RETINOIDS, REXINOIDS AND THEIR COGNATE NUCLEAR RECEPTORS: CHARACTER AND THEIR ROLE IN CHEMOPREVENTION OF SELECTED MALIGNANT DISEASES

BACKGROUND
Retinoids, rexinoids and their biologically active derivatives are involved in a complex arrangement of physiological and developmental responses in many tissues of higher vertebrates. Both retinoids and rexinoids are either natural or synthetic compounds related to retinoic acids that act through interaction with two basic types of nuclear receptors belonging to the nuclear receptor superfamily: All-trans retinoic acid receptors (RARalpha, RARbeta, and RARgamma) and retinoid X receptors (RXRalpha, RXRbeta and RXRgamma) as retinoid-inducible transcription factors.


AIM
Summarization of selected effects of biologically active natural or synthetic retinoids and rexinoids and their exploitation in chemoprevention of various types of cancer.


RESULTS
Retinoid receptors play a role as ligand-activated, DNA-binding, trans-acting, transcription-modulating proteins involved in a general molecular mechanism responsible for transcriptional responses in target genes. They exert both beneficial and detrimental activity; they have tumour-suppressive activity but on the other hand they are teratogenic. A number of nuclear receptor selective retinoids and rexinoids, have been successfully tested using a variety of cell lines or animal models. Retinoids inhibit carcinogenesis, suppress premalignant epithelial lesions and tumour growth and invasion in a variety of tissues.


CONCLUSIONS
Natural and synthetic retinoids exert important biological effects due to their antiproliferative and apoptosis-inducing effects. They are also known to cause redifferentiation or to prevent further dedifferentiation of various tumour tissues.


INTRODUCTION
Retinoids and their natural metabolites and synthetic derivatives are polyisoprenoid compounds containing a cyclohexenyl ring.Their parent compound is all-trans retinol (vitamin A) and its subsequent oxidation product is all-trans retinal (retinaldehyde).From retinaldehyde alltrans retinoic acid (ATRA) is formed irreversibly 1 .ATRA is considered to be the main biologically active form of vitamin A in a variety of functions in vertebrates.9-cis retinoic acid (9cRA) has a diff erent spectrum of biological activity in comparison with that of all-trans retinoic acid.9cRA is a high affi nity ligand for the 9-cis retinoic acid (retinoid X or rexinoid) receptors (RXRs), as well as the all-trans retinoic acid receptors (RARs) 2 .Retinoids are involved in the complex arrangements of physiological and developmental responses in many tissues of higher vertebrates that include embryonic development, vision, reproduction, bone formation, haematopoiesis, metabolism, growth and diff erentiation of a variety of cell types, apoptosis and processes of carcinogenesis [1][2][3] .It is well known that retinoids are also teratogens and the therapeutic doses of them are contraindicated during pregnancy 4 .This article summarizes selected eff ects of biologically active natural or synthetic retinoids and rexinoids, acting through their cognate nuclear receptors, and their use in chemotherapy and chemoprevention of various types of cancer.We would like to apologize to all authors whose original references have not been mentioned in this article due to our intention to limit the references to a reasonable number.

NUCLEAR RECEPTORS -LIGAND INDUCIBLE TRANSCRIPTION FACTORS
A breakthrough in the fi eld of nuclear receptors was discovery of the nuclear receptor superfamily that comprises nuclear receptors for steroid hormones, retinoids, thyroid hormones, vitamin D 3 , nuclear receptors for other biologically important compounds, and a number of "orphan" nuclear receptors for which the ligands remain still unknown 5 .It is generally accepted that nuclear receptors represent a superfamily of ligand-inducible transcription factors.Dysfunction of nuclear receptor signalling leads to proliferative, reproductive and metabolic diseases.The nuclear receptor superfamily of 48 identifi ed nuclear receptors in human 6 is shown in Table 1 and 2.
Proteins of the nuclear receptor superfamily represent single polypeptide chains with several major domains Testosterone, fl utamide (Fig. 1).The amino terminal A/B-domain of the nuclear receptor molecule contains a constitutive activation function independent on ligand (AF-1).The central C-domain is a cysteine-rich DNA-binding region consisting of two highly conserved zinc fi ngers.The D-domain represents a highly fl exible structure, and it plays a role as a hinge of the receptor molecule.8][9] ).All nuclear receptors are known to modulate gene transcription, although among them marked diff erences do exist, predominantly in the mechanisms through which it is accomplished [10][11][12] .Retinoids, rexinoids and their cognate nuclear receptors: Character and their role in chemoprevention of selected malignant diseases The diversity of retinoic acid-induced signalling pathway is associated with existence of at least three subtypes for RAR (α, β and γ) and three subtypes for RXR (α, β and γ) with distinct amino-and carboxy-terminal domains.Later on, two major isoforms for RARα (α1 and α2) and for RARγ (γ1 and γ2) and four major isoforms for RARβ (β1, β2, β3 and β4) have been found.Similarly, two major isoforms for RXRα (α1 and α2), RXRβ (β1 and β2), and RXRγ (γ1 and γ2) have been identifi ed up to now 5,13 .All-trans retinoic acid receptors upon all-trans retinoic acid binding act as all-trans retinoic acid-inducible transcription factors by directly interacting as heterodimers with 9-cis retinoic acid receptor.The RXR/RAR heterodimer interacts with specifi c DNA response elements of target genes and its eff ect on transcription is mediated also through recruitment of a number of coregulators  (corepressors and coactivators) 5,10,11 .Ligand occupancy of both receptors in RXR/RAR heterodimes was found to synergistically increase transcriptional activity 14,15 .RARs bind both all-trans retinoic acid and 9-cis retinoic acid with similar affi nities while RXRs bind only 9-cis-retinoic acid.RXR/RAR heterodimers bind to specifi c DNA sequence -retinoic acid response elements (RAREs), characterized by direct repeats of two hexamers AGGTCA separated predominantly by two nucleotides (DR+2) or fi ve nucleotides (DR+5) (Fig. 2).In the absence of all-trans retinoic acid, the RAR/RXR heterodimer recruits nuclear receptor corepressor proteins, e.g.nuclear receptor corepressor (N-CoR) or silencing mediator of retinoid and thyroid hormone receptor (SMRT), and associated enzymes such as histone deacetylases or DNA-methyl transferases that lead to an inactive condensed chromatin structure preventing transcription process.All-trans retinoic acid binding leads to the dissociation of corepressor proteins and enables association of coactivator proteins (histone acetyltransferases or histone arginine methyltransferases) with liganded receptor complex yielding in activation of gene transcription 6,[16][17][18] .Thus, the retinoid receptors are considered to be ligand-activated, DNA-binding, trans-acting, transcription-modulating nuclear proteins involved in a general molecular mechanism responsible for transcriptional responses in target genes 6 .
The retinoids selective for specifi c binding to RXRs are called rexinoids 19 , and RXRs play an crucial role in the nuclear receptor mediated transcription processes for their ability to heterodimerize with many other members of nuclear receptor superfamily, including RARs, thyroid hormone receptors (TRs), vitamin D 3 receptor (VDR), peroxisome proliferator-activated receptors (PPARs), liver X receptors (LXRs), farnesoid X receptor (FXR), pregnan X receptor (PXR), and constitutively activated receptors (CARs) (ref. 6,20,21 )RXRs thus behave as promiscuous dimerization partners for a large number of nuclear receptors, and thus play an integrative and crucial role in nuclear receptor mediated pathways 6,22,23 .While RAR ligand (agonist) autonomously activates transcription through RXR/RAR heterodimer, RXRs are unable to respond to RXR ligands (rexinoids) in the absence of RAR ligand.Rexinoid binding to RXR is incapable to induce the dissociation of corepressor molecule from the RXR/ RAR heterodimers 24 moreover, no interaction was found between RXRs and corepressors NCoR and SMRT 25 .
Genetic strategies in the mouse in order to determine the function of both RAR and RXR subtypes have shown following results.RARα null mutant males have been found to be sterile due to degeneration of the seminiferous epithelium causing inhibition of spermatogenesis [26][27][28] , RARβ null mice display abnormalities in the vitreous body in eyes and impaired abilities in locomotion and motor coordination, and ablation of RARγ causes both skeletal and epithelial defects 29 .The loss of RXRα was found to be lethal during fetal life due to hypoplasia of the myocardium that appears to be the principal cause of animal death.Moreover, fetuses lacking RXRα have ocular malformation.Also, it has been shown that RXRα is involved in the mediation of a teratogenic eff ect due to administration of exogenous retinoids [30][31][32] .The ablation of RXRβ led to approximately 50 % in utero lethality.Males lacking RXRβ are sterile and exhibit testicular defects and abnormal spermatid maturation, leading to defects of spermatozoa.Moreover, RXRβ mutation causes abnormal lipid metabolism in Sertoli cells, suggesting functional interactions of RXRβ with other nuclear receptors that control lipid metablism [32][33][34] .RXRγ null mutants are fertile but they have higher serum levels of both L-thyroxine and thyroid-stimulating hormone (TSH), and increased metabolic rate when compared to wild type animals 32,35 .Retinoids, rexinoids and their cognate nuclear receptors: Character and their role in chemoprevention of selected malignant diseases Thus, RXR-RAR heterodimers play an important role in transducing in vivo the retinoid signal and that specifi c heterodimers are involved in many developmental and metabolic processes 28,32 .

RETINOID SIGNALLING IN CHEMOPREVENTION OF SELECTED MALIGNANT DISEASES
In animal models, retinoids or rexinoids have been found to suppress carcinogenesis or induce diff erentiation of malignant cells through their cognate nuclear receptors in a variety of tissues 36 .Altered expression of all-trans or 9cis retinoic acid receptors may be associated with the processes of malignant transformation of animal or human cells in a variety of tissues.Aberrant retinoid signalling mechanisms is oftentimes linked to cancer, and at present, the retinoids are used to a progressively increasing extent in a variety of chemopreventive and chemotherapeutic settings 28 .

Acute promyelocytic leukaemia and exploitation of retinoids
The most direct implication of RAR in human disease is given by acute promyelocytic leukaemia (APL), which is characterized by selected expansion of immature myeloid precursors or malignant myeloid cells blocked at the promyelocytic stage of hemopoietic development 36,37 .Retinoic acids as retinoid inducible transcription factors are involved in myeloid diff erentiation through nuclear receptor heterodimer (RXR/RARα) to modulate the expression of target genes.Molecular studies have identifi ed the fusion of the RARα gene to partner genes as the crucial event in APL pathogenesis [38][39][40] .APL has been found to be associated with reciprocal translocation involving the RARα gene on chromosome 17 that translocates and fuses to genes including promyeolocytic leukemia gene (PML), promyelocytic leukemia zinc fi nger gene, nucleophosmin gene, nuclear mitotic apparatus gene, and the Stat5b gene 3,37,41 .RARα usually fuses to the PML gene on chromosome 15 yielding the PML -RARα chimeric gene, which is expressed in all APL patients with t(15;17) 39,42 .Thus, chimeric receptors are responsible for development of leukemias by interfering with the functions of RARs, RXRs and PML expressed by the normal alleles present in the same cell 41 .In addition, it has been shown that PML -RARα is capable to form homodimer through the coiledcoil motif of PML and thus to compete with the RARα for binding to retinoic acid responsive element of target genes 39 .The all-trans retinoic acid-induced diff erentiation of cells is based on important fi ndings that it can cause a degradation of PML -RARα, induce dissociation of corepressors from PML -RARα 37 .All-trans retinoic acid (ATRA) eff ects are also accompanied by cell cycle arrest at G1 phase and inhibition of cell growth.Results based on in vitro studies enabled the treatment of APL patients with all-trans retinoic acid that caused the malignant cells to diff erentiate towards mature granulocytes, thus regaining most functions of their normal counterparts 39 .In the meantime, ATRA (Tretinoin, Vasanoid) was approved by the FDA for diff erentiation therapy of patients with acute promyelocytic leukemia and the successful application of all-trans retinoic acid for treatment of APL might be considered as a start of the retinoid renaissance period, since it rekindled interest in the use of retinoids or rexinoids for treatment of a variety of established malignancies.In addition, high concentration of ATRA is able to induce postmaturation apoptosis through the induction of the tumour-selective death ligand tumour necrosis factor-related apoptosis-inducing ligand TRAIL, also called Apo2L, a most promising molecule in cancer research 28,43,44 .

Retinoids or rexinoids in breast cancer chemoprevention
Breast cancer incidence, morbidity and mortality remain a major problem in the most of countries.Breast cancer development is associated with deregulation of cell growth and cell death.It has been shown that retinoids are able to inhibit mammary gland cancer in animal models and human breast cancer 36,[45][46][47] .They are eff ective inhibitors of breast cancer cells at the early stages of tumour progression and their eff ectiveness diminishes as tumours become more aggressive 48 .Retinoid receptors are expressed diff erently in normal and malignant epithelial cells and are critical for normal development 49 .Recently, we have also shown marked diff erences between nonlactating and postlactating mammary gland in rat.RARα, RXRα N-CoR, SMRT in rat were signifi cantly increased in postlactating mammary gland when compared to that of nonlactating mammary tissue.Postlactating mammary glands were found to express all RAR and RXR subtypes studied when compared to nonlactating mammary tissues that express exclusively RARα and RXRα subtypes.Enhanced expression of a number of nuclear hormone receptors, their coregulators in mammary tissue of postlactating rats in comparison with nonlactating animals identify a potential role for retinoid signalling pathways also after lactation period 50 .Growth inhibition of breast cancer cells by retinoic acid has been associated with induction of the expression of RARβ, which may act as a tumour suppressor and appears to be down-regulated in breast cancer tissue and cell lines, and conversely, up regulated in normal mammary epithelian cells 51 .9-cis retinoic acid, a high affi nity ligand for RXRs as well as LGD1069 (Targretin, Bexarotene) (Fig. 3), a synthetic RXR-selective ligand have been shown to have effi cacy superior to ATRA as a chemopreventive compound in the 1-methyl-1-nitrosourea (MNU)-induced rat mammary gland carcinoma model.Moreover, Targretin was very well tolerated during chronic therapy with no classic signs of "retinoid-associated" toxicities 52 .In animal mammary gland carcinoma models, additional promising retinoids are retinyl acetate or N-(4-hydroxyphenyl)retinamide (Fenretinide) 53 (Fig. 3).As mentioned before, lack of RARβ gene expression is a typical feature for human breast cancer and may be one of the major factors responsible for retinoid resistance in those neoplasias.Examination of the expression of all the subtypes of RARs and RXRs in a number of hormonedependent and hormone-independent breast cancer cells J. Brtko have shown similar expression of RARα, RARγ, RXRα and RXRβ.However, all-trans retinoic acid was found to be a strong inductor of the RARβ gene expression in several hormone-dependent breast cancer cell lines.Due to up-regulation of RARβ by all-trans retinoic acid or other retinoids, the expression of RARβ apparently plays a critical role in increasing retinoid sensitivity.These data indicate that the defi ciency of the RARβ expression and a defi cient responsiveness of retinoids via RARβ may account for the ineff ective treatment with retinoids in patients with advanced breast tumour 51 .The potency of retinoids to inhibit proliferation of breast cancer cells was seen when retinoids were administered either alone or in combination with antiestrogens.Tamoxifen and fenretinide combination therapy has been proven to be an active treatment regimen in metastatic breast cancer patients, however, not in estrogen receptor negative metastatic breast cancer or in patients whose disease had progressed on tamoxifen 54 .Retinoids do not require estrogen receptors for their action, they may aff ect neoplastic transformation in estrogen-negative cells, in contrast to tamoxifen, whose primary mechanism of action is through estrogen receptors.In spite of that, retinoids and rexinoids seem to be more active in estrogen positive than in estrogen negative precancerous tissue 55 .Furthermore, it is known that retinoids can promote apoptosis in breast cancer cells as well as in other types of neoplasias and that induction is predominantly mediated by RARβ 56 .In spite of a number of important fi ndings achieved recently by many prestigious laboratories, the precise mechanism(s) by which natural or synthetic retinoids inhibit breast cancer cell growth has not been completely elucidated.Great interest has been recently focused on rexinoids, when combined with selective estrogen receptor modulators (SERMs), they dramatically inhibit breast cancer cell growth inducing apoptosis even with intermittent administration 49 .

Retinoids in thyroid gland cancer treatment
Thyroid cancer is a relatively common malignancy.A minority of patients suff ers from poorly diff erentiated thyroid carcinoma that is unresponsive to radioiodine therapy.Rediff erentiation agents that "reprogram" these thyroid tumours in order to concentrate radioiodine would be of great value in treating patients with advanced thyroid cancer [57][58][59] .The biologically active retinoids or rexinoids are extensively studied from that aspect.In follicular thyroid cancer cells, 13-cis retinoic acid has been found to induce radioiodine avidity of cells formerly unable to accumulate radioiodine 60 .In human thyroid carcinoma cell lines, natural or synthetic retinoids were found to induce expression of type I iodothyronine-5'-deiodinase, sodium/ iodide-symporter, the thyroid diff erentiation markers [61][62][63] .However, approximately 30 % of the thyroid tumors may start to dediff erentiate and thus fi nally develop into highly malignant anaplastic thyroid carcinomas 64 .Non-operable thyroid follicular tumors, which due to loss of thyroid specifi c functions also fail to take up radioiodine, have been treated with 13-cis retinoic acid.The benefi cial outcome of this treatment was interpreted as partial rediff erentiation of thyroid cancer cells 64 .This eff ect markedly requires existence of functional nuclear retinoid/rexinoid receptors.The presence of high-affi nity and limited capacity of nuclear all-trans retinoic acid binding sites has been shown in follicular thyroid carcinoma cell lines 65 .The ability of retinoid receptors in thyroid carcinoma cell lines to bind to their responsive elements has been evaluated by electrophoretic mobility and supershift assays 66 .In clinical pilot studies patients with poorly diff erentiated, inoperable thyroid carcinoma and lacking or insuffi cient iodine uptake have been treated with oral doses of 1.0-1.5 mg 13-cis retinoic acid per kg body weight and day over 5 weeks.The results revealed an increased radioiodide uptake in 30 %-50 % of patients 64 .In a recent study a fl uorine-18 fl uorodeoxyglucose positron emission tomography ( 18 F-FDG PET) was used for the evaluation and monitoring of therapy with 13-cis retinoic acid in thyroid cancer.In 6 out of 21 patients, radioiodine accumulation was newly induced after 6-9 months, and in 3 patients a residual iodine accumulation was enhanced 67 .The data correspond well with fi ndings described previously indicating retinoids as promising therapeutic agents for the treatment of thyroid cancer 59 .

Possible use of retinoids or rexinoids in other organs cancer chemoprevention
Retinoids as previously reviewed 36 have been implicated in induction of cell death in many tumour-derived cultured cell systems or tumour tissues, e.g., neuroblastoma cells, ovarian cancer, cervical cancer cells, prostate cancer, head and neck squamous cell carcinoma, liver cancer, pancreatic cancer, lung cancer and many other types of carcinomas.Relationship between biologically active retinoids or rexinoids, which can act as eff ective cancer chemopreventive agents or potential chemotherapeutic compounds and the regulation of the processes such as cell diff erentiation and apoptosis is very complex and requires identifi cation of new target genes that might be involved in those processes.

CONCLUSION
A variety of biologically active retinoids or rexinoids has been tested on diff erent types of cancer in order to evaluate the precise mechanism of action.In a number of diff erent types of tumours both retinoids or rexinoids are capable to induce both apoptosis and diff erentiation, and they represent a potentially powerful alternative to present chemotherapeutic treatment of late stages of cancer 36 .
In conclusion, a number of novel chemical compounds, receptor selective retinoids and rexinoids, have been synthesized up to now and tested both in vitro and in vivo.In spite of that rapid progress novel synthetic retinoids and rexinoids with greater retinoid receptor selectivity, reduced teratogenic and other side eff ects are still highly required.

Table 2 .
Photoreceptor-specifi c nuclear receptor Chicken ovalbumin upstream promoter-transcription factor I Chicken ovalbumin upstream promoter-transcription factor II ErbA2-related gene-2 Estrogen receptor-related receptor α NGF-induced factor B Nur-related factor 1 Neuron-derived orphan receptor 1 Steroidogenic factor 1 Liver receptor homologous protein 1 Germ cell nuclear factor DSS-AHC critical region on the chromosome, gene 1 Short heterodimeric partner

Fig. 1 .
Fig. 1.Structural and functional organization of nuclear receptor specifi c domains

Table 1 .
Biologically active ligands and human nuclear receptors.