Dana Macejova

Expression of nuclear retinoic acid receptor in peripheral blood mononuclear cells (PBMC) of healthy subjects

In vertebrates, both nuclear all-trans and 9-cis retinoic acid receptors (RAR and RXR) belonging to the steroid/thyroid/retinoid nuclear receptor superfamily play a crucial role in the vitamin A action. Qualitative analysis of all known RAR or RXR subtypes in both pooled and non-pooled peripheral blood mononuclear cells (PBMC) from healthy human subjects has been performed by reverse transcription and polymerase chain reaction (RT-PCR). Our data, based on qualitative RT-PCR analysis has shown that human PBMC are capable to express RAR alpha, RAR gamma, RXR alpha, and RXR beta.

Expression, protein stability and transcriptional activity of retinoic acid receptors are affected by microtubules interfering agents and all-trans-retinoic acid in primary rat hepatocytes.

Cellular signaling by glucocorticoid receptor and aryl hydrocarbon receptor is restricted by microtubules interfering agents (MIAs). This leads to down-regulation of drug metabolizing enzymes and drug interactions. Here we investigated the effects of all-trans-retinoic acid (ATRA) and MIAs, i.e. colchicine, nocodazole and taxol on the regulation of retinoic acid receptor (RAR) genes in primary cultures of rat hepatocytes. ATRA (1microM) down-regulated RARalpha and RARgamma mRNAs (decrease 23% and 41%, respectively) whereas it up-regulated RARbeta mRNA (4.3-fold induction). All MIAs diminished the expression of RARs in dose-dependent manner; the potency of MIAs increased in order NOC<COL<TAX and the extent of inhibition increased in order RARalpha<RARgamma<RARbeta. The levels of RARalpha protein were decreased by both MIAs and ATRA. The effects of ATRA were reversed by proteasome inhibitor MG-132, implying ligand-dependent RARalpha degradation. In contrast, the effects of MIAs were proteasome-independent and decrease in RARalpha protein content was due to RARalpha gene down-regulation. We monitored transcriptional activity of RARalpha. For this purpose, we measured catalytic activity of trans-glutaminase-target gene of RARalpha. trans-Glutaminase activity was increased by ATRA (1.23-fold increase) and decreased by colchicine (decrease 51%). Co-treatment with proteasome inhibitor MG-132 partly reversed inhibitory effect of colchicine, and it further augmented the increase of trans-glutaminase activity by ATRA. We have also observed decrease of RARalpha protein level and inhibition of RARs mRNAs expression in HeLa cells by MIAs. In conclusion, our data indicate that microtubules play the role in regulation of RARs activity and expression. Our data are the first report on the effects of ATRA and MIAs on RARs regulation in quiescent cells.

MNU-induced carcinogenesis of rat mammary gland: Effect of thyroid hormone on expression of retinoic acid receptors in tumours of mammary gland

The rat model of N-methyl-N-nitrosourea (MNU)-induced mammary carcinomas is well-established animal model for breast cancer. This study was carried out to investigate whether hypothyroid (thyroidectomy or PTU treatment) or hyperthyroid status of female rats would affect MNU-induced mammary carcinogenesis with specific focus on both retinoid and rexinoid receptor expression in mammary tumours. Application of PTU before and during MNU-induced mammary gland carcinogenesis yielded in a marked decrease of the number and volume of tumours per animal, however, there was no effect of hypothyroid state in thyroidectomized rats as well as hyperthyroid state concerning the number and volume of tumours. Mammary tumours of in euthyroid group of MNU animals showed that there was no tumour, in which all of subtypes of retinoid and rexinoid receptors were expressed. A different pattern of expression of retinoid or rexinoid receptors was found either in MNU-induced mammary carcinomas in both hypothyroid and hyperthyroid rats.

Proteomic analysis of changes in the protein composition of MCF-7 human breast cancer cells induced by all-trans retinoic acid, 9-cis retinoic acid, and their combination.

Retinoic acid (all-trans and 9-cis) isomers represent important therapeutic agents for many types of cancers, including human breast cancer. Changes in protein composition of the MCF-7 human breast cancer cells were induced by all-trans retinoic acid, 9-cis retinoic acid, and their combination and subsequently proteomic strategies based on bottom-up method were applied. Proposed approach was used for the analysis of proteins extracted from MCF-7 human breast cancer cell line utilizing a commercially manufactured kit RIPA and separated on two dimensional (2D) sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) after treatment with both retinoic acid isomers. We found significant differences in occurrence of proteins probably affecting the cell migration process in tumour cells. Heat shock protein 27, ribonucleoprotein SmD3, and cofilin-1 were significantly upregulated after treatment with combination of individual retinoic acid isomers. On the other hand, AP-5 complex subunit beta-1 shows the different response. Thus, the results might help to find the answer to important medical questions on (i) the identification of signaling pathways affected by retinoic acid isomers or (ii) how the observed proteomic pattern might reflect the effectiveness of retinoic acids treatment.

Age-related change in the retinoid X receptor beta gene expression in peripheral blood mononuclear cells of healthy volunteers: Effect of 13-cis retinoic acid supplementation

The regulation of cell growth and differentiation and also expression of a number of genes by retinoids are mediated by nuclear retinoid receptors (RARs and/or RXRs). In this study we investigated age-related alteration in both RAR and RXR receptor subtypes gene expression and tissue transglutaminase (tTG) activity before and after supplementation with 13-cis retinoic acid (13cRA) in human peripheral blood mononuclear cells (PBMCs). Healthy men (40) were divided in two groups according to their age (young group: 26.1+/-4.1 years and old group: 65.4+/-3.8 years). Each volunteer received 13cRA (Curacné), 0.5mg/(kgday)) during a period of 4 weeks. We have shown that RXRbeta expression was decreased significantly (p=0.0108) in PBMCs of elderly men when compared to that of young volunteers. Distribution of retinoic acid receptor subtype expression in PBMCs was found in the order: RXRbeta>RARgamma>RXRalpha>RARalpha. The tTG activity in PBMCs reflected a trend to be enhanced after 13-cis retinoic acid supplementation. In conclusion, we demonstrate a significant decrease in the expression of RXRbeta subtype of rexinoid receptors in PBMCs of healthy elderly men. Our data suggest that in healthy elderly men reduction of RXRbeta expression in PBMCs might be a common feature of physiological senescence.

MNU-induced mammary gland carcinogenesis: Chemopreventive and therapeutic effects of vitamin D and Seocalcitol on selected regulatory vitamin D receptor pathways

The effects of administration of vitamin D₃ and Seocalcitol on MNU-induced carcinogenesis of mammary gland in Sprague-Dawley rats have been investigated. Administration of both substances in a weekly dose of 7 μg/kg caused prolonged latency of mammary gland tumors. The latency of tumors was markedly prolonged for 30-40 days by Seocalcitol. Using PET analysis, reduction in [¹⁸F]2-fluoro-2-deoxy-d-glucose (FDG) uptake or tumor volume in tumors chemopreventively treated with vitamin D₃ were detected in MNU-induced tumors, vitamin D₃ reduced expression of 25-hydroxylase (25OHase) (p<0.01) and 24-hydroxylase (24OHase) (p<0.01) and Seocalcitol 24OHase. Positive regulation of 25OHase mRNA level after the treatment with vitamin D₃ was observed in liver, while in kidney, vitamin D₃ and Seocalcitol induced expression of 24OHase was significant. Our observations indicate a cross talk between respective pathways of VDR, RARs/RXRs, TRs and ERs in carcinogenesis process.

Radioligand binding assay for accurate determination of nuclear retinoid X receptors: A case of triorganotin endocrine disrupting ligands

Nuclear 9-cis retinoic acid receptors (retinoid X receptors, RXR) are promiscuous dimerization partners for a number of nuclear receptors. In the present study, we established a novel in vitro method for quantitative determination of the nuclear retinoid X receptors in rat liver. One type of high affinity and limited capacity RXR specific binding sites with the Ka value ranging from 1.011 to 1.727×10(9)l/mol and the Bmax value ranging from 0.346 to 0.567pmol/mg, was demonstrated. Maximal 9-cis retinoic acid (9cRA) specific binding to nuclear retinoid X receptors was achieved at 20°C, and the optimal incubation time for the 9cRA-RXR complex formation was 120min. From a number of endocrine disruptors, tributyltins and triphenyltins are known as RXR ligands. Our data confirmed the property of tributyltin chloride or triphenyltin chloride to bind to a high affinity and limited capacity RXR binding sites. Described optimal conditions for ligand binding to RXR molecules enabled us to calculate maximal binding capacity (Bmax) and affinity (Ka) values. This study provides an original RXR radioligand binding assay that can be employed for investigation of novel RXR ligands that comprise both drugs and endocrine disruptors.

Agonistic effect of selected isoflavones on arylhydrocarbon receptor in a novel AZ-AhR transgenic gene reporter human cell line.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that controls the expression of a diverse set of genes. Structurally diverse compounds bind to AhR and act as AhR agonists. Well characterised family of natural AhR ligands are isoflavones, which are compounds found predominantly in soy beans or red clover. In this study we have examined agonistic effect of selected isoflavones (genistein, daidzein, biochanin A, formononetin and equol) on AhR in the novel transgenic gene reporter human cell line AZ-AhR, a stably transfected AhR-responsive cell line allowing rapid and sensitive assessment of AhR transcriptional activity. We demonstrated that biochanin A, formononetin and genistein at concentration 10(-4) mol/l exerted agonistic effects on AhR with fold activation of 309- fold, 108-fold and 27-fold, which is about 84.8%, 29.6% and 7.4%, respectively, of the value attained by 2,3,7,8-tetrachlorodibenzo-p-dioxin. Daidzein and equol did not show any significant effects on AhR.

Nuclear receptors - target molecules for isoflavones in cancer chemoprevention

Breast cancer is the most occurring type of cancer among women. In Slovakia, there are yearly diagnosed about 1900 new cases of this disease. Breast cancer treatment is very expensive, psychic stressful and in some cases ineffective. Therefore, it is essential to search for new and/or alternative methods for breast cancer treatment, since nuclear receptors are considered to be a central goal for maximizing treatment opportunities in breast cancer. Among natural ligands for estrogen receptors (ERα and ERβ), which are member of nuclear receptors superfamily, belongs also isoflavones. These natural compounds have similar structure to main female hormon-17β estradiol. A rich source of isoflavones is soy and its products. Three aglycones form of isoflavones (genistein, daidzein, glycitein) are predominantly found in soybean and red clover. Among other important isoflavones belongs also biochanin A and formononetin.

AT1 receptor and ACE mRNA are increased in chemically induced carcinoma of rat mammary gland

Angiotesin II has except of strong vasoconstrict effect also ability to potentiate protein synthesis and cellular growth. The aim of this study was to investigate the gene expression of components of the renin-angiotensin system, angiotensinogen, renin, angiotensin-converting enzyme and AT(1) receptor, in rat mammary gland and in chemically induced carcinoma of this gland. Retinoids are known to inhibit cell proliferation and induce cell differentiation so they are considered as a promising chemopreventive agents. We studied the effect of 13-cis-retinoic acid on the gene expression of mentioned elements of the renin-angiotensin system in tumour tissue. The expressions in control and carcinoma tissue were investigated using RT-PCR and activity of angiotensin-converting enzyme was measured. The amount of angiotensin-converting enzyme and AT(1) receptor mRNA and angiotensin-converting enzyme activity always showed a significant increase in the carcinoma tissue in comparison with the control. Administration of 13-cis-retinoic acid to rats with induced mammary gland carcinoma was without significant effect on either tumour numbers or tumour burden and volume. Similarly, 13-cis-retinoic acid did not change the angiotensin-converting enzyme expression and activity. The AT(1) receptor gene expression displayed a clear tendency to decrease in tumour tissue after retinoic acid treatment. Our results demonstrate the presence of angiotensin-converting enzyme and AT(1) receptor in control and carcinoma tissue of mammary gland. We assume that both proteins might play a role in development of tumour cells and vasculature.