell nucleus. Because of these wide-ranging and powerful biological activities, retinoids have emerged as therapeutic candidates of enormous potential. However, their use has been limited, to date, due to a lack of understanding of the complex and intricate signaling pathways that they control. We have designed and synthesized a family of synthetic retinoids that exhibit strong, intrinsic, solvatochromatic fluorescence as multifunctional tools to interrogate these important biological activities. We utilized the unique photophysical characteristics of these fluorescent retinoids to develop a novel in vitro fluorometric binding assay to characterize and quantify their binding to their cellular targets, including cellular retinoid binding protein II (CRABPII). The dihydroquinoline retinoid, DC360, exhibited particularly strong binding ( Kd = 34.0 ± 2.5 nM), and we further used X-ray crystallography to determine the structure of the DC360-CRABPII complex to 1.8 Å, which showed that DC360 occupies the known hydrophobic retinoid binding pocket. Finally, we used confocal fluorescence microscopy to image the cellular behavior of the compounds in cultured human epithelial cells, highlighting a fascinating nuclear localization, and used RNA sequencing to confirm that the compounds regulate cellular processes similar to those of ATRA. We anticipate that the unique properties of these fluorescent retinoids can now be used to cast new light on the vital and highly complex retinoid signaling pathway.

Synthesis of phenyl analog of retinoic acid methyl ester proceeding from 3-(bromomethyl)but-3-enal diethylacetal

Aromatic analog of retinoic acid methyl ester was prepared using a convenient prenylating agent, 3-(bromomethyl)but-3-enal diethylacetal, and Barbier reaction in the key stage of building up the carbon chain of target molecules. A version is offered of the synthesis of methylidene analog of aromatic retinoic acid.

The molecular basis of the interactions between synthetic retinoic acid analogues and the retinoic acid receptors.

All-trans-retinoic acid (ATRA) and its synthetic analogues EC23 and EC19 direct cellular differentiation by interacting as ligands for the retinoic acid receptor (RARα, β and γ) family of nuclear receptor proteins. To date, a number of crystal structures of natural and synthetic ligands complexed to their target proteins have been solved, providing molecular level snap-shots of ligand binding. However, a deeper understanding of receptor and ligand flexibility and conformational freedom is required to develop stable and effective ATRA analogues for clinical use. Therefore, we have used molecular modelling techniques to define RAR interactions with ATRA and two synthetic analogues, EC19 and EC23, and compared their predicted biochemical activities to experimental measurements of relative ligand affinity and recruitment of coactivator proteins. A comprehensive molecular docking approach that explored the conformational space of the ligands indicated that ATRA is able to bind the three RAR proteins in a number of conformations with one extended structure being favoured. In contrast the biologically-distinct isomer, 9-cis-retinoic acid (; 9CRA), showed significantly less conformational flexibility in the RAR binding pockets. These findings were used to inform docking studies of the synthetic retinoids EC23 and EC19, and their respective methyl esters. EC23 was found to be an excellent mimic for ATRA, and occupied similar binding modes to ATRA in all three target RAR proteins. In comparison, EC19 exhibited an alternative binding mode which reduces the strength of key polar interactions in RARα/γ but is well-suited to the larger RARβ binding pocket. In contrast, docking of the corresponding esters revealed the loss of key polar interactions which may explain the much reduced biological activity. Our computational results were complemented using an in vitro binding assay based on FRET measurements, which showed that EC23 was a strongly binding, pan-agonist of the RARs, while EC19 exhibited specificity for RARβ, as predicted by the docking studies. These findings can account for the distinct behaviour of EC23 and EC19 in cellular differentiation assays, and additionally, the methods described herein can be further applied to the understanding of the molecular basis for the selectivity of different retinoids to RARα, β and γ.

Fenretinide (4-HPR) Targets Caspase-9, ERK 1/2 and the Wnt3a/β-Catenin Pathway in Medulloblastoma Cells and Medulloblastoma Cell Spheroids.

Medulloblastoma (MB), a neuroectodermal tumor arising in the cerebellum, represents the most frequent childhood brain malignancy. Current treatments for MB combine radiation and chemotherapy and are often associated with relevant side effects; novel therapeutic strategies are urgently needed. N-(4-Hydroxyphenyl) retinamide (4-HPR, fenretinide), a synthetic analogue of all-trans retinoic acid, has emerged as a promising and well-tolerated cancer chemopreventive and chemotherapeutic agent for various neoplasms, from breast cancer to neuroblastoma. Here we investigated the effects of 4-HPR on MB cell lines and identified the mechanism of action for a potential use in therapy of MB. Flow cytometry analysis was performed to evaluate 4-HPR induction of apoptosis and oxygen reactive species (ROS) production, as well as cell cycle effects. Functional analysis to determine 4-HPR ability to interfere with MB cell migration and invasion were performed. Western Blot analysis were used to investigate the crucial molecules involved in selected signaling pathways associated with apoptosis (caspase-9 and PARP-1), cell survival (ERK 1/2) and tumor progression (Wnt3a and β-catenin). We show that 4-HPR induces caspase 9-dependent cell death in DAOY and ONS-76 cells, associated with increased ROS generation, suggesting that free radical intermediates might be directly involved. We observed 4-HPR induction of cell cycle arrest in G1/S phase, inactivated β-catenin, and inhibition of MB cell migration and invasion. We also evaluated the ability of 4-HPR to target MB cancer-stem/cancer-initiating cells, using an MB spheroids model, followed by flow cytometry and quantitative real-time PCR. 4-HPR treatment reduced DAOY and ONS-76 spheroid formation, in term of number and size. Decreased expression of the surface markers CD133+ and ABCG2+ as well as Oct-4 and Sox-2 gene expression were observed on BTICs treated with 4-HPR further reducing BITIC invasive activities. Finally, we analyzed 4-HPR ability to inhibit MB tumor cell growth in vivo in nude mice. Taken together, our data suggest that 4-HPR targets both parental and MB tumor stem/initiating cell-like populations. Since 4-HPR exerts low toxicity, it could represent a valid compound in the treatment of human MB.

Cell Line-Dependent Variability of Coordinate Expression of p75NTR and CRABP1 and Modulation of Effects of Fenretinide on Neuroblastoma Cells.

Neuroblastoma is a childhood neural crest tumor. Fenretinide, a retinoic acid analogue, induces accumulation of mitochondrial reactive oxygen species and consequent apoptosis in neuroblastoma cells. The p75 neurotrophin receptor (p75NTR) enhances the antineuroblastoma cell efficacy of fenretinide in vitro. We examined the role of the retinoid binding protein, CRABP1, in p75NTR-mediated potentiation of the efficacy of fenretinide. Knockdown and overexpression, respectively, of either p75NTR or CRABP1 were effected in neuroblastoma cell lines using standard techniques. Expression was determined by qRT-PCR and confirmed at the protein level by Western blot. Metabolic viability was determined by Alamar blue assay. While protein content of CRABP1 correlated roughly with that of p75NTR in the three neuroblastoid or epithelioid human neuroblastoma cell lines studied, manipulation of p75NTR expression resulted in cell line-dependent, variable change in CRABP1 expression. Furthermore, in some cell lines, induced expression of CRABP1 in the absence of p75NTR did not alter cell sensitivity to fenretinide treatment. The effects of manipulation of p75NTR expression on CRABP1 expression and the effects of CRABP1 expression on fenretinide efficacy are therefore neuroblastoma cell line-dependent. Potentiation of the antineuroblastoma cell effects of fenretinide by p75NTR is not mediated solely through CRABP1.

Retinoic acid ortho-hydroxy aniline amide promotes neurotrophin mediated cell growth and proliferation in nerve cells

In the present study, effect of retinoic acid ortho-hydroxy aniline amide (RAA), an analog of retinoic acid (RA) was investigated on neurotrophin mediated cell growth and proliferation in hippocampus-derived stem cell clones. The results revealed that retinoic acid ortho-hydroxy aniline amide enhanced neuronal differentiation, increased NeuroD expression and p21 compared to proliferating cells stimulated by FGF-2. Retinoic acid ortho-hydroxy aniline amide treatment prevented decrease in RNA for trkC, enhanced expression of trkB RNA and p75NGFR. Retinoic acid ortho-hydroxy aniline amide treatment for 5 days increased the baseline expression of c- fos mRNA following stimulation with NGF, BDNF, and NT-3. Treatment of cultures with retinoic acid ortho-hydroxy aniline amide for 5 days followed by 5 days in serum alone increased Map2ab-positive cells compared to that of control. Therefore, retinoic acid ortho-hydroxy aniline amide is an effective agent for inducing neuronal differentiation and proliferation and is even more potent than the parent retinoic acid.

A novel retinoic acid analog, 4-amino-2-trifluoromethyl-phenyl retinate, inhibits gastric cancer cell growth

Retinoic acid (RA) analogs have been used in the treatment of a variety of cancers; however, their application is limited due to serious therapy-related sequelae. In the present study, the effects of a novel RA analog, 4-amino-2-trifluoromethyl-phenyl retinate (ATPR), on the growth of gastric cancer cells were evaluated. Three gastric cancer cell lines, AGS, MKN-74 and SC-M1, were treated with either all‑trans retinoic acid (ATRA) or ATPR, and their growth and distribution in different cell cycle phases were assessed using an MTT assay and propidium iodide (PI) staining followed by flow cytometry. The binding affinity of ATPR to the retinoic acid receptors, retinoic acid receptor-α (RAR-α) and retinoid X receptor-α (RXR-α), was determined using ligand-binding assays. Activator protein-1 (AP-1) activity was measured using a luciferase reporter assay. Western blot analysis was used to determine cyclin E, Bcl-2 and Bax protein expression. ATPR preferentially bound RXR-α (0.04 nM) as compared with RAR-α (20.96 nM). Although both ATRA and ATPR inhibited the growth of AGS, MKN-74 and SC-M1 cells in a dose-dependent manner, a significantly greater inhibitory effect was observed with treatment with 5 and 500 µM ATPR for 3 days (P<0.05). In addition, ATPR (50 µM), but not ATRA, significantly increased the population of AGS and MKN-74 cells in the subG1 phase and decreased the Bcl-2/Bax ratio (P<0.05). Furthermore, in MNK-74 and SC-M1 cells treated with 12-O-tetradecanoylphorbol-13-acetate (TPA) and 5 or 10 µM of ATPR significantly suppressed the activity of the AP-1 reporter as compared to treatment with ATRA (P<0.05). Thus, ATPR inhibits cancer cell proliferation to a greater extent compared to ATRA, possibly through the RXR-mediated inhibition of AP-1 activity.

Application of synthetic photostable retinoids induces novel limb and facial phenotypes during chick embryogenesisin vivo

We have recently developed a range of synthetic retinoid analogues which include the compounds EC23 and EC19. They are stable on exposure to light and are predicted to be resistant to the normal metabolic processes involved in the inactivation of retinoids in vivo. Based on the position of the terminal carboxylic acid groups in the compounds we suggest that EC23 is a structural analogue of all-trans retinoic acid (ATRA), and EC19 is an analogue of 13-cis retinoic acid. Their effects on the differentiation of pluripotent stem cells has been previously described in vitro and are consistent with this hypothesis. We present herein the first description of the effects of these molecules in vivo. Retinoids were applied to the anterior limb buds of chicken embryos in ovo via ion-exchange beads. We found that retinoid EC23 produces effects on the wing digits similar to ATRA, but does so at two orders of magnitude lower concentration. When larger quantities of EC23 are applied, a novel phenotype is obtained involving production of multiple digit 1s on the anterior limb. This corresponds to differential effects of ATRA and EC23 on sonic hedgehog (shh) expression in the developing limb bud. With EC23 application we also find digit 1 phenotypes similar to thumb duplications described in the clinical literature. EC23 and ATRA are shown to have effects on the entire proximal–distal axis of the limb, including hitherto undescribed effects on the scapula. This includes suppression of expression of the scapula marker Pax1. EC23 also produces effects similar to those of ATRA on the developing face, producing reductions of the upper beak at concentrations two orders of magnitude lower than ATRA. In contrast, EC19, which is structurally very similar to EC23, has novel, less severe effects on the face and rarely alters limb development. EC19 and ATRA are effective at similar concentrations. These results further demonstrate the ability of retinoids to influence embryonic development. Moreover, EC23 represents a useful new tool to investigate developmental processes and probe the mechanisms underlying congenital abnormalities in vertebrates including man.