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Selective retinoids and rexinoids in cancer therapy and chemoprevention
Abstract
Natural and synthetic retinoids are effective inhibitors of tumor cell growth in vitro and in vivo. However, the toxicity of natural derivatives of vitamin A limits their therapeutic use. Recently, synthetic compounds selective for the different retinoid receptor isotypes have been generated that circumvent pan-retinoid toxicity. The tumor-suppressive activity of selective retinoid and/or rexinoid ligands has been established preclinically, and emerging clinical trials are supportive of the chemotherapeutic and chemopreventive potential of these compounds in multiple oncology indications, with reduced toxicity. Moreover, the combination of retinoids and/or rexinoids with chemotherapeutic agents for the synergistic modulation of specific pathways could also be of benefit in cancer therapy.
... The study of the relationships between structure and activity (SAR) has established the structure template of ATRA derivatives as a hydrophobic region and a polar region connected via a linker ( Figure 1) [6][7][8]. Further SAR has revealed that the nature of the linker is crucial for the compounds to attain RAR-isotype selectivity and that the amide linker group is a key structural feature for RARα-specificity, presumably due to a favorable hydrogen-bonding interaction between the amide group of the ligand and the hydroxyl group of serine 232 residue present in the ligand binding pocket of RARα [9,10]. ...
... To date, most of the developed ATRA derivatives contain a tri-/tetra-methylated six-membered rigid ring in their hydrophobic region. Since it has been reported that the size of the hydrophobic part of the ligands can significantly affect the activity [10], we were interested in studying the impact of a smaller ring system on the activity of derivatives by replacing the hydrophobic part of AM80 with mono-/di-substituted indene derivatives. Specifically, compound 5 was designed by incorporating small alkoxyl or alkyl groups into the indane structure. ...
... Melting points were measured with a B-540 melting-point apparatus (Büchi, Flawil, St. Gallen, Switzerland) and are uncorrected. (10). To a solution of 2,3-dihydro-1H-inden-1-one (9, 1.32 g, 10.0 mmol) in concentrated sulfuric acid (10 mL), KNO3 (1.21 g, 12.0 mmol) in concentrated sulfuric acid (10 mL) was added dropwise at −5 °C in 30 min. ...
A series of novel indene-derived retinoic acid receptor α (RARα) agonists have been designed and synthesized. The use of receptor binding, cell proliferation and cell differentiation assays demonstrated that most of these compounds exhibited moderate RARα binding activity and potent antiproliferative activity. In particular, 4-((3-isopropoxy-2,3-dihydro-1H-inden-5-yl)-carbamoyl) benzoic acid (36d), which showed a moderate binding affinity, exhibited a great potential to induce the differentiation of NB4 cells (68.88% at 5 μM). Importantly, our work established indene as a promising skeleton for the development of novel RARα agonists.
... Spośród ko-represorów poznane zostały ko-represor receptorów jądrowych, milczący mediator dla receptorów retinoidowych i hormonów tarczycy oraz deacetylazy białek histonowych. Natomiast w obecności kwasu retinowego lub innego liganda zmienia się konformacja receptora, następuje odłączenie korepresora i przyłączenie wielobiałkowego kompleksu aktywującego, który zawiera w swoim składzie między innymi acetylotransferazę białek histonowych, dzięki czemu po procesie acetylacji zostaje zapoczątkowana transkrypcja genów (rycina 2), [2,5,6,7]. Ważne jest, że ekspresja receptorów retinoidowych nie jest równomierna w całym organizmie. Obecne są one jedynie w niektórych tkankach i komórkach, jak: naskórek, mieszki włosowe, gruczoły łojowe, czy komórki układu immunologicznego (na przykład komórki Langerhansa). ...
... Jako naturalny ligand receptorów jądrowych wpływa na transkrypcję określonych genów uczestnicząc w: -procesach odpornościowych, -różnicowaniu się komórek, -reprodukcji, jako czynnik wpływający na wytwarzanie komórek rozrodczych, a także na embriogenezę i rozwój płodu, -utrzymaniu właściwego stanu naskórka, poprzez regulację procesów złuszczania i wymiany zewnętrznych warstw komórek [6,12]. [7,12,13,14]. ...
... Beksaroten natomiast w postaci doustnych kapsułek (75 mg) oraz żelu (1%) znalazł zastosowanie w terapii zaawansowanego chłoniaka skórnego z komórek T. Dodatkowo znajduje się on w trzeciej fazie badań klinicznych leczenia niedrobnokomórkowego raka płuc w terapii skojarzonej ze środkami cytotoksycznymi. Fenretynid (rycina 4), tak zwany atypowy retinoid, badany jest jako środek w leczeniu nowotworów u dzieci [2,3,7,15]. ...
Retinoids are compounds, which
show the activity of vitamin A. Both naturally occurred (retinol,
ratinaldehyde, retinoic acid) and synthetic (eg. acitretin, adapalene,
tazarotene, bexarotene) compounds belong to this group. They
show their activity through nuclear receptors which bind to DNA
in promoter regions of genes regulated by retinoids. Some of them
are ligands of the receptors while other need to be metabolized to
active derivatives. Naturally occurred in the organism retinoids take
part in such processes as proliferation and differentiation of cells,
reproduction, maintenance of epithelial tissue, immune function and
vision (retinaldehyde). Considering their mechanism of action they
are used in the therapy of various disorders like acne, psoriasis or
neoplasms. They can be administered orally (acitretin) or topically
(alitretinoin, motretinide, adapalene, tazatorene) and some of them
both routes (tretinoin, isotretinoin, bexarotene). They are also active
constituents of cosmeceuticals. The usage of retinoids is limited
by adverse effects caused by them to which belong teratogenicity,
mucocutaneous and skeletal toxicity.
... Biological activities of retinoic acids and retinoids (derivatives of retinoic acid) in the target cells are mediated through specific representatives of the NR's "superfamily" -receptors for all-trans retinoic acid (RAR) and receptors for 9-cis retinoic acid (RXR) (ref. 31 ). ...
... In contrast, 9-cis retinoic acid binds and activates all three subtypes of RAR and RXR but with a different affinity for each receptor and its subtypes. RAR and RXR are ligand-inducible transcription factors that function predominantly as RAR-RXR heterodimers, positively or negatively affecting the specific genetic expression 30,31,33 . The all RAR subtypes bind all-trans and 9-cis retinoic acid with similar affinity, whereas RXRs bind only 9-cis retinoic acid. ...
... In humans, retinoids exhibit strong potential for the treatment of precancerous epithelial lesions and in the prevention of some primary solid tumours -lung, liver and breast 4,36 . Despite the positive effects of retinoids, their teratogenic effect, which limits their use in women of child-bearing period cannot be overlooked 3,31,35,37 . ...
Renal cell carcinoma (RCC) is a urologic malignancy with a steady rise in incidence and high mortality rate. Between 60 to 70% of patients with renal cell carcinoma can only be cured with surgery but despite advances in early diagnostis, in around 20-30% of cases there is metastasis. For these patients, chemotherapy and radiotherapy are ineffective and hence the prognosis is poor. Retinoids are biologically active compounds of either natural or synthetic origin that are involved in complex physiological and developmental processes in many tissues including cell proliferation and activation of tumour suppression genes. This article reviews the role of retinoids and their cognate nuclear retinoid/rexinoid receptors in relation to renal cell carcinoma.
A literature search using ScienceDirect and Medline with a focus on the relationship between renal cell carcinoma and nuclear retinoid/rexinoid receptors.
Use of retinoids/rexinoids in the treatment of locally advanced and metastatic RCC significantly prolongs median time of tumour progression and overall survival of patients. Combination therapy with other preparations has greater efficacy than treatment with retinoids alone. Patient survival can be predicted on the basis of the expression of different all-trans retinoic acid receptor (RAR) and 9-cis retinoic acid receptor (RXR) subtypes.
Since nuclear retinoid receptors play a crucial role as ligand-activated, DNA binding, trans-acting, transcription-modulating proteins involved in a general molecular mechanism responsible for transcriptional responses in target genes, retinoids might be an alternative approach for the treatment of renal cell carcinoma.
... To date, more than 1500 different related compounds have been discovered and tested. Retinoids have raised interest within the scientific community thanks to their beneficial effects in vision [5], skin disorders (acne, psoriasis, and keratinization disorders) [6], and cancer [7][8][9]. Specifically, in the oncology field, retinoids attracted researchers' attention due to their known anti-tumor properties. In particular, they were demonstrated Table 1. ...
... Retinoids are a class of natural and synthetic compounds derived from vitamin A, which have raised interest within the scientific community thanks, among others, to their beneficial effects in cancer [7][8][9]. More specifically, the synthetic retinoic acid derivative fenretinide (4-HPR) is endowed with anti-tumor properties and characterized by a favorable pharmacological profile, with lower systemic toxicity and better tissue distribution compared to its natural analog [24]. ...
Retinoids are a class of natural and synthetic compounds derived from vitamin A. They are involved in several biological processes like embryogenesis, reproduction, vision, growth, inflammation, differentiation, proliferation, and apoptosis. In light of their important functions, retinoids have been widely investigated for their therapeutic applications. Thus far, their use for the treatment of several types of cancer and skin disorders has been reported. However, these therapeutic agents present several limitations for their widespread clinical translatability, i.e., poor solubility and chemical instability in water, sensitivity to light, heat, and oxygen, and low bioavailability. These characteristics result in internalization into target cells and tissues only at low concentration and, consequently, at an unsatisfactory therapeutic dose. Furthermore, the administration of retinoids causes severe side-effects. Thus, in order to improve their pharmacological properties and circulating half-life, while minimizing their off-target uptake, various retinoids delivery systems have been recently developed. This review intends to provide examples of retinoids-loaded nano-delivery systems for cancer treatment. In particular, the use and the therapeutic results obtained by using fenretinide-loaded liposomes against neuroectodermal-derived tumors, such as melanoma, in adults, and neuroblastoma, the most common extra-cranial solid tumor of childhood, will be discussed.
... Retinoidler, heterodimerik reseptörlere bağlanıp aktive ederek, belirli genlerin transkripsiyonel düzenlenmelerini, yani genin kodladığı protein sentezini doğrudan etkileyebilirler (12). Hem RAR, hem de RXR nükleer reseptörleri için biyolojik aktif ligandlar mevcut olmakla birlikte (11,13), tamamen reseptör-spesifik olan, RXR'a bağlanıp aktive eden, fakat RAR ile etkileşim göstermeyen doğal ve sentetik RXR ligandları bulunmaktadır (13,14,15). Retinoid X reseptörlerinin yapısında, ligandların yüksek ve spesifik bir affinite ile bağlanabileceği küçük bir cep bulunmaktadır. ...
... Retinoidler, heterodimerik reseptörlere bağlanıp aktive ederek, belirli genlerin transkripsiyonel düzenlenmelerini, yani genin kodladığı protein sentezini doğrudan etkileyebilirler (12). Hem RAR, hem de RXR nükleer reseptörleri için biyolojik aktif ligandlar mevcut olmakla birlikte (11,13), tamamen reseptör-spesifik olan, RXR'a bağlanıp aktive eden, fakat RAR ile etkileşim göstermeyen doğal ve sentetik RXR ligandları bulunmaktadır (13,14,15). Retinoid X reseptörlerinin yapısında, ligandların yüksek ve spesifik bir affinite ile bağlanabileceği küçük bir cep bulunmaktadır. ...
... Moreover, in vitro studies have revealed that certain cellular phenomena, such as differentiation, proliferation and apoptosis, require the activation of a specific RAR isotype, even though others are also expressed. Examples include the differentiation of leukaemic cells and the inhibition of proliferation of certain breast-cancer cell lines by RARα agonists 31 . It is believed that in these cases RARα responds to its ligand as a heterodimer with an RXR partner, as RXR-selective ligands ('rexinoids'), which are inactive on their own, synergize with RAR-selective ligands 32 . ...
... Retinoids and rexinoids have been discussed as potential candidates for cancer chemoprevention in at-risk patients, particularly with respect to the prevention of oral cancers, and cancers of the head and neck, and lung 31,[72][73][74] . Moreover, in a randomized placebo-controlled study, the loss of RARβ expression, which is considered to be a biomarker of pre-neoplasia 75 , could be significantly reversed in former smokers by treatment with 9-cis retinoic acid, but not with 13-cis retinoic acid 76 . ...
... Various synthetic retinoids have been produced to identify cellular responses to retinoid signaling. 9,10 Bristol-Myers Squibb (BMS) and other companies developed a series of lowmolecular-weight 'arotinoid' compounds that function as RAR antagonists by blocking ATRA binding and activation of transcription of RAR target genes ( Figure 1). [11][12][13] Using systematically modified dosing regimens, we demonstrated that low levels of RAR pan-antagonist BMS-189453 (1) inhibited spermatogenesis in mice without any observable side-effects. ...
... Oral doses of 2.0 and 10 mg/kg for 7 days were chosen to minimize possible bioavailability differences. 10,16 Surprisingly, detailed morphological analysis of testes obtained one-month after the 7-day dosing period revealed no effect on spermatogenesis ( Figure 3). Specifically, alignment of step 16 spermatids at the lumen in stage VIII tubules and sperm release at stage IX into the lumen appeared normal in testes from mice treated with both compounds in both 2-mg (data not shown) and even with 10mg group (3, Figure 3B; 2, Figure 3E), compared with control ( Figure 3A and 3D). ...
Oral administration of a retinoic acid receptor (RAR) pan-antagonist reversibly inhibits spermatogenesis. Given the importance of RARα in regulating spermatogenesis, we identified two RARα-selective antagonists by transactivation and transactivation competition assays and asked whether they effectively inhibit spermatogenesis. Although these two antagonists were potent in vitro, they displayed poor in vivo activity in mice when administered orally. Testicular weights were normal and morphological analysis revealed normal spermatid alignment and sperm release. In vitro drug property analyses were performed with one of these antagonists and compared with the pan-antagonist. We showed that the discrepancies may be explained by several factors, including high plasma protein binding, faster hepatic metabolism relative to the pan-antagonist, and only moderate permeability. The conclusion of poor oral bioavailability was supported by more pronounced defects in mice when the antagonist was administered intravenously versus intraperitoneally. These results are crucial for designing new RARα-selective antagonists for pharmaceutical application.
... Therapies based on 9cRA and other retinoids have two limitations: (1) a high dose is needed to achieve a biological response; 19,20 and (2) a variety of side effects are observed including hypothyroidism, liver toxicity and teratogenicity. [21][22][23][24][25] Rexinoids are RXR-specific ligands having the potential to function as cancer-preventive agents without the serious side effects associated with retinoids. One rexinoid, bexarotene (2), is used to treat cutaneous T-cell lymphoma and holds promise as a chemopreventive agent against various cancers. ...
... A common characteristic of RXR agonists is the presence of an acidic moiety, the conjugate base of which can bind to the positively charged Arg316. 22,26,[30][31][32] The indenoisoquinoline 3 (Figure 1) was synthesized in our laboratory and found to bind to RXRα and induce apoptosis in MCF-7 breast cancer cells in a dose-dependent manner. 33 The rexinoid activity of the lead compound 3 was discovered through its inclusion in a 5000-compound library that was screened for RXR agonist activity in an RXRE-luciferase reporter gene assay, and it proved to be the only active compound in the whole array. ...
Nuclear receptors, such as the retinoid X receptor (RXR), are proteins that regulate a myriad of cellular processes. Molecules that function as RXR agonists are of special interest for the prevention and control of carcinogenesis. The majority of these ligands possess an acidic moiety that is believed to be key for RXR activation. This communication presents the design, synthesis, and biological evaluation of both acidic and nonacidic indenoisoquinolines as new RXR ligands. In addition, a comprehensive structure-activity relationship study is presented that identifies the important features of the indenoisoquinoline rexinoids. The ease of modification of the indenoisoquinoline core and the lack of the necessity of a carboxyl group for activity make them an attractive and unusual family of RXR agonists. This work establishes a structural foundation for the design of new and novel rexinoid cancer chemopreventive agents.
... For example, a nuclear receptor co-repressor and a short heterodimer partner bind to RARα and suppress its function [6,7] while SP1 and the B-cell translocation gene 1 activate RARα via direct interaction [8,9]. Since retinoids produce severe side effects including tetracarcinogenesis, mucocutaneous cytotoxicity, and hypertriglyceridemia [10], elucidating the mechanism underlying retinoid-facilitated transcriptional regulation is crucial in developing next-generation retinoid-acting drugs with an improved specificity. ...
Retinoids exert antitumor effects through the retinoic acid receptor α (RARα). In the present study, we sought to identify the factors involved in the RARα-mediated transcriptional regulation of the tumor suppressor gene and the tissue factor pathway inhibitor 2 (TFPI2) in hepatocellular carcinoma (HCC). All-trans-retinoic acid (ATRA) was used in the in vitro experiments. Cell invasiveness was measured using trans-well invasion assay. ATRA significantly increased TFPI2 expression through RARα in a human HCC cell line known as HuH7. TFPI2 was vital in the ATRA-mediated suppression of HuH7 cell invasion. The musculo-aponeurotic fibrosarcoma oncogene homolog B (MAFB) significantly enhanced the activation of the TFPI2 promoter via RARα while MAFF inhibited it. The knockdown of RARα or MAFB counteracted the ATRA-mediated suppression of HuH7 cell invasion while the knockdown of MAFF inhibited the invasion. TFPI2 expression in HCC tissues was significantly downregulated possibly due to the decreased expression of RARβ and MAFB. Patients with HCC expressing low MAFB and high MAFF levels showed the shortest disease-free survival time. These results suggest that MAFB and MAFF play critical roles in the antitumor effects of retinoids by regulating the expression of retinoid target genes such as TFPI2 and can be promising for developing therapies to combat HCC invasion.
... Treatment with retinoic acid (RA) can inhibit tumor outgrowth by suppressing cell proliferation, promoting cellular differentiation, or by inducing apoptotic cell death [1]. RAs exert these effects through two main groups of intranuclear receptors belonging to the steroid/thyroid hormone receptor family: retinoic acid receptors (RARs) and retinoid X receptors (RXRs) [2,3]. RARs interact with all-trans retinoic acid (ATRA) and 9-cis retinoic acid (9-cis RA), while RXRs bind 9-cis RA [4,5]. ...
Retinoic acids are natural derivatives of vitamin A, and play important roles in modulating tumor cell growth by regulating differentiation, thus suggesting the potential use of these derivatives in cancer therapy and prevention. To visualize the intranuclear responses of functional retinoic acid receptors, we have developed a dual-imaging reporter gene system based on the use of sodium/iodide symporter (NIS) and luciferase in cancer cell lines. NIS and luciferase genes were linked with an internal ribosome entry site, and placed under the control of an artificial cis-acting retinoic acid responsive element (pRARE/NL). After retinoic acid treatment, I-125 uptake by pRARE/NL transfected cells was found to have increased by up to about five times that of nontreated cells. The bioluminescence intensity of pRARE/NL transfected cells showed dose-dependency. In vivo luciferase images showed higher intensity in retinoic acid treated SK-RARE/NL tumors, and scintigraphic images of SK-RARE/NL tumors showed increased Tc-99m uptake after retinoic acid treatment. The NIS/luciferase imaging reporter system was sufficiently sensitive to allow the visualization of intranuclear retinoic acid receptor activity. This cis-enhancer imaging reporter system may be useful in vitro and in vivo for the evaluation of retinoic acid responses in such areas as cellular differentiation and chemoprevention.
... The biological activities of retinoids are mediated in target cells through two different classes of nuclear receptors: RARs and RXRs, which are encoded by separate sets of genes [3]. There are three subtypes of RARs and of RXRs, which are designated as α, β, and γ. ...
Introduction:
Differentiation therapy using all-trans retinoic acid (ATRA) revolutionised the treatment of acute promyelocytic leukaemia to such an extent that it is now one of the most curable types of leukaemia, with ATRA and anthracycline-based chemotherapy providing cure rates above 80% (reviewed in [1]). Isotretinoin is used to treat chronic acne. Here, we examine the information described in recent patents and the extent to which new findings are influencing extending retinoid-based differentiation therapy to other cancers, as well as the development of new therapies for other disorders.
Areas covered:
A search has been performed on the literature and worldwide patents filed during 2014 to the present time, focusing on synthetic agonists and antagonists of retinoic acid receptors and novel compositions for the delivery of these agents.
Expert opinion:
New potential therapeutic applications have been described, including lung, breast and head and neck cancers, T cell lymphoma and neurodegenerative, metabolic, ophthalmic, muscle, and inflammatory disorders. Recent patents have described the means to maximise retinoid activity. Two decades of efforts to extend retinoid-based therapies have been disappointing and new synthetic retinoids, target diseases and modes of delivery may well resolve this long standing issue.
... Retinoidal aktivitelerin birçoğu, RXR-RAR heterodimerlerinin RAR bölgesine retinoidlerin bağlanması ile gerçekleşmektedir. Retinoidler, heterodimerik reseptörlere bağlanıp aktive ederek, belirli genlerin transkripsiyonel düzenlenmelerini, yani genin kodladığı protein sentezini doğrudan etkileyebilirler (12). Hem RAR, hem de RXR nükleer reseptörleri için biyolojik aktif ligandlar mevcut olmakla birlikte (11,13), tamamen reseptör-spesifik olan, RXR'a bağlanıp aktive eden, fakat RAR ile etkileşim göstermeyen doğal ve sentetik RXR ligandları bulunmaktadır (13, 14,15). ...
Docking studies of the previously synthesized retinoid analogues were performed using the AutoDock Tools - 1.5.4 in order to study the interactions between the retinoid compounds and RXR-alpha (RXRα) receptor protein. The RXRα receptor protein was taken from the Protein Databank in*.pdb format (3FUG). The binding energies of the interaction in different conformations were obtained and the best conformation fit was chosen for the representation of the each compound. Compound 17 appears as the most strongly linked molecule (ΔG = -10.90 kcal/mol) to the RXRα receptor protein.
... Since the synergistic effect of demethylation and histone acetylation in re-expression of genes was first reported by using TSA and 5-aza-dC (Cameron and Bachman et al., 1999), DNMTi 5-aza-dC and HDACi TSA have been used to partially erase the pre-existing epigenetic marks of donor cells to improve subsequent in vitro development of cloned embryos (Tsuji and Kato et al., 2009), and more recently to improve the efficiency of iPSC generation (Wernig and Zhao et al., 2008). Unlike 5-aza-dC and TSA, ATRA does not regulate gene expression directly, instead, after interacting with its target receptors the complex activates HAT and inhibits HDAC and activate gene expression (Zusi and Lorenzi et al., 2002;Wei, 2003). ATRA has also been applied to reduce methylation of cytosine in somatic cells (Eilertsen and Power et al., 2007). ...
... In 1995, the Food and Drug Administration (FDA) approved all-trans-retinoic acid for the treatment of a rare leukemia, acute promyelocytic leukemia, which is caused by translocations of the retinoic acid receptor-α gene (Tang and Gudas, 2011). However, the clinical utilization of retinoids has been limited by their adverse effects, including varying degrees of teratogenicity, mucocutaneous cytotoxicity, chondrogenesis inhibition and hypertriglyceridemia (Zusi et al., 2002). Possibly, the combination of retinoids with other agents may allow obtaining therapeutic efficacy with lower doses through a synergism of action. ...
Melanoma incidence is dramatically increasing and the available treatments beyond partial efficacy have severe side effects. Retinoids are promising anticancer agents, but its clinical use has been limited by their toxicity, although a combination with other agents can possibly generate a therapeutic action at lower dosage. Thus, we investigated the effects of all-trans-retinoic acid combined with the antiestrogen endoxifen on melanoma cell proliferation and the effects were compared with its pro-drug tamoxifen. Moreover, we evaluated the effects of these combinations on non-neoplasic cells and assessed mitochondrial bioenergetic functions, to predict their potential toxicity. Individually, all-trans-retinoic acid and the antiestrogens endoxifen and tamoxifen decreased melanoma cell biomass, cell viability and DNA synthesis, without increased cell death, suggesting that the compounds inhibited cell proliferation. Noteworthy, endoxifen decreased cell proliferation more efficiently than tamoxifen. The combination of endoxifen with all-trans-retinoic acid enhanced the antiproliferative effects of the compounds individually more potently than tamoxifen, which did not enhance the effects induced by all-trans-retinoic acid alone, and blocked cell cycle progression in G1. Moreover, the combination of all-trans-retinoic acid with endoxifen significantly decreased melanoma cells migration, whereas the combination with tamoxifen did not present significant effects. At the concentrations used the compounds did not induce cytotoxicity in non-neoplasic cells and liver mitochondrial bioenergetic function was not affected. Altogether, our results show for the first time that a combined treatment of all-trans-retinoic acid with endoxifen may provide an anti-proliferative and anti-migration effect upon melanoma cells without major toxicity, offering a powerful therapeutic strategy for malignant melanoma.
... Synthetic compounds selective for the diff e rent re t i n o i d receptor isotypes are currently undergoing clinical evaluation. In addition, the combination of retinoids with other chemotherapeutic agents may also be of value in cancer therapy [11] . By analogy to the SERM concept, it has been suggested that PPA Rγ modulators (SPA R M S ) , designed to have desired effects on specific genes and target tissue without undesirable effects on others, will be clinically important in the future for chemoprevention and chemotherapy of c a n c e r [ 1 2 ] . ...
INTRODUCTION The need for better cancer treatment is evident. In the developed world, approximately one in three persons contracts cancer and around one in four of these dies from the disease. The worldwide incidence of cancer is predicted to double from 10 to 20 million over the next two decades and the death rate will increase from 6 to 10 million. Advances in treatment with surgery, radiotherapy and chemotherapy have had a limited impact on m o r t a l i t y. Cures can be achieved in childhood cancers, testicular cancer and lymphoma, and improvements in survival rates have been made as a result of the adjuvant drug treatment of breast and colorectal cancer. However, the majority of human cancers are difficult to treat, especially in their advanced, metastatic forms. The need for new and effective forms of systemic therapy is pressing and the discovery of novel, mechanism-based agents directed against the molecular pathology of cancer is of enormous potential [1] . It has been known for many years that cancer has a genetic component and it is clear that there is a multistage pro g ression to malignancy. The application of modern molecular techniques to study cancer over the last 2 decades has led to the identification of 4 major groups of genes which are involved in tumourigenesis – oncogenes, tumour s u p p ressor (TS), cell cycle control (CCC) and mismatch repair (MMR). Cellular oncogenes (p roto-oncogenes) encode proteins, which are important in the control of cell pro l i f e r a t i o n , differentiation, cell cycle control and apoptosis. Mutations in these genes act dominantly and lead to a gain of function. In contrast, TS genes inhibit cell proliferation by arresting progression through the cell cycle and block differentiation. CCC genes a re involved in the positive and negative regulation of the cell cycle and they interact with oncogenes and TS genes, and in some cases may be considered to be such in their own right. To ensure that DNA replication is complete and that any damaged DNAis repaired, cells must pass through specific checkpoints and MMR genes ensure that damaged DNA is repaired. There is compelling evidence for the importance of these genes in the etiology of many human tumours.
... Additionally, vitamin A has been researched as a chemotherapeutic and chemopreventive agent for a variety of malignancies. Currently, it is utilized to treat acute promyelocytic leukemia (Zusi et al., 2002). The mechanism of vitamin A's inhibition of tumor growth is thought to work through modulation of gene expression in cell growth, differentiation, and apoptosis (Zanardi et al., 2006). ...
... [16] The study of Newman et al. [17] also demonstrated the differentiation capability of RA. [17] Vitamin A and related carotenoid compounds have antioxidant and immune stimulatory properties, thus have a serious role in cancer prevention. [18,19] Especially, retinoids and related compounds such as atRA may have therapeutic value in the treatment of human malignant gliomas as it is remarked in the article of Young-II et al. [18] . Also, the studies of Rotan [20] and Bouterfa et al. [21] showed that retinoids strongly inhibit proliferation and migration in primary cultures of human glioblastomas multiforme. ...
Polymeric biomaterials are being investigated for the last several years because of their controllable properties. In this study, it was aimed to prepare and evaluate the atRA carrying system using Poly(lactide-co-glycolide) (PLGA). Microparticles were characterized in terms of morphology and encapsulation efficiency. Release studies were performed for the evaluation of drug release rates. Cytotoxicity tests were implemented on MCF-7 Human breast cancer cell line for the investigation of drug and polymer toxicity. The microparticles were found smooth and spherical in shape. However, as the loaded drug amount increased, the sizes of microparticles also increased and the size distribution became less uniform. The sizes of atRA-loaded microparticles ranged between 1-10 µm. The encapsulation efficiency of atRetinoic acid (all-trans-Retinoic acid) was achieved approximately %90. Approximately, 45% of atRA was released from atRA-loaded microparticles by the end of 4-5 days. Cell growth inhibition was observed after 4 days of incubation of cells with PLGA microparticles.
... RA has been widely used in cancer chemotherapy because of its property to suppress growth and to induce differentiation and apoptosis (Meyskens and Manetta, 1995; Zusi et al., 2002; Ravandi, 2009). Several cytosolic elements can influence the biological effects of RA. ...
Medulloblastoma cells exhibit varied responses to therapy by all-trans retinoic acid (RA). The underlying mechanism for such diverse effects however remains largely unclear. In this study, we attempted to elucidate the molecular basis of RA resistance through the study of RA signaling components in both RA-sensitive (Med-3) and RA-resistant (UW228-2 and UW228-3) medulloblastoma cells. The results revealed that RARα/β/γ and RXRα/β/γ were found in the three cell lines. Expression of CRABP-I and CRABP-II was seen in Med-3 cells, up-regulated when treated with RA, but was absent in UW228-2 and UW228-3 cells regardless of RA treatment. Bisulfite sequencing revealed 8 methylated CG sites at the promoter region of CRABP-II in UW228-2 and UW228-3 but not in Med-3 cells. Demethylation by 5-aza-2'-deoxycytidine recovered CRABP-II expression. Upon restoration of CRABP-II expression, both UW228-2 and UW228-3 cells responded to RA treatment by forming neuronal-like differentiation, synaptophysin expression, β-III tubulin upregulation, and apoptosis. Furthermore, CRABP-II specific siRNA reduced RA sensitivity in Med-3 cells. Tissue microarray-based immunohistochemical staining showed variable CRABP-II expression patterns among 104 medulloblastoma cases, ranging from negative (42.3%), partly positive (14.4%) to positive (43.3%). CRABP-II expression was positively correlated with synaptophysin (rs = 0.317; p = 0.001) but not with CRABP-I expression (p > 0.05). In conclusion, aberrant methylation in CRABP-II reduces the expression of CRABP-II that in turn confers RA resistance in medulloblastoma cells. Determination of CRABP-II expression or methylation status may enable a personalized RA therapy in patients with medulloblastomas and other types of cancers.
... In addition, it has also found use in cancer therapy and prevention for leukemia or AIDS-related Kaposi's sarcoma. [6][7][8] RA has two common isomers: Alltrans-retinoic acid (all-trans RA) and 9-cis-retinoic acid (9-cis RA) (shown in Figure 1), which usually bind to two classes of nuclear receptors. In the presence of RA, superfamily members of the nuclear receptor, i.e. the retinoid X receptor (RXR) and the retinoic acid receptor (RAR), activates the transcription of vitamin A and its biologically active derivatives. ...
Retinoic acid (RA) is a vitamin A derivative, which modifies the appearance of fine wrinkles and roughness of facial skin and treats acne and activates gene transcription by binding to heterodimers of the retinoic acid receptor (RAR) and the retinoic X receptor (RXR). There are series of protein bound RA complexes available in the protein databank (PDB), which provides a broad range of information about the different bioactive conformations of RA. To gain further insights into the observed bioactive RA conformations we applied quantum mechanic (QM)/molecular mechanic (MM) approaches to re-refine the available RA protein-ligand complexes. MP2 complete basis set (CBS) extrapolations single energy calculations are also carried out for both the experimental conformations and QM optimized geometries of RA in the gas as well as solution phase. The results demonstrate that the re-refined structures show better geometries for RA than seen in the originally deposited PDB structures through the use of QMs for the ligand in the X-ray refinement procedure. QM/MM re-refined conformations also reduced the computed strain energies found in the deposited crystal conformations for RA. Finally, the dependence of ligand strain on resolution is analyzed. It is shown that ligand strain is not converged in our calculations and is likely an artifact of the typical resolutions employed to study protein-ligand complexes.
... The search for isotype-selective modulators [15,[34][35][36] is pursued to first, reduce side effects associated with current retinoid therapy; second, elicit more specific biological responses, because the tissue distribution of retinoid receptors isotypes is not uniform, third, better define the physiological role of each retinoid receptor isotype in the control of certain biological phenomena such as proliferation, differentiation and apoptosis [15]. ...
BMP-4 plays a crucial role not only in the formation of the anterior pituitary during embryo development but also in the pathogenesis of pituitary tumors in adults. In tumor cells, BMP-4 promotes prolactin secretion and lactotroph cell proliferation through a Smad-estrogen receptor crosstalk but it inhibits ACTH production and cell proliferation of corticotrophs. In addition, BMP-4 increases GH secretion in rat pituitary tumor somatolactotroph GH3 cells and FSHbeta subunit gene transcription in the murine gonadotroph cell line, LbetaT2. Therefore, BMP-4 has a differential role on different types of pituitary tumors: it promotes pituitary prolactinoma while it inhibits corticotroph pathogenesis in Cushing's disease. The modulation of BMP-4 also plays an important role in the therapeutic mechanism of action of bromocriptine, somatostatin analogs and retinoic acid.
... The ability of RA to promote hepatic differentiation may be exploited as chemotherapeutic and/or chemopreventive measures for liver cancer (53)(54)(55)(56)(57)(58)(59)(60)(61)(62). Interestingly, it has been reported that exogenous RA may be teratogenic, depending on the dose and on the developmental stage at the time of exposure (63,64). ...
Hepatic progenitor cells (HPCs) can be isolated from fetal liver and extrahepatic tissues. Retinoic acid (RA) signalling plays an important role in development, although the role of RA signalling in liver-specific progenitors is poorly understood.
We sought to determine the role of RA in regulating hepatic differentiation.
RNA was isolated from liver tissues of various developmental stages. Liver marker expression was assessed by reverse transcriptase-polymerase chain reaction and immunofluorescence staining. Reversibly immortalized HPCs derived from mouse embryonic day 14.5 (E14.5) liver (aka, HP14.5) were established. Albumin promoter-driven reporter (Alb-GLuc) was used to monitor hepatic differentiation. Glycogen synthesis was assayed as a marker for terminal hepatic differentiation.
Retinoic acid receptor (RAR)-alpha, retinoid X receptor (RXR)-alpha and RXR-gamma expressed in E12.5 to postnatal day 28 liver samples. Expression of RAR-beta and RXR-beta was low perinatally, whereas RAR-gamma was undetectable in prenatal tissues and increased postnatally. Retinal dehydrogenase 1 and 2 (Raldh1 and Raldh2) were expressed in all tissues, while Raldh3 was weakly expressed in prenatal samples but was readily detected postnatally. Nuclear receptor corepressors were highly expressed in all tissues, while expression of nuclear co-activators decreased in perinatal tissues and increased after birth. HP14.5 cells expressed high levels of early liver stem cell markers. Expression of RA signalling components and coregulators was readily detected in HP14.5. RA was shown to induce Alb-GLuc activity and late hepatocyte markers. RA was further shown to induce glycogen synthesis in HP14.5 cells, an important function of mature hepatocytes.
Our results strongly suggest that RA signalling may play an important role in regulating hepatic differentiation.
A cosmeceutical should be represented a topical preparation sold as a cosmetic but with an observable pharmaceutical action. Retinoids define a class of substances comprising vitamin A (retinol) and its naturally and synthetic derivatives. Retinoic acid is oxidized to a less active metabolite, 4‐oxoretinoic acid, or converted to retinoyl glucuronide, whereas retinol is converted to retinyl glucuronide. Vitamin A is present in the epidermis as free and esterified retinol at an average concentration of 1–2 nmol/g. Natural retinoids may be efficient when used in topical application, but they are irritant and unstable following exposure to the sunlight. The genomic effects of topical retinoids are the consequence of the modulation of gene expression following their binding to nuclear retinoic acid receptor or retinoid X receptors. Retinol and retinyl esters have been incorporated into many skin products. Cosmeceutical retinoids may be useful in the treatment of skin aging, acne, and pigmentation disorders.
In an attempt to improve anticancer activity, a series of retinoids–chromene hybrids was described. The novel heterocyclic chromene–retinoids hybrid including oxygen as a heteroatom in a six‐membered cyclic ring (2H‐chromene or 2H‐1‐benzopyran) was designed and synthesized by introducing different groups such as an aromatic or styrylphenyl ring in 6‐position of 2H‐chromene. These novel compounds were synthesized by using the efficient cascades one‐pot process involving Wittig–Horner–Emmons reaction and Suzuki–Miyaura cross‐coupling pallado‐catalyzed reactions with 60% to 90% overall yields. These new compounds were tested against glioblastoma multiforme brain cancer, medulloblastoma, neuroblastoma cell lines, and breast cancer MCF‐7 cell lines. Two of them exhibited an appreciable anti‐tumor activity in the low micromolar range, which opens new perspectives for therapeutic application on humans.
Medulloblastoma (MB) cells exhibit different responses to retinoid acid (RA) for reasons that are poorly understood. RA signaling can be transduced by two approaches that are mediated by cellular retinoic acid-binding protein 2 (CRABP-II) as a tumor-suppressive pathway, and by fatty acid-binding protein 5 (FABP5) as a tumor-promoting pathway. The biological effects of RA on cancer cells are largely determined by the patterns of CRABP-II and FABP5 expression. This study aims to profile the statuses of CRABP-II and FABP5 expression in MB and to evaluate their correlation with RA sensitivities using RA-sensitive (Med-3) and RA-insensitive (UW228-2, UW228-3) MB cells. Our results show that CRABP-II is distinctly expressed and the level of FABP5 is extremely low in Med-3 cells, while the patterns of CRABP-II and FABP5 expression are reversed in UW228-2 and UW228-3 cells. RA up-regulates CRABP-II expression in Med-3 cells, whereas it up-regulates FABP5 expression in the other two cell lines. The FABP5-specific inhibitor BMS309403 increases the RA sensitivity of UW228-2 cells (p < 0.01). Tissue microarray-based immunohistochemical staining showed CRABP-II/FABP5 expression patterns in MB that were variable (CRABP-II−/FABP5−, CRABP-II−/FABP5+, CRABP-II+/FABP5− and CRABP-II+/FABP5+) and imbalanced (CRABP-II↑/FABP5↓ and CRABP-II↓/FABP5↑). MB cases exhibited patterns ofCRABP-II−/FABP5− (12.24%, 6/49), CRABP-II−/FABP5+ (30.61%, 15/49) or CRABP-II↓/FABP5↑ (12.24%, 6/49), implicating unresponsiveness or insensitivity to RA. In conclusion, the ratios of CRABP-II/FABP5 levels are closely related to the RA sensitivities of MB cells. The differential CRABP-II and FABP5 expression patterns are prospective parameters, and of potential value in personalized RA therapy for MB.
Retinoids have a dominant role in topical acne therapy and to date, only RARβ and RARγ dual agonists have reached the market. Given the tissue distribution of RAR isoforms, it was hypothesized that developing RARγ -selective agonists could yield a new generation of topical acne treatments that would increase safety margins while maintaining the robust efficacy of previous drugs. Structural knowledge derived from the X-ray structure of known γ-selective CD437, suggested the design of a novel triaryl series of agonists which was optimized and ultimately led to the discovery of Trifarotene/CD5789.
Retinoids are natural and synthetic vitamin A derivatives. As lipophilic molecules they cross plasma membranes. Their biologically active forms modulate the expression of genes involved in cellular differentiation and proliferation, and inhibit transcription factors involved in tumorigenesis. Cis/trans isomers of retinoic acid and various synthetic retinoids are used for therapeutic purposes, whereas retinaldehyde, retinol and retinyl esters, due to their limited conversion to retinoic acid, can be used as cosmeceuticals. These natural “soft” retinoids are expected to be helpful in (i) renewing epidermal cells; (ii) filtering UV radiations; (iii) preventing oxidative stress, (iv) controlling cutaneous bacterial flora, and (v) improving skin aging and photoaging. Retinol and retinyl esters are not irritant, but have only a modest clinical efficiency. On the other hand retinaldehyde, which is fairly well tolerated, seems to be the most efficient cosmeceutical retinoid: it is efficient toward oxidative stress, cutaneous bacterial flora, epidermal renewing and photoaging.
Retinoid receptors (RARs and RXRs) transduce the signals of their natural and synthetic ligands (retinoids and rexinoids) to cellular transcriptional machinery to induce gene programs that control diverse biological and physiological effects on organisms. All-trans-retinoic acid, the natural ligand for RARs, is used therapeutically for the treatment of acute promyelocytic leukemia (APL), whereas the synthetic rexinoid bexarotene (a representative member of the aromatic retinoids or arotinoids) is approved for the treatment of cutaneous T-cell lymphoma (CTCL). Other retinoids have found applications in the topical treatment of skin disorders. In continuation of previous work on the naphthalene-based arotinoid scaffold, we synthesized a new series of (3-halo)benzoic acids connected to C5- or C8-substituted naphthyl rings via (E)-ethenyl and amide and, for the C5 series, (E)-chalcone linkers. These compounds were evaluated as RAR modulators in comparison with previously described dihydronaphthalene arotinoids with the same substitution pattern. Transactivation studies in this series revealed an absence of synergy between small halogen atoms (F, Cl) at C3 and the groups at C5 or C8, as had been observed on some of the dihydronaphthalene analogues. Instead, non-halogenated 4-(2-naphthamido)benzoic acid derivatives transactivated toward the RARβ subtype in preference to the paralogues. The derivatives with bulkier substituents at C8 were characterized as dual RARβ/RARα antagonists, and (E)-4-[(8-(phenylethynyl)naphthalene-2-yl)ethenyl]benzoic acid (11 c), with an ethenyl connector, was shown to be a potent antagonist of RARα.
© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
Retinoids possess anti‑proliferative properties, which suggests that they possess chemopreventive and therapeutic potential against cancer. In the current study, genes modulated by rexinoids (retinoid X receptor (RXR)‑pan agonists, LGD1069 and LG100268; and the RXRα agonist, Ro25‑7386) were identified using an Affymetrix microarray in normal and malignant breast cells. It was observed that LGD1069, LG100268 and Ro25‑7386 suppressed the growth of breast cells. Secondly, several rexinoid‑regulated genes were identified, which are involved in cell death, cell growth/maintenance, signal transduction and response to stimulus. These genes may be associated with the growth‑suppressive activity of rexinoids. Therefore, the identified genes may serve as biomarkers and novel molecular targets for the prevention and treatment of breast cancer.
Resumo Os retinóides são um grupo de substâncias reguladoras do crescimento que incluem o ácido trans-retinóico (tRA), um derivado metabólico natural da vitamina A (retinol), o seu isômero 9-cis (cAR) e uma série de derivados naturais ou sintéticos. O tAR exerce efeitos importantes durante o desenvolvimento embrionário de vertebrados e na vida adulta, e desempenham papéis importantes na modulação do crescimento de células normais e tumorais, através da regulação da diferenciação ou apoptose. As atividades biológicas dos tAR e o cAR são mediadas através da ligação e ativação receptores nucleares específicos (RARá,â,ã) e receptores X de retinóides (RXRá,â,ã). Esses receptores são produtos de genes distintos, pertencem à superfamília de receptores nucleares de hormônios esteróides e atuam como reguladores de transcrição dependente do ligante. Essas moléculas são objeto de vários estudos científicos e clínicos que estão baseados tanto em suas propriedades químicas quanto em seu potencial em induzir apoptose e (ou) diferenciação celular, constituindo, assim, uma alternativa em potencial para o tratamento de diversas formas de câncer. Os avanços no conhecimento sobre a ativação dos RARs e RXRs deu origem uma nova geração de retinóides sintéticos, os quais têm demonstrado igualmente uma ação efetiva contra células cancerígenas de diferentes origens, seja inibindo o crescimento, seja induzindo apoptoses ou diferenciação celular.
Retinoids are a class of synthetic and natural compounds structurally related to retinoic acid. In a search for discovery of a new class of heterocycle-bridged and conformationally constrained retinoids, here we report the synthesis of 4-(7,8,9,10-tetrahydro-7,7,10,10-tetramethyl-4H-benzo[6,7]chromeno[4,3-d]thiazole-2-yl)benzoic acid (10) starting from 5,6,7,8-tetrahydro-5,5,8,8-tetramethylnaphthalen-2-ol (13). Several approaches was attempted to obtain target compound 10. Structure elucidation of synthesized compounds has been made on the basis of elemental analysis and spectral data (1H NMR, IR and MS).
All-trans-retinoic acid (RA) is a promising agent for breast cancer treatment, but it induces several adverse effects and the few clinical trials performed up to now in breast cancer patients have provided disappointing results. The combination of RA and antiestrogenic compounds, such as tamoxifen, synergistically decreases the proliferation of breast cancer cells and an interplay between retinoid and estrogen signaling has begun to be unraveled, turning these combinations into an appealing strategy for breast cancer treatment. This review focus on the current knowledge regarding the interplay between retinoid and estrogen signaling in breast cancer and the combinations of RA with antiestrogens, aiming their future utilization in cancer therapy.
Vitamin A is an important constituent of the epidermis, where it plays a crucial role in epidermal turnover. A deficiency of epidermal vitamin A may be the consequence of nutritional vitamin A deficiency, exposure to sunlight or any UV source, oxidative stress or chronological ageing. As a consequence, any treatment aiming at increasing epidermal vitamin A would exert a protective effect against these deleterious conditions. Retinoids may counteract some deleterious actions of UV radiation by physical and biological mechanisms. Topical natural retinoic acid precursors such as retinaldehyde or retinol are less irritant than acidic retinoids and may prevent epidermal vitamin A deficiency due to nutritional deficiency, exposure to sunlight or any condition leading to free radical production. Retinoids may be combined with other compounds with complementary actions against ageing, nutritional deficiency and cancer, such as antioxidants, to potentiate their beneficial effects in the skin. © 2014 S. Karger AG, Basel.
Aims:
The clinical utilization of the combinations of all-trans-retinoic acid (RA) with antiestrogens, which present synergism of action in breast cancer, has been limited by RA adverse effects, including hepatotoxicity, which may be related with mitochondrial damage. This work evaluated the effects of RA alone and in combination with the antiestrogen endoxifen (EDX) on liver mitochondria.
Main methods:
Mitochondrial permeability transition (MPT) was assessed by using Calcium Green-5N fluorescence and a tetraphenylphosphonium selective electrode. Oxidative stress was evaluated by oxygen consumption and thiobarbituric acid method. Mitochondrial bioenergetic was monitored by measuring oxygen consumption and mitochondrial membrane potential (ΔΨ). Osmotic volume changes of mitochondria were followed at 540nm.
Key findings:
EDX prevents the MPT induced by RA, allowing mitochondria pre-incubated with RA to accumulate Ca(2+) and inhibiting the depolarization of ΔΨ. RA above 10 nmol/mg protein depresses the phosphorylation capacity of mitochondria, as shown by the increase in the time required for ADP phosphorylation as well as by the decrease in state 3 respiration. At 20 nmol/mg protein, RA decreases the ΔΨ and increases the state 4 respiration, suggesting that high concentrations of RA permeabilize the membrane to protons, possibly due to a proton leak through the Fo fraction of complex V. Moreover, the effects of RA on mitochondrial bioenergetics are not changed by EDX.
Significance:
RA-induced hepatotoxicity may be related with induction of MPT and alterations in bioenergetic parameters; the combination with EDX, which reduces mitochondrial dysfunction and synergistically potentiates the anticancer activity, may provide a safer therapeutic strategy.
Vitamin A (retinol) and its related metabolites like retinoic acid (RA) have great potential in their roles as prostate cancer chemopreventive and chemotherapeutic agents by exerting regulation on cell growth and differentiation. Several studies have shown that there is a reduction in retinoid levels and retinoid receptors (e.g. RARBeta2) in prostate cancer. RA is being used to treat patients with prostate cancer and has been shown to inhibit tumor growth and reverse the events of carcinogenesis in animal models of prostate cancer. There is a disparity in prostate cancer among the African-American population and we hypothesize that more severe disruptions of retinoid signaling occur, contributing to this disparity. The purpose of this study is to examine the underlying causes for the clinical behavior of prostate cancer in African-Americans as compared to Caucasian patients. Immunohistochemical analysis has shown the expression of LRAT, an enzyme responsible for retinol esterification and storage as retinyl esters, to be reduced in tumor tissue specimens from prostate cancer patients as compared to adjacent nonmalignant tissue. Understanding the role of retinoid signaling in prostate carcinogenesis will lead to improved chemoprevention strategies and to the development of novel therapies for this disease.
Retinoids, derivatives of vitamin A (Retinol), are required for the appropriate differentiation of normal human prostate epithelial cells. Human prostate cancer cell contain much lower levels of vitamin A and its metabolites than normal cells. We hypothesize that aberrant metabolism of vitamin A and dysregulation of gene expression in prostate tumor cells are related to the abnormal growth properties of the tumor cells. A rationale for using retinoids in prostate cancer chemotherapy is further supported by the effectiveness of ATRA (all-trans Retinoic Acid), a vitamin A metabolite, in the treatment of acute promyelocytic leukemia (APL).
Background: The superfamily of nuclear receptors (NRs) comprises ligand-gated and ligand-independent transcription factors, modulating genetic programs mostly by binding to specific DNA elements. NRs not only control many aspects of cellular homeostasis but also play crucial roles in numerous human diseases. As the activity of NRs can be modulated by small molecules, these are successfully used in the clinics for the treatment of metabolic and cardiovascular diseases as well as cancer. Objective/methods: This review provides an overview on the molecular classification and disease relevance of NRs, as well as on recent advances in pharmacologic (pre)clinical targeting approaches and patent applications. Patent, drug approval and bibliographic searches were carried out using various databases and websites, including Freepatentsonline, CenterWatch Clinical Trials Listing Service and the PubMed database. Results/conclusion: To fully exploit and market the tremendous potential of NRs for the treatment of human diseases, a systematic understanding of the structure–function relationship of NRs is urgently needed. Novel insights into the network of NR biology together with the recent advancements in medicinal chemistry may lead to novel therapeutics with improved efficacy, selectivity and safety.
Activation of the retinoid X receptor (RXR), which is involved in cell proliferation, differentiation, and apoptosis, is a strategy for cancer chemotherapy and chemoprevention, and 3-amino-6-(3'-aminopropyl)-5H-indeno[1,2-c]isoquinoline-5,11-(6H)dione (AM6-36) (3) is among the few RXR ligands known. The presently reported studies of 3 include its binding to human plasma proteins, metabolic stability using human liver microsomes, metabolism by human liver microsomes and hepatocytes, and in vivo disposition in rat serum, liver, and mammary tissue. Compound 3 was 75% bound to human plasma proteins, and its metabolic stability was much greater than propranolol. One phase I metabolite was formed by human liver microsomes, seven phase I and II metabolites were formed by human hepatocytes, and five metabolites were detected in rat serum and liver after oral administration. The putative metabolites predicted using LC-MS-MS were synthesized to confirm their structures and to provide sufficient material for investigation of induction of RXRE transcriptional activity and inhibition of NFκB.
The effect of all-trans retinoic acid (ATRA) on cutaneous squamous cell carcinomas (c-SCC) has been poorly described. Because the imbalance of CRABP-II-mediated anticancer signalling and FABP5-mediated growth-promoting signalling was supposed to be related with ATRA sensitivities of cancer cells, COLO16 human c-SCC cell line was selected to check underlying mechanism leading to ATRA resistance by multiple experimental approaches. The results revealed that COLO 16 cells were resistant to 15 μm ATRA treatment. FABP5 as well as the elements related with CRABP-II signalling (CYP26A1, CYP26B1, CRABP-I, RARα/β/γ and RXRα/β/γ) and with FABP5 signalling (PPARβ/δ) were expressed, but CRABP-II was undetectable in COLO 16 cells. 5-Aza treatment enhanced CRABP-II expression but further bisulfite sequencing PCR-DNA sequencing revealed no methylation in CRABP-II promoter region. Transfection of CRABP-II-expressing plasmids or FABP5 siRNA or both successfully manipulated the level(s) of target gene expression but failed to overcome ATRA resistance in the transfectants. In conclusion, CRABP-II and FABP5 expression were imbalanced in ATRA-resistant COLO 16 cells. 5-Aza-enhanced CRABP-II expression and unmethylation in CRABP-II promoter region suggest the methylation of certain CRABP-II regulatory gene(s) in COLO 16 cells. As neither restoration of CRABP-II expression nor the increased CRABP-II versus FABP5 ratio can overcome ATRA resistance of COLO 16 cells, additional ATRA-resistant mechanism(s) may present in human c-SCCs and COLO 16 cells would be of value in addressing this issue.
Following the discovery that defective retinoid signaling directly contributes to tumorigenesis, and, that retinoids have an anti-cancer effect in vitro and in vivo, retinoids have become part of the routine care in children with neuroblastoma at the stage of minimal residual disease. However, many patients still relapse following retinoid therapy, demonstrating the need for more effective retinoids and better assays to predict retinoid sensitivity in cancer cells. Recent evidence suggests that the copper metabolism gene, ATP7A, is retinoid-regulated and an important component of the retinoic acid receptor β (RARβ) anticancer effect in neuroblastoma cells. To highlight and further develop the concept of using ATP7A as a target in retinoid therapy, and combination therapy with copper chelators in neuroblastoma, the current literature and abstracts related to the clinical application of retinoids, the function of ATP7A and the clinical application of copper chelators are summarized. We propose that strategies targeting the copper export protein, ATP7A, in combination therapy with retinoids and copper depletion therapy, may have great therapeutic potential in the clinical treatment of neuroblastoma and other malignancies.
In this review a discussion of the usual procedures used to synthesize retinoids is followed by an overview of the structure-activity relationships of these molecules. The discussion is then focused on the role and impact of retinoids on metabolic disorders with a particular emphasis on obesity, diabetes, and the metabolic syndrome. In these areas, both natural and synthetic retinoids that are being studied are reviewed and areas where likely future research will occur are suggested. This article is part of a Special Issue entitled Retinoid and Lipid Metabolism.
Here we investigated a pharmacological approach to inhibit spermatogenesis in the mouse model by manipulating retinoid signaling using low doses of the pan-retinoic acid receptor (RAR) antagonist BMS-189453. Spermatogenesis was disrupted, with a failure of spermatid alignment and sperm release and loss of germ cells into lumen, abnormalities that resembled those in vitamin A-deficient and RARα-knockout testes. Importantly, the induced sterility was reversible. Enhanced efficacy and a lengthened infertility period with full recovery of spermatogenesis were observed using systematically modified dosing regimens. Hematology, serum chemistry, and hormonal and pathological evaluations revealed no detectable abnormalities or adverse side effects except the distinct testicular pathology. Our results suggest that testes are exquisitely sensitive to disruption of retinoid signaling and that RAR antagonists may represent new lead molecules in developing nonsteroidal male contraceptives.
9cUAB30 is a synthetic analog of 9-cis-retinoic acid with chemopreventive activity in cell lines and in animal models. The purpose of this first-in-human evaluation of 9cUAB30 was to evaluate the single-dose pharmacokinetic profile and toxicity of the compound in healthy volunteers at 3 dose levels. This study enrolled 14 patients to receive a single dose of 5, 10, or 20 mg of 9cUAB30. Plasma and urine samples were collected to assess 9cUAB30 concentrations by a validated LC/MS MS method. 9cUAB30 was well tolerated, with 1 patient experiencing grade 2 toxicity and no grade 3 or 4 toxicities reported. T(max) occurred approximately 3 hours after dose administration with the plasma half-life ranging from 2.79 to 7.21 hours. AUC increased linearly across the examined dose range of 5 to 20 mg; C(max) was proportional to the log of the dose. The plasma clearance ranged from 25 to 39 L/h compared to the renal clearance which ranged from 0.018 to 0.103 L/h. 9cUAB30 has a favorable toxicity and pharmacokinetic profile, with oral availability and primarily hepatic metabolism. Further dose ranging studies with once a day dosing are underway.
The synthetic retinoic acid analog, 9-cis-UAB30 [(2E,4E,6Z,8E)-8-(3',4'-dihydro-1'(2'H)-naphthalen-1'-ylidene)-3,7-dimethyl-2,4,6-octatrienoic acid], is a specific ligand for the retinoid X receptor. Murine oncogenicity and pharmacokinetics studies were performed as part of the preclinical development of 9-cis-UAB30 for breast cancer chemoprevention. In the oncogenicity study, TSG-p53((+/-)) (p53 knockout) mice (25 per sex per group) received daily gavage exposure to 9-cis-UAB30 doses of 0 (control), 30, 100, or 300 mg/kg/d for 6 months. Positive controls received p-cresidine (400 mg/kg/d) for 6 months. 9-cis-UAB30 had no biologically significant effects on survival, body weight, body weight gain, clinical signs, hematology, or clinical chemistry but induced dose-related hepatomegaly in both sexes and decreased thymus weights in high-dose females. Gross and microscopic pathology provided no evidence of 9-cis-UAB30 toxicity or oncogenicity; by contrast, p-cresidine induced urinary bladder neoplasms in more than 60% of male and female mice. It was concluded that 9-cis-UAB30 is not oncogenic in p53((+/-)) mice. In the pharmacokinetics study, C57BL/6 mice received daily gavage exposure to 9-cis-UAB30 (100 or 300 mg/kg/d) for 1 or 7 days. Pharmacokinetic parameters were similar after 1 and 7 days of dosing. Dose-related peak plasma levels of 9-cis-UAB30 were seen between 0.25 and 3 hours; volume of distribution was comparable at both dose levels. Increases in area under the curve were less than proportional to dose and were associated with an increased rate of apparent clearance and decreased elimination half-life. These results suggest decreased absorption and/or possible induction of clearance mechanisms. Enzyme induction may underlie the hepatomegaly seen in mice treated with 9-cis-UAB30 for 6 months in the oncogenicity study.
The synthesis and biological evaluation of the entire series of C3-halogenated derivatives and bulkier substituents at the C8″ position of the parent stilbene-based RARβ-selective agonist BMS641 4c was undertaken. The synthesis uses an E-selective Horner-Wadsworth-Emmons (HWE) condensation of C8-substituted C5-dimethyl dihydronaphthaldehyde and the benzylic phosphonates derived from the C3-halogenated benzoates to construct the stilbene skeleton. Transactivation studies revealed the synergistic effect of small halogen atoms at C3 (F, Cl) and the moderately bulky phenyl group at C8″ (in 4b and 4c) to achieve RARβ selectivity. Our results, supported by computational studies, provide a structural rationale for the mixed agonist-antagonist activities of these arotinoids, which are potent agonists of the RARβ subtype and antagonists of the RARα paralogue. Moreover, transitions from partial agonists to inverse agonists and antagonists can be accomplished with the incorporation of the same halogen atoms into the structures of known modulators BMS701 (5a) and BMS493 (6a), which have bulkier substituents than phenyl (p-tolyl and phenylethynyl, respectively) at C8″. Conversely, incorporation of halogen atoms in 6a converted the ligand from an RARβ inverse agonist (6b) to an antagonist (6c) or an agonist (6d). Amazingly, 6a-c commonly acted as inverse agonists for RARα, while 6d and 6e acted as regular RARα antagonists, not affecting co-repressor interaction. In the case of the mixed agonist/antagonist 5a, C3-halogenation yields inverse RARα and RARβ agonists (5b-d) with the exception of iodinated 5e, which is a regular antagonist for both these receptors. Because RARβ gene expression is frequently deleted or epigenetically silenced in several tumor cells, the novel repertoire of receptor and function-selective RAR agonists, mixed agonist/antagonists, regular antagonists, and inverse agonists will be useful in the elucidation of the mechanism of tumor suppression by retinoids.
A series of retinoids designed to interfere with the repositioning of H12 have been synthesized to identify novel RARgamma antagonists based on the structure of known RARgamma agonists. The transcriptional activities of the novel ligands were revealed by cell-based reporting assays, using engineered cells containg RAR subtype-selective fusions of the RAR ligand-binding domains with the yeast GAL4 activator DNA-binding domain and the cognate luciferase reporter gene. Whereas none of the ligands exhibited features of a selective RARgamma antagonist, some of them are endowed with interesting activities. In particular 24a acts as a pan-RAR agonist that induces at high concentration a higher transactivation potential on RARalpha than TTNPB and synergizes at low concentration with TTNPB-bound RARalpha but not RARbeta or RARgamma. Similarly, 24c synergizes with TTNPB-bound RARgamma and exhibits RARalpha,beta antagonist activity. Compounds 24b and 25b are strong RARalpha,beta-selective antagonists without agonist or antagonist activities for RARgamma. Compounds 24b and 24c display weak RXR antagonist activity. In addition several pan-antagonists and partial agonist/antagonists have been defined.
Peroxisome proliferator-activated receptor gamma (PPAR-gamma) and retinoic acid receptors (RAR/RXR) belong to the nuclear steroid receptor family. In vitro studies have suggested that PPAR-gamma ligands are highly effective in preventing mammary tumours and these effects are enhanced by some retinoids. However, in vivo anti-initiator and anti-promoter efficacies of this combination are not clear.
The present study aimed to investigate the chemopreventive efficacies of the PPAR-gamma ligand rosiglitazone (200 microg/kg/day), synthetic retinoid fenretinide (0.3 mg/kg/day) and their combination on a DMBA-induced rat mammary carcinogenesis model.
In the rosiglitazone group, no malignant tumour developed, apart from the lowest proliferative mammary lesions. In the fenretinide group, 30% developed a malignant tumour but there were no benign tumours. Cancer incidences were 61.5% and 10% in the control and combination groups respectively.
Our results showed that the PPAR-gamma ligand rosiglitazone and synthetic retinoid fenretinide have potent chemopreventive properties against in vivo mammary carcinogenesis; however, the efficacies were not enhanced by their combination.
On the basis of the observations that chalcone 7 (MX781) and some related adamantyl arotinoids (AdArs) inhibit IkappaB alpha kinase beta (IKKbeta) activity, inhibit cell growth, and induce apoptosis in cancer cells, a new series of AdArs structurally related to 7 have been designed and synthesized. Modifications were intended to reduce or eliminate RAR activity, and we evaluated the effect of the novel analogues of 7 on IKKbeta activity and proliferation of a variety of cancer cell lines (leukemia, Jurkat; prostate, PC-3; breast carcinomas, T47D, MDA-MB-468). Consistent with the design principles, the biological activities of these AdArs do not appear to be RAR-mediated, since most analogues are unable to activate RAR-mediated transactivation and exhibit significantly diminished antagonist activity. All compounds are capable of inducing apoptosis in Jurkat cells, as demonstrated by elevated DEVDase activity and externalization of phosphatidylserine. Several of the analogues elicit stronger growth inhibitory activity against prostate (PC-3) and breast (MDA-MB-468) carcinoma cells, which contain elevated basal IKK activity; this antiproliferative activity correlates with increased inhibition of recombinant IKKbeta in vitro, suggesting that the anticancer activities of these AdArs might be related to the inhibition of IKK/NFkappaB signaling.
A large number of physiological processes in the adult liver are regulated by nuclear receptors that require heterodimerization
with retinoid X receptors (RXRs). In this study, we have usedcre-mediated recombination to disrupt the mouse RXRα gene specifically in hepatocytes. Although such mice are viable, molecular
and biochemical parameters indicate that every one of the examined metabolic pathways in the liver (mediated by RXR heterodimerization
with PPARα, CARβ, PXR, LXR, and FXR) is compromised in the absence of RXRα. These data demonstrate the presence of a complex
circuitry in which RXRα is integrated into a number of diverse physiological pathways as a common regulatory component of
cholesterol, fatty acid, bile acid, steroid, and xenobiotic metabolism and homeostasis.
The 2.0-A crystal structure of the ligand-binding domain (LBD) of the human retinoic acid receptor (RAR)-gamma bound to all-trans retinoic acid reveals the ligand-binding interactions and suggests an electrostatic guidance mechanism. The overall fold is similar to that of the human RXR-alpha apo-LBD, except for the carboxy-terminal part which folds back towards the LBD core, contributing to the hydrophobic ligand pocket and 'sealing' its entry site. We propose a 'mouse trap' mechanism whereby a ligand-induced conformational transition repositions the amphipathic alpha-helix of the AF-2 activating domain and forms a transcriptionally active receptor.
Using retinoic acid receptor (RAR) reporter cells specific for either RAR alpha, beta or gamma, we have identified synthetic retinoids which specifically induce transactivation by RAR beta, while antagonizing RA-induced transactivation by RAR alpha and RAR gamma. Like RA, these synthetic retinoids allow all three RAR types to repress AP1 (c-Jun/c-Fos) activity, demonstrating that the transactivation and transrepression functions of RARs can be dissociated by properly designed ligands. Using AP1 reporter cells, we also show that glucocorticoids or vitamin D3, together with either RA or these 'dissociating' synthetic retinoids, can synergistically repress phorbol ester-induced AP1 activity. RA, but not these 'dissociating' retinoids, induced transcription of an interleukin-6 promoter-based reporter gene transiently transfected into HeLa cells together with RARs. Using Ki-ras-transformed 3T3 cells as a model system, we show that both RA and the 'dissociating' retinoids inhibit anchorage-independent cell proliferation, suggesting that retinoid-induced growth inhibition may be related to AP1 transrepression.
The crystal structure of the human retinoid-X receptor RXR-alpha ligand-binding domain reveals a previously undiscovered fold of an antiparallel alpha-helical sandwich, packed as dimeric units. Two helices and one loop form the homodimerization surface, and hydrophobic heptad repeats participate in stabilizing the fold. The existence of a ligand-binding pocket is proposed that would allow 9-cis retinoic acid to interact with different functional modules, including the AF-2 activating domain. Several lines of evidence indicate that the overall structure is a prototype fold of ligand-binding domains of nuclear receptors.
Utilizing a strategy involving domain exchange between retinoic acid receptors alpha and beta (RAR alpha and RAR beta) and monitoring the transcriptional activity of the resulting chimeric receptors with receptor-selective retinoids, we identified a 70-aa region within the N-terminal portion of the RAR alpha and -beta domain E which is important for an RAR alpha- or RAR beta-specific response. Two amino acid residues within this region, serine-232 (S232) and threonine-239 (T239) in RAR alpha and the corresponding alanine-225 (A225) and isoleucine-232 (I232) in RAR beta, were found to be essential for this effect. In addition, binding studies using the chimeric receptors expressed in Escherichia coli showed that the N-terminal portion of domain E was also important for the characteristic binding profile of t-RA and various retinoids with RAR alpha or RAR beta. Structural predictions of the primary amino acid sequence in this region indicate the presence of an amphipathic helix-turn-helix structure with five hydrophobic amino acids that resemble a leucine zipper motif. The amino acid residues identified by domain swapping, S232 and T239 in RAR alpha and A225 and I232 in RAR beta, were found within the hydrophobic face of an alpha-helix in close proximity to this zipper motif, suggesting that the ligand may interact with the receptor in the region adjacent to a surface involved in protein-protein interactions. This finding may link ligand binding to other processes important for transcriptional activation.
The binding of endogenous retinoids and stereoisomers of retinoic acid (RA) to the retinoid nuclear receptors, RA receptor (RARs) and retinoid X receptors (RXRs), was characterized using nucleosol preparations from transiently transfected COS-1 cells. Among several stereoisomers of RA tested, including 7-cis-, 9-cis-, 11-cis-, 13-cis-, and all-trans-RA, only 9-cis-RA effectively competes with 9-cis-[3H]RA binding to the RXRs. Additionally, the endogenous retinoid trans-didehydro-RA (t-ddRA) does not interact with RXRs, whereas the 9-cis form of ddRA competes effectively. RXRs (alpha, beta, and gamma) bind 9-cis-RA with dissociation constants (Kd) of 15.7, 18.3, and 14.1 nM, respectively. In contrast to the selectivity of RXRs for 9-cis-RA, RARs bind both t-RA and 9-cis-RA with high affinity, exhibiting Kd values in the 0.2-0.7 nM range for both ligands. Unlike RARs, the cellular RA binding proteins CRABPI or CRABPII bind t-RA but do not bind 9-cis-RA. Consistent with the binding data, 9-cis-RA and 9-cis-ddRA transcriptionally activate both GAL4-RXR and GAL4-RAR chimeric receptors with EC50 values of 3-20 nM for 9-cis-RA and 9-cis-ddRA, whereas t-RA and t-ddRA efficiently activate only GAL4-RAR chimeric receptors. Thus, 9-cis forms of endogenous retinoids can contribute to the pleiotropic effects of retinoids by interacting with both the RARs and RXRs.
The expression of the retinoic acid receptor beta (RAR beta) mRNA is absent or down-regulated in most human breast cancer cell lines. To investigate the role RAR beta may have in regulating the proliferation of breast cancer cells, we used retroviral vector-mediated gene transduction to introduce the human RAR beta gene into two RAR beta-negative breast tumor cell lines, MCF-7 and MDA-MB-231. RAR beta-transduced clones underwent growth inhibition associated with G1 arrest when treated with 1 microM all-trans-retinoic acid (RA). Moreover, the MCF7-RAR beta transduced clones also underwent apoptosis after 4 to 6 days of RA treatment. The RA-induced growth arrest in MDA231-RAR beta transduced cells is associated with c-myc mRNA down-regulation, whereas the RA-mediated apoptosis of MCF7-RAR beta transduced cells is not associated with c-myc down-regulation. These observations suggest a critical role for RAR beta in mediating growth arrest and apoptosis in breast cancer cells.
Retinoids play an important role in development, differentiation, and homeostasis. The discovery of retinoid receptors belonging to the superfamily of nuclear ligand-activated transcriptional regulators has revolutionized our molecular understanding as to how these structurally simple molecules exert their pleiotropic effects. Diversity in the control of gene expression by retinoid signals is generated through complexity at different levels of the signaling pathway. A major source of diversity originates from the existence of two families of retinoid 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. The possibility of cross-modulation (cross-talk) with cell-surface receptors signaling pathways, as well as the finding that RARs and RXRs interact with multiple putative coactivators and/or corepressors, generates additional levels of complexity for the array of combinatorial effects that underlie the pleiotropic effects of retinoids. This review focuses on recent developments, particularly in the area of structure-function relationships.
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 agonists) and/or antiestrogens may have utility in the treatment of breast cancer.
The transcriptional response mediated by retinoic acid involves a complex series of events beginning with ligand recognition by a nuclear receptor. To dissect the amino acid contacts important for receptor-specific ligand recognition, a series of retinoic acid receptor (RAR) mutants were constructed. Transcriptional studies revealed that serine 232 (Ser²³²) in RARα and methionine 272 (Met²⁷²) in RARγ are critical residues for the recognition of their respective receptor-selective analogs.
The identification of these key amino acids in the ligand binding pocket is confirmed by the reported crystal structure of RARγ. Interestingly, the serine at position 232 in RARα gives an explanation for the observed differences in the affinity of the naturally occurring ligand, all-trans-retinoic acid (t-RA), in this receptor compared with that for the other receptors, since hydrogen bonding would not be permitted between the hydroxyl of serine and the hydrophobic linker of t-RA. Using this model, a molecular mechanism for the transcriptional antagonism of a synthetic analog is suggested that involves an alteration in the structure of the receptor protein in the region around the AF2 domain in helix 12.
All-trans retinoic acid (ATRA), a potent differentiating drug for acute promyelocytic leukemia (APL), induces a high incidence of complete remission (CR) in patients with APL and is now established as a first-line therapy. However, ATRA resistance has become a clinical problem. Patients who relapsed after ATRA-induced CR have had difficulty in obtaining a second CR with ATRA therapy. Although several mechanisms have been postulated, treatment strategies to overcome resistance have not been established. We used a new synthetic retinoid, Am-80, as reinduction therapy for APL relapse after from ATRA-induced CR. Am-80 was several times more potent than ATRA in inducing differentiation in vitro. At a 6 mg/m2 dose, there were 24 evaluable patients; 14 (58%) achieved CR between days 20 and 58 (median, 37 days). Clinical response correlated with the in vitro response to Am-80. Adverse effects included retinoic acid syndrome (n = 1), hyperleukocytosis (n = 1), xerosis (n = 9), cheilitis (n = 8), hypertriglyceridemia (n = 16), and hypercholesterolemia (n = 15). Am-80 is active in APL after relapse from ATRA-induced CR. Further clinical trials are needed to establish strategies to overcome ATRA resistance.
Four candidate retinoid antagonists (LE135, LE511, LE540, and LE550) were designed on the basis of the ligand superfamily
concept and synthesized. Analysis of these related retinoids by transient transfection assay demonstrated that LE135, LE540,
and LE550 are effective retinoic acid receptor (RAR) antagonists, whereas LE511 selectively induced RARβ transcriptional activity.
Both LE135 and LE540 inhibited retinoic acid (RA)-induced transcriptional activation of RARβ, but not RARα, RARγ or retinoid
X receptor α (RXRα), on a variety of RA response elements. The retinoid antagonists also inhibited all-trans-RA-induced transcriptional activation of RARβ/RXRα heterodimers, although they did not show any effect on transactivation
activity of RXR/RXR homodimers. In ZR-75-1 human breast cancer cells, cotreatment of LE135 and LE540 with all-trans-RA inhibited all-trans-RA-induced apoptosis of the cells, further demonstrating that RARβ plays a role in RA-induced apoptosis of breast cancer cells.
We also evaluated the effect of these retinoids on AP-1 activity. Our data showed that LE135 and LE540 strongly repressed
12-O-tetradecanoylphorbol-13-acetate-induced AP-1 activity in the presence of RARβ and RXRα. Interestingly, LE550 induced AP-1
activity when RARβ and RXRα were expressed in HeLa cells but not in breast cancer cells. These results demonstrate that LE135
and LE540 were a novel class of RARβ-selective antagonists and anti-AP-1 retinoids and should be useful tools for studying
the role of retinoids and their receptors.
F9 teratocarcinoma cell lines, carrying one or two disrupted alleles of the RARβ2 gene, were generated by homologous recombination to study the role of RARβ2 in mediating the effects of retinoids on cell growth and differentiation. Retinoic acid (RA) does not induce growth arrest
of the RARβ2−/− cells, whereas the F9 WT and RARβ2+/− heterozygote lines undergo RA-induced growth arrest. The RARβ2+/− lines also exhibit a faster cell cycle transit time in the absence of RA. The RARβ2−/− stem cells exhibit an altered morphology when compared with the F9 WT parent line, and after RA treatment, the RARβ2−/− cells do not exhibit a fully differentiated cell morphology. As compared with F9 WT cells, the RARβ−/− cells exhibited
a markedly lower induction of several early RA-responsive genes and no induction of laminin B1, a late response gene. The
induction of RA metabolism in the F9 RARβ2−/− cells following differentiation was not impaired. The research presented here, and prior research suggest that RARβ is
required for RA-induced growth arrest in a variety of cell types and that RARβ also functions in mediating late responses
to RA. These findings are significant in view of the reduced expression of RARβ transcripts in a number of different types
of human carcinomas.
N-(4-Hydroxyphenyl)retinamide (4HPR) is currently used in cancer prevention and therapy trials. It is thought that its effects result from induction of apoptosis. 4HPR-induced apoptosis in human cervical carcinoma C33A cells involves enhanced generation of reactive oxygen species (ROS). In this study we explored the mechanism by which 4HPR increases ROS and induces apoptosis in these cells. 4HPR induced cytochrome c release from mitochondria to cytoplasm, activated caspase-3, and caused a membrane permeability transition (MPT). All these 4HPR's effects, as well as the induction of apoptosis, were inhibited by antioxidants, which decrease ROS. Thenoyltrifluoroacetone, a mitochondrial respiratory chain (MRC) complex II inhibitor, and carbonylcyanide m-chlorophenyl hydrazone, which uncouples electron transfer and ATP synthesis and inhibits ROS generation by MRC, inhibited 4HPR-induced ROS generation very effectively. Rotenone, an MRC complex I inhibitor was less effective and azide, an MRC complex IV inhibitor, exhibited a marginal effect. In contrast, antimycin A, an MRC complex III inhibitor, enhanced 4HPR-induced ROS generation. These findings suggest that 4HPR enhances ROS generation by affecting a target between complex II and complex III, presumably coenzyme Q. This effect is followed by release of cytochrome c, increased caspase-3 activity, induction of MPT and eventual DNA fragmentation and cell death.
The crystal structure of the human retinoid-X receptor RXR-alpha ligand-binding domain reveals a previously undiscovered fold of an antiparallel alpha-helical sandwich, packed as dimeric units. Two helices and one loop form the homodimerization surface, and hydrophobic heptad repeats participate in stabilizing the fold. The existence of a ligand-binding pocket is proposed that would allow 9-cis retinoic acid to interact with different functional modules, including the AF-2 activating domain. Several lines of evidence indicate that the overall structure is a prototype fold of ligand-binding domains of nuclear receptors.
Retinoids in clinical use today are known to induce hypertriglyceridemia as one of their major side effects. The purpose of the present study was to determine, in an appropriate animal model, if retinoid-induced hypertriglyceridemia is mediated by retinoic acid receptors (RARs) and/or by retinoid X receptors (RXRs). Oral gavage of male Fischer rats with 13-cis-retinoic acid for 6 days caused a rapid and sustained increase in serum triglycerides that was reversible within 4 days posttreatment. In subsequent experiments, rats were treated by gavage once daily for 3 days with various retinoids, and serum triglyceride levels were determined 24 hr after the last treatment without fasting. All-trans- and 13-cis-retinoic acid, which can be converted to both RAR and RXR agonists, and 9-cis-retinoic acid, an RAR/RXR pan-agonist, caused dose-dependent increases in serum triglycerides at doses that did not cause weight loss or mucocutaneous toxicity. Ro 13-6298 and AGN 190121, two RAR-specific agonists, caused dose-dependent increases in serum triglycerides, although Ro 13-6298 only induced hypertriglyceridemia at weight-suppressive doses. Two RXR-selective agonists, LG100268 and AGN 191701, failed to induce hypertriglyceridemia or weight loss up to the highest doses tested. A structural isomer of AGN 190121 that does not activate RARs or RXRs, AGN 190727, did not induce hypertriglyceridemia. Hypertriglyceridemia induced by AGN 190121 was significantly inhibited by cotreatment with an RAR-selective antagonist, AGN 193109. Taken together, these data provide strong evidence that retinoid-induced hypertriglyceridemia is mediated, at least in part, by RARs. These data also suggest that RXR-specific agonists may have reduced potential to induce hypertriglyceridemia relative to RAR-active retinoids..
Transcription of the retinoic receptor β (RARβ) gene is activated in a ligand-dependent manner by the retinoic acid receptor α. Reduced RARβ gene expression and loss of ligand inducibility are frequently observed in human carcinoma cells indicating that such alterations might contribute to carcinogenesis. In this study we have analyzed the influence of RARβ on cervical cancer cell growth. Transfection of HeLa cells with RARβ expression plasmids resulted in reduced clonal cell growth in the presence of retinoic acid (RA). RA-induced growth inhibition in HeLa × fibroblast hybrid cells was partially relieved by a dominant-negative RARβ mutant. HeLa clones stably expressing a RARβ transgene under control of the human β-actin promoter [HeLa(RARβ)] were established and analyzed for transgene-mediated growth alterations in vitro and in vivo. Anchorage-independent growth of the HeLa(RARβ) lines was indistinguishable from that of control cells in the absence of RA, but strongly impaired after RA treatment. Reduced tumor growth of HeLa(RARβ) clones was associated with high RARβ protein levels. Somatic cell fusion experiments revealed that the loss of ligand inducibility of RARβ gene expression in HeLa cells cannot be complemented by fusion with other cervical cancer cell lines. Our data indicate, firstly, that RARβ is a negative regulator of tumor cell growth and, secondly, that cancer-associated defects in RARβ gene expression are caused by stable, non-complementable silencing mechanisms. Int. J. Cancer 85:289–295, 2000. ©2000 Wiley-Liss, Inc.
A series of potent retinoid X receptor (RXR) selective ligands was designed and prepared. The lead compound 7a showed good binding (Ki; 20–50 nM) and transactivation (EC50; 40–50 nM) to the RXR subfamily of retinoid receptors. More importantly, small variations in the geometry of the cyclopentane ring moiety led to 9, one of the most potent RXR agonists to date (Ki: 3–8 nM; EC50: 3–4 nM).
Among various chemical classes of compounds with retinoidal activity, the retinobenzoic acid system having a 5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl moiety and a benzoic acid moiety at opposite ends of the molecule has proved to be one of the most promising leads. Many retinoidal-active compounds with various kinds of chemical moieties as the linking group between the above two groups were synthesized, and amide moieties proved to be excellent linkages for potent retinoidal activities. The activities, however, were diminished by methylation at the amide nitrogen. To elucidate the reasons for the loss of the activities, we performed X-ray crystal structure analyses of three free amides and the corresponding three N-methylamide compounds. It was proved that all the free amide molecules take an extended trans conformation, whereas all the N-methylamide molecules take a folded cis conformation in the crystalline state. In combination with spectroscopic studies (UV and NMR), the above results suggested that these N-methylamides adopt remarkable folded conformations compared to the free amide molecules in solution. These facts strongly indicate that all these N-methylamides take cis conformations not only in the crystal but also in solution. It is the extended trans conformation that is required for specific binding to the retinoidal receptor macromolecule. Consequently, the loss of activities in N-methylamides seem to be ascribed to conformational factors but not to steric hindrance by the methyl group which might prevent binding to the target receptor.
In the course of our studies on retinoic acid receptor (RAR) agonists, we have designed and synthesized a series of quinoxaline derivatives. One of them, 4-[5-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-quinoxalinyl)-1H-2-pyrrolyl]benzoic acid (3a), which possesses a 2,5-disubstituted pyrrole moiety, showed selectivity for the RARα receptor and exerted highly potent cell-differentiating activity on HL-60 cells.
A series of potent retinoid X receptor (RXR) selective ligands were designed and prepared. The lead compound 6a, showed good binding (Kd; 3–7 nM) and transactivation (EC50; 19–24 nM) to the RXR subfamily of retinoid receptors. More importantly, a small variation on the aromatic ring moiety led to 6b, which had less residual RAR agonist activity with RXR binding and potency of 4–5 nM and 5–13 nM, respectively.
The biological effects of vitamin A are mediated in part by retinoic acid (RA) modulation of gene transcription. In this study, we examined whether normal human mammary epithelial cells (HMECs) are biologically responsive to retinol (ROH), the metabolic precursor of RA. While both ROH and tRA resulted in time- and dose-dependent decreases in total cell number, tRA was markedly more potent. Metabolically, treatment of HMECs with physiological doses of ROH resulted in rapid uptake and subsequent production of both retinyl esters and tRA. Although a comparatively minor metabolite, tRA levels peaked at 6 h and remained above endogenous levels for up to 72 h in proportion to cellular ROH concentrations. In HMECs transfected with an RA-responsive luciferase reporter gene, treatment with 3 μM ROH resulted in an increase in luciferase activity to a level intermediate between that observed with 0.001 and 0.01 μM tRA. Citral, an RA-synthesis inhibitor, was also used to examine the biological activity of ROH. Compared to ROH alone, ROH plus citral treatment resulted in three-fold less tRA synthesis and a > 65% attentuation of RA-responsive reporter gene activity which persisted through 72 h. Citral also significantly attenuated the extent of ROH-mediated reductions in total HMEC number. Thus, treatment with physiological concentrations of ROH results in fewer total numbers of HMECs and this response is a consequence of cellular tRA synthesis which can induce RA-responsive gene expression. © 2001 Wiley-Liss, Inc.
Repression by nuclear receptors plays important roles in acute promyelocytic leukemia and other diseases. Nuclear receptor corepressor (N-CoR) and SMRT (silencing mediator of retinoic acid and thyroid hormone receptor) are corepressor proteins whose modular structure facilitates receptor interaction as well as transduction of repression signals involving histone deacetylation, alterations in chromatin structure and direct interactions with the basal transcription machinery. Interactions between nuclear receptors and corepressor complexes have multiple determinants. This allows regulation, and potentially therapeutic manipulation, of receptor, corepressor, cell-type and target-gene specificity.
The action of retinoids on gene regulation is mediated by three distinct nuclear retinoic acid receptor (RAR) subtypes called RAR alpha, beta and gamma. Since RAR gamma is predominantly expressed in adult skin, specific ligands for this subtype could (i) represent valuable tools to evaluate the biological role of RAR gamma in skin and (ii) provide therapeutic entities with a higher therapeutic index at lower teratogenic risk. Using in vitro binding studies and a functional transactivation assay, we have identified three compounds with high RAR gamma selectivity.
Two types of aromatic amides, terephthalic monoanilides and (arylcarboxamido)benzoic acids, have been shown to possess potent retinoidal activities and can be classified as retinoids. The structure-activity relationships of these amides are discussed on the basis of differentiation-inducing activity on human promyelocytic leukemia cells HL-60. In generic formula 4 (X = NHCO or CONH), the necessary factors to elicit the retinoidal activities are a medium-sized alkyl group (isopropyl, tert-butyl, etc.) at the meta position and a carboxyl group at the para position of the other benzene ring. The bonding of the amide structure can be reversed, this moiety apparently having the role of locating the two benzene rings at suitable positions with respect to each other. Substitution at the ring position ortho to the amide group or N-methylation of the amide group caused loss of activity, presumably owing to the resultant change of conformation. It is clear that the mutual orientation of the benzylic methyl group(s) and the carboxyl group and their distance apart are essential factors determining the retinoidal activity. Among the synthesized compounds, 4-[(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)carbamoyl]benz oic acid (Am80) and 4-[(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)carboxamido] benzoic acid (Am580) were several times more active than retinoic acid in the assay. They are structurally related to retinoic acid, as is clear from the biological activity of the hybrid compounds (M2 and R2).
Retinoids, which are derivatives of vitamin A, have a variety of effects on normal cellular differentiation and on the process of carcinogenesis. A number of novel endogenous retinol metabolites have been identified recently. The response of many cell types to retinoid treatment is mediated by retinoid receptors, and involves changes in gene expression, cell growth and cell differentiation. The gene encoding one of the retinoic acid receptors is disrupted by the chromosome translocations associated with acute promyelocytic leukemia, and the expression of another is altered in epithelial tumors; both of these findings have important implications for the use of retinoids as anti-carcinogenic agents. It has been demonstrated recently that certain homeobox genes are regulated by retinoids; these genes may also prove to be useful agents for anti-carcinogenic therapies.
Two series of potent retinoid X receptor (RXR)-selective compounds were designed and synthesized based upon recent observation that (E)-4-[2-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-2-naphthalenyl)-1- propenyl]benzoic acid (TTNBP) binds and transactivates only the retinoic acid receptor (RAR) subtypes whereas (E)-4-[2-(3,5,5,8,8-pentamethyl-5,6,7,8- tetrahydro-2-naphthalenyl)-1-propenyl]benzoic acid (3-methyl TTNPB) binds and transactivates both the RAR and RXR subfamilies. Addition of functional groups such as methyl, chloro, bromo, or ethyl to the 3 position of the tetrahydronaphthalene moiety of 4-[(5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-2-naphthyl)carbonyl]benzoic acid (5a) and 4-[1-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-2- naphthyl)ethenyl]benzoic acid (6a) results in compounds which elicit potent and selective activation of the RXR class. Such RXR-selective compounds offer pharmacological tools for elucidating the biological role of the individual retinoid receptors with which they interact. Activation profiles in cotransfection and competitive binding assays as well as molecular modeling calculations demonstrate critical structural determinants that confer selectivity for members of the RXR subfamily. The most potent compound of these series, 4-[1-(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydro-2-naphthyl)ethenyl]ben zoi c acid (6b), is the first RXR-selective retinoid (designated as LGD1069) to enter clinical trials for cancer indications.
Retinoic acid receptor beta (RAR beta), which codes for a nuclear receptor for retinoic acid, is localized in a chromosomal region frequently deleted in lung cancer cells. The gene is expressed in normal lung tissue and in the majority of the cell lines derived from lung tumors but not in most of the lines derived from lung tumors with epidermoid characteristics. To study the possible role of RAR beta in growth control of epidermoid lung tumor-derived cells, transfectants expresing RAR beta were generated from nonexpressing epidermoid tumor-derived cell lines. Four clones were derived from line CALU-1, three of which showed a 20-60% increase in doubling time in the presence of retinoic acid. Parental and control-transfected cells were unaffected or slightly stimulated. All four clones expressing RAR beta were less tumorigenic in nude mice than were the untransfected or control-transfected cells, with about a 50% incidence of take vs. 95%. When tumors did develop from RAR beta-positive cells, they showed a reduced rate of growth, an increased latency, and, in six of seven tumors tested, a much reduced level of RAR beta expression. Transfectants derived from a second tumor line, H157, also showed a markedly reduced incidence of take in nude mice. Together with the known effects of retinoic acid on differentiation and carcinogenesis, our results support the hypothesis that RAR beta functions as a tumor suppressor gene in epidermoid lung tumorigenesis.
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The retinoic acid receptors (RARs) transduce retinoid dependant gene regulation, and many biological effects of retinoids are mediated through binding and activation of three closely related receptor subtypes (RAR alpha, RAR beta, and RAR gamma). In order to investigate the role of receptor subtypes, we have carried out a chemical synthesis program to seek selective retinoids for these receptors. We measured receptor binding affinity using recombinant RAR alpha, -beta, and -gamma proteins and assessed cellular differentiating activity in F9 murine teratocarcinoma cells (F9 cells). This research has identified the 4-substituted-3-(1-adamantyl)phenyl moiety as a new pharmacophore which can replace the beta-cyclogeranylidene ring of the naturally occurring all-trans-retinoic acid. Two chemical series derived from the general structures 6-(3-tertioalkylphenyl)-2-naphthoic acid (series I) and 4-[(E)-2-(3-tertioalkylphenyl)propenyl]benzoic acid (series II) were developed. In particular, we have obtained the RAR gamma selective derivatives 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthoic acid (7) [Ki(RAR alpha) = 6500 nM, Ki(RAR beta) = 2480 nM, Ki(RAR gamma) = 77 nM] and 4-[(E)-2-[3-(1-adamantyl)-4-hydroxyphenyl]propenyl]benzoic acid (19) [Ki(RAR alpha) = 1,144 nM, Ki(RAR beta) = 1245 nM, Ki(RAR gamma) = 53 nM]. In series I, the presence of a phenol group, irrespective of the nature of tertioalkyl group, imparted at least partial RAR gamma selectivity, whereas in series II, the presence of both adamantyl and phenol groups is needed to confer RAR gamma selectivity. The RAR gamma selective ligands induce differentiation in F9 cells (7, AC50 = 33 nM; 19, AC50 = 66 nM). From series I, a mixed RAR beta-gamma agonist with potent cellular differentiating activity was selected for development as a topical antiacne agent, 6-[3-(1-adamantyl)-4-methoxyphenyl]-2-naphthoic acid (5, CD 271) [Ki(RAR alpha) = 1100 nM, Ki-(RAR beta) = 34 nM, Ki(RAR gamma) = 130 nM, AC50(F9) = 37 nM]. Finally, from series II, we have obtained a weak antagonist in the F9 cellular differentiation assay, 4-[(E)-2-(3-tert-butyl-4-hydroxyphenyl)propenyl]benzoic acid (15, IC50 = 700 nM).
Metabolic activation of retinol into the hormone retinoic acid and metabolism of retinoic acid entail essential aspects of retinoid biology that seem interdependent with functions of retinoid binding-proteins. Cellular retinol binding protein and cellular retinoic acid binding protein enjoy widespread expression and, where expressed, their liganded forms represent the major physiological forms of retinol and retinoic acid, respectively. These retinoid binding proteins may protect cells from the amphipathic properties of retinoids and protect the structurally sensitive retinoids from the cellular milieu. Starting from the perspective that the enzymes most likely to metabolize retinoids in vivo might recognize the major forms of retinoids that occur in vivo, several laboratories have produced results that support a model of retinoid metabolism with prominent roles for the cellular retinoid binding proteins. In this model, liganded cellular retinoid binding proteins serve as substrates for the metabolism of some retinoids (retinol, retinoic acid), restricting access to those enzymes that recognize both the binding protein and the retinoid. Other retinoids (3,4-didehydroretinol, 4-oxo-retinoic acid) liganded to binding-proteins have their metabolism arrested. In its unliganded form, at least one retinoid binding protein (cellular retinol binding protein) serves as a retinoid concentration-sensitive modulator of enzymes that catalyze retinol metabolism. This review will describe the model and the intrinsic relationships among retinoid-specific enzymes and retinoid binding proteins.
N-(4-Hydroxyphenyl) retinamide (4HPR) and retinoyl beta-glucuronide (RAG) are two derivatives of all-trans retinoic acid (RA) that show properties both similar to as well as different from their parent compound, RA. Both retinoids possess the important property of showing much-reduced toxicity relative to RA while maintaining significant biological activity. 4HPR, a synthetic derivative, is active in the prevention and treatment of a variety of neoplasms in animals, and by inducing apoptosis, shows growth inhibitory activity against many human tumor cell types in vitro. In humans, 4HPR reduces the incidence of new occurrences of leukoplakia and is currently being tested as a preventive agent for breast cancer. RAG, a naturally occurring metabolite of RA, effectively stimulates the growth of vitamin A-deficient animals, induces the differentiation of epithelial cells in vivo and in vitro, and is effective in the topical treatment of acne in humans. Unlike RA, RAG is nontoxic when applied to the skin and is nonteratogenic when given orally to rats. Possible mechanisms of action of both compounds are discussed. These two derivatives of retinoids show interesting physiologic effects and potentially beneficial pharmacologic actions.
Retinoid X receptor (RXR) plays a central role in the regulation of many intracellular receptor signalling pathways and can mediate ligand-dependent transcription, acting as a homodimer or as a heterodimer. Here we identify an antagonist towards RXR homodimers which also functions as an agonist when RXR is paired as a heterodimer to specific partners, including peroxisome proliferator-activated receptor and retinoic acid receptor. This dimer-selective ligand confers differential interactions on the transcription machinery: the antagonist promotes association with TAF110 (TATA-binding protein (TBP)-associated factor 110) and the co-repressor SMRT, but not with TBP, and these properties are distinct from pure RXR agonists. This unique class of RXR ligands will provide a means to control distinct target genes at the level of transcription and allow the development of retinoids with a new pharmacological action.
Retinoids in clinical use today are known to induce hypertriglyceridemia as one of their major side effects. The purpose of the present study was to determine, in an appropriate animal model, if retinoid-induced hypertriglyceridemia is mediated by retinoic acid receptors (RARs) and/or by retinoid X receptors (RXRs). Oral gavage of male Fischer rats with 13-cis-retinoic acid for 6 days caused a rapid and sustained increase in serum triglycerides that was reversible within 4 days posttreatment. In subsequent experiments, rats were treated by gavage once daily for 3 days with various retinoids, and serum triglyceride levels were determined 24 hr after the last treatment without fasting. All-trans- and 13-cis-retinoic acid, which can be converted to both RAR and RXR agonists, and 9-cis-retinoic acid, an RAR/RXR pan-agonist, caused dose-dependent increases in serum triglycerides at doses that did not cause weight loss or mucocutaneous toxicity. Ro 13-6298 and AGN 190121, two RAR-specific agonists, caused dose-dependent increases in serum triglycerides, although Ro 13-6298 only induced hypertriglyceridemia at weight-suppressive doses. Two RXR-selective agonists, LG100268 and AGN 191701, failed to induce hypertriglyceridemia or weight loss up to the highest doses tested. A structural isomer of AGN 190121 that does not activate RARs or RXRs, AGN 190727, did not induce hypertriglyceridemia. Hypertriglyceridemia induced by AGN 190121 was significantly inhibited by cotreatment with an RAR-selective antagonist, AGN 193109. Taken together, these data provide strong evidence that retinoid-induced hypertriglyceridemia is mediated, at least in part, by RARs. These data also suggest that RXR-specific agonists may have reduced potential to induce hypertriglyceridemia relative to RAR-active retinoids.
Recently, 9-cis retinoic acid, a high affinity ligand for retinoic acid receptors and retinoid X-receptors (RXRs), was shown to have efficacy superior to all-trans retinoic acid as a chemopreventive agent in the N-nitroso-N-methylurea-induced rat mammary carcinoma model. To further explore the specific contribution RXR activation may play in suppression of carcinogenesis, the efficacy of LGD1069 (Targretin), an RXR-selective ligand, in the N-nitroso-N-methylurea-induced rat mammary tumor model was studied. LGD1069-treated animals showed a 90% reduction in tumor burden and tumor incidence compared with vehicle-treated rats with an efficacy similar to that achieved with tamoxifen. LGD1069 was very well tolerated during 13 weeks of chronic therapy with no classic signs of "retinoid-associated" toxicities. These data demonstrate that LGD1069, an RXR-selective ligand, can act as a highly effective and benign chemopreventive agent for mammary carcinoma.
The retinoid response is mediated by nuclear receptors, including retinoic acid receptors (RARs) and retinoid "X" receptors (RXRs). All-trans retinoic acid (RA) binds only RARs, while 9-cis RA is an agonist for both RARs and RXRs. Recently, LGD1069 was identified as a highly selective RXR agonist with low affinity for RARs. We undertook a dose-ranging study to examine the safety, clinical tolerance, and pharmacokinetics of LGD1069 in patients with advanced cancer.
Fifty-two patients received. LGD1069 administered orally once daily at doses that ranged from 5 to 500 mg/m2 for 1 to 41 weeks. Treatment proceeded from a starting dose of 5 mg/m2. Pharmacokinetic sampling was performed on selected patients on days 1, 15, and 29.
Reversible, asymptomatic increases in liver biochemical tests were the most common dose-limiting adverse effect. Less prominent reactions included leukopenia, hypertriglyceridemia, and hypercalcemia. Characteristic retinoid toxicities, such as cheilitis, headache, and myalgias/arthralgias, were mild or absent. Two patients with cutaneous T-cell lymphoma experienced major antitumor responses. Pharmacokinetic studies obtained in 27 patients at eight dose levels showed that the day 1 area under the plasma concentration-times-time curves (AUCs) were proportional to dose. At all doses studied, the day 1 AUCs were similar to those on days 15 and 29, indicating a lack of induced metabolism.
LGD1069 is a unique compound that exploits a newly identified pathway of retinoid receptor biology that may be relevant to tumor-cell proliferation and apoptosis. Further investigation of this drug is warranted. Based on the results of this study, a dose of 300 mg/m2 is recommended for single-agent trials.
To characterize the dimerization of retinoid receptors in solution, RAR alpha homodimers and RAR alpha-RXR alpha heterodimers, formed in the absence or the presence of a naturally occurring RA response element (betaRARE) under different ionic conditions, were analyzed by size-exclusion fast protein liquid chromatography and sucrose density gradient sedimentation. In the presence of [3H]RA both RAR alpha and RXR alpha existed primarily as monomers of 50 kDa in solutions containing 80 mM KCl. However, when betaRARE was included in these incubations, a 40-fold increase in the occurrence of both the RAR alpha homodimers and the RAR alpha-RXR alpha heterodimers (125 kDa) was observed. The presence of RAR alpha and RXR alpha in the betaRARE-associated homo- and heterodimers was confirmed by the positive interaction of the receptors with the specific antibodies. Both RAR alpha homodimers and RAR alpha-RXR alpha heterodimers bound betaRARE even in the absence of the ligand RA with the heterodimer showing a 2- to 4-fold greater affinity than the homodimer for the DNA binding element. When the receptors were incubated in solutions of increasing ionic concentration (50-300 mM KCl), a decrease in the amount of both RAR alpha homodimers and RAR alpha-RXR alpha heterodimers was accompanied by a corresponding increase in the monomeric fraction even in the presence of betaRARE, suggesting that the high salt concentrations inhibit the surface to surface interactions between the monomers. These observations suggest that in vivo, as in solution, the formation of a stable retinoid receptor dimer complex is dependent upon both receptor-receptor and receptor-RARE interactions.
The syntheses and full retinoid receptor characterization of a novel series of retinoic acid receptor alpha (RAR alpha) antagonists, 1-5, are described. These compounds bind with high affinity to RAR alpha but were completely inactive in gene transactivation. They were also potent and effective antagonists of retinoic acid (RA) induced gene transcription at RAR alpha. Compounds 1-5 exhibited varying degrees of selectivity for RAR alpha relative to RAR beta/gamma, with compound 5 being the most selective in both binding and functional antagonism assays. These compounds will be invaluable tools in delineating the physiological roles of RAR alpha in development and in the adult animal and may themselves be useful therapeutic agents in human diseases associated with RAR alpha.
Unliganded bacterially expressed RXRα lacking the N-terminal region AB (apo-RXRαΔAB) was found in solution as an apparent mixture of 165 kDa tetramers and 42 kDa monomers which could be quantitatively separated by gel filtration and non-denaturing gel electrophoresis. Under identical conditions both liganded (holo-) and apo-RARαΔAB were present as single monomeric species. apo-RXRαΔAB tetramers, as well as dimers of the apo-RXR ligand binding domain (apo-LBD), dissociated readily into monomers when exposed to their cognate ligand 9-cis retinoic acid (9c-RA). The apo-RXRαΔAB tetramer bound only transiently to a cognate DR1 response element, and was converted into DR1-apo-RXRαΔAB homodimer complexes indistinguishable from those generated by cooperative DNA binding of apo-RXRαΔAB monomers. In the absence of DNA, the addition of 9c-RA greatly accelerated the formation of hetero dimers with the apo-RARαΔAB heterodimerization partner. No RXRαΔAB or RARαΔAB homodimers could be observed in solution, but upon mixing of the two receptor monomers stable heterodimers could be isolated which bound to DR5 response elements in a highly cooperative manner. In these heterodimers, RXRαΔAB interacted with its cognate ligand as efficiently as in RXRαΔAB homodimers. The presence of ligand did not alter the stability of RXRαΔAB homodimer or RXRαΔAB-RARαΔAB heterodimer complexes on DR1 and DR5 response elements, respectively. These in vitro data support a model in which RXR tetramers could serve as an inactive pool with the dual function of: (i) rapidly supplying large amounts of RXR heterodimerization partners upon 9c-RA generation; and (ii) allowing RXR homodimer formation on 'accessible' cognate response elements in the absence of 9c-RA. These events may represent a ligand-dependent regulatory mechanism controlling the availability of the promiscuous RXR dimerization partner that is engaged in multiple nuclear receptor signalling pathways.
Recently, we reported that LGD1069, a high-affinity ligand for the retinoid X receptors (RXRs), was shown to have an efficacy equivalent to that of tamoxifen (TAM) as a chemopreventive agent in the N-nitroso-N-methylurea-induced rat mammary carcinoma model. Furthermore, LGD1069 was very well tolerated during 13 weeks of chronic therapy with no classic signs of "retinoid-associated" toxicities. Due to the high efficacy and benign profile of this RXR agonist as a suppressor of carcinogenesis, we examined its role as a therapeutic agent on established mammary carcinomas. In the rat mammary carcinoma model, N-nitroso-N-methylurea was used to induce tumors, and the tumors were allowed to grow to an established size prior to initiation of treatment. LGD1069-treated animals showed complete regression in 72% of treated tumors and had a reduced tumor load compared to control. In addition, the combination of LGD1069 and TAM showed increased efficacy over either agent alone. Histopathological analysis showed a reduction of LGD1069-treated tumor malignancy, an increase in differentiation, and a sharp decrease in cellular proliferation compared to vehicle-treated control tumors. These data demonstrate that the RXR-selective ligand LGD1069 is a highly efficacious therapeutic agent for mammary carcinoma and enhances the activity of TAM.
A number of studies have suggested that the active derivative of vitamin A, retinoic acid (RA), may be important for early development of mammalian embryos. Severe vitamin A deprivation in rodents results in maternal infertility, precluding a thorough investigation of the role of RA during embryogenesis. Here we show that production of RA by the retinaldehyde dehydrogenase-2 (Raldh2) enzyme is required for mouse embryo survival and early morphogenesis. Raldh2 is an NAD-dependent aldehyde dehydrogenase with high substrate specificity for retinaldehyde. Its pattern of expression during mouse development has suggested that it may be responsible for embryonic RA synthesis. We generated a targeted disruption of the mouse Raldh2 gene and found that Raldh2-/- embryos, which die at midgestation without undergoing axial rotation (body turning), exhibit shortening along the anterioposterior axis and do not form limb buds. Their heart consists of a single, medial, dilated cavity. Their frontonasal region is truncated and their otocysts are severely reduced. These defects result from a block in embryonic RA synthesis, as shown by the lack of activity of RA-responsive transgenes, the altered expression of an RA-target homeobox gene and the near full rescue of the mutant phenotype by maternal RA administration. Our data establish that RA synthesized by the post-implantation mammalian embryo is an essential developmental hormone whose lack leads to early embryo death.
The synthesis and biological activity of a series of structurally related retinoids with different RAR subtype selectivities are described. These retinoids bind to all three RAR subtypes but in functional transactivation assays, they show RARbeta or RARbeta,gamma selectivity with weak RARalpha activity. The subtype selectivity of these retinoids was found to correlate with their efficacy (ODC inhibition) and toxicity (topical irritation and teratogenicity) profiles. The degree of RARgamma transactivation activity correlates with their topical toxicity and teratogenicity as measured by the inhibition of chondrogenesis. Of the RARbeta selective retinoids reported here, retinoid 12 is the most promising, as it is completely devoid of two common retinoid related toxicities, namely topical irritation and teratogenesis.
Many synthetic retinoids have been generated that exhibit a distinct pattern of agonist/antagonist activities with the three retinoic acid receptors (RARalpha, RARbeta and RARgamma). Because these retinoids are selective tools with which to dissect the pleiotropic functions of the natural pan-agonist, retinoic acid, and might constitute new therapeutic drugs, we have determined the structural basis of their receptor specificity and compared their activities in animal and yeast cells.
There are only three divergent amino acid residues in the ligand binding pockets (LBPs) of RARalpha, RARbeta and RARgamma. We demonstrate here that the ability of monospecific (class I) retinoid agonists and antagonists to bind to and induce or inhibit transactivation by a given isotype is directly linked to the nature of these residues. The agonist/antagonist potential of class II retinoids, which bind to all three RARs but depending on the RAR isotype have the potential to act as agonists or antagonists, was also largely determined by the three divergent LBP residues. These mutational studies were complemented by modelling, on the basis of the three-dimensional structures of the RAR ligand-binding domains, and a comparison of the retinoid agonist/antagonist activities in animal and yeast cells.
Our results reveal the rational basis of RAR isotype selectivity, explain the existence of class I and II retinoids, and provide a structural concept of ligand-mediated antagonism. Interestingly, the agonist/antagonist characteristics of retinoids are not conserved in yeast cells, suggesting that yeast co-regulators interact with RARs in a different way than the animal cell homologues do.
We have previously shown that a retinoid X receptor (RXR)-selective ligand (a rexinoid), called LGD1069, is highly efficacious in both the chemoprevention and the chemotherapy for N-nitrosomethylurea-induced rat mammary carcinomas. To evaluate a possible role for rexinoids in breast cancer therapy further, we have designed and characterized a novel carcinogen-induced model to mimic the clinical situation in which the tumors of patients stop responding to tamoxifen therapy and develop resistance to this drug.
Rats with experimentally induced mammary tumors were treated with tamoxifen to select a population with primary tumors that failed to respond completely to the drug. Once the failure of tamoxifen therapy had been established, LGD1069 was added to the treatment regimen, and the tumors in these animals were compared with tumors in a group of animals that remained on tamoxifen alone.
LGD1069 in combination with tamoxifen for up to 20 weeks yielded an overall objective response rate of 94% (95% confidence interval [CI] = 86%-100%) (includes complete and partial responses) in primary tumors compared with a rate of 33% (95% CI = 11%-56%) in primary tumors treated with tamoxifen alone, a statistically significant difference (two-sided P<.0001). In addition, the LGD1069 and tamoxifen combination was associated with a statistically significant decrease in total tumor burden (two-sided P =.03). In a second study, tumors that failed to respond to tamoxifen therapy exhibited a 51% (95% CI = 34%-71%) objective response rate when treated with LGD1069 alone for 6 weeks after tamoxifen therapy was withdrawn.
We have demonstrated that the RXR-selective ligand LGD1069 in combination with tamoxifen is a highly efficacious therapeutic agent for tumors that fail to respond completely to tamoxifen. This finding suggests that rexinoid therapy offers a novel approach to the treatment of breast tumors that may have developed resistance to antihormonal therapies such as tamoxifen.