Consequences of the natural retinoid/retinoid X receptor ligands action in human breast cancer MDA-MB-231 cell line: Focus on functional proteomics.

The main intention of this study was the investigation of impact of natural biologically active ligands of nuclear retinoid/retinoid X receptors (all-trans and 9-cis retinoic acid) on proteomic pattern in human estrogen receptor negative breast cancer cell line MDA-MB-231. For this purpose, proteomic strategies based on bottom-up method were applied. The total cell proteins were extracted utilizing a commercially Radio-Immunoprecipitation Assay (RIPA) buffer and separated on 2D sodium dodecyl sulfate polyacrylamide gel electrophoresis (2D SDS-PAGE). The proteins were subsequently digested in-gel by trypsin and their characterization was achieved by MALDI-TOF/TOF. By employing PDQuest™ software, we identified more than 50 proteins affected by retinoic acid isomers. For more information, 9 proteins which are associated with tumor process were selected. We determined that derivatives of retinoic acid led to significantly reduced level of proteins belonging to metabolic pathway (e.g. glyceraldehyde-3-phosphate dehydrogenase or pyruvate kinase 2) or to other cellular processes as apoptosis, regulation of transcription process or epithelial-mesenchymal transition (e.g. annexins, nucleoside diphosphate kinase B, vimentin). On the other hand all-trans retinoic acid treatment indicates up-regulated effect for heterogeneous nuclear ribonucleoprotein A2/B1.

The role of retinoic acid receptors and their cognate ligands in reproduction in a context of triorganotin based endocrine disrupting chemicals.

Retinoic acid (RA), an active form of vitamin A, regulates the embryonic development, male and female reproduction and induces important effects on the cell development, proliferation, and differentiation. These effects are mediated by the retinoid (RAR) and rexinoid nuclear receptors (RXR), which are considered to be a ligand-activated, DNA-binding, trans-acting, and transcription-modulating proteins, involved in a general molecular mechanism responsible for the transcriptional responses in target genes. Organotin compounds are typical environmental contaminants and suspected endocrine disrupting substances. They may affect processes of reproductive system in mammals, predominantly via nuclear receptor signaling pathways. Triorganotins, such as tributyltin chloride (TBTCl) and triphenyltin chloride (TPTCl), are capable to bind to RXR molecules, and thus represent potent agonists of RXR subtypes of nuclear receptors not sharing any structural characteristics with endogenous ligands of nuclear receptors. Th is article summarizes selected effects of biologically active retinoids and rexinoids on both male and female reproduction and also deals with the effects of organotin compounds evoking endocrine disrupting actions in reproduction.

Anticancer effects of tributyltin chloride and triphenyltin chloride in human breast cancer cell lines MCF-7 and MDA-MB-231.

Triorganotin compounds induce hormonal alterations, i.e., endocrine-disrupting effects in mammals, including humans. Tributyltin chloride (TBT-Cl) and triphenyltin chloride (TPT-Cl) are known to function as nuclear retinoid X receptor (RXR) agonists. Their cytotoxic effects in ER(+) luminal human breast cancer cell line MCF-7 and ER(-) basal-like human breast cancer cell line MDA-MB-231 were examined. We observed significantly higher toxicity of TBT-Cl in comparison with TPT-Cl in both cell lines. Comparable apoptosis-inducing concentrations were 200 and 800 nM, respectively, as shown by PARP cleavage and FDA staining. Both compounds activated executive caspases in the concentration-dependent manner in MDA-MB-231 cells, but the onset of TPT-Cl-induced caspase-3/7 activation was delayed in comparison with TBT-Cl. Both compounds slowed down the migration of these highly invasive cells, which was accompanied by RARbeta upregulation. Other RAR and RXR expressions were differentially modulated by studied organotins in both cell lines.

Triorganotin compounds--ligands for "rexinoid" inducible transcription factors: biological effects.

We review trialkyltin and triaryltin compounds, representing a class of organometallic compounds that function as nuclear retinoid X receptors (RXR) agonists due to their capability to bind to the ligand-binding domain of RXR subtypes and function as transcriptional activators. RXRs act predominantly as heterodimers with other nuclear receptors as permissive heterodimers with peroxisome proliferator-activated receptors, liver X receptors, farnesoid X receptor, pregnane X receptor and constitutive androstan receptor or as non-permissive heterodimer with vitamin D receptor, and as conditional heterodimers with retinoid receptors, and thyroid hormone receptors. RXR - "partner" receptor heterodimers are considered to be ligand-activated, DNA-binding, trans-acting, transcription-modulating proteins involved in a general molecular mechanism responsible for transcriptional responses in target genes. Tributyltin at even pico- or nanomolar concentrations may cause the superimposition of male genitalia on female in several aquatic organisms, since they are DNA-targeted, mitotic, and their actions are occurring through target gene(s)-mediated pathways. They may cause molecular interactions with reproductive system in mammals, and as potent environmental obesogens, they promote adipocyte differentiation. Organotin compounds become known also for their immunotoxicity, neurotoxicity, for their effects on reproduction and/or development. We also review effects of organotins with respect to levels and activities of hepatic P450s and aromatase activity.

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.

Effect of 9-cis retinoic acid and all-trans retinoic acid in combination with verapamil on P-glycoprotein expression in L1210 cells.

The development of the most common multidrug resistance (MDR) phenotype is associated with a massive overexpression of P-glycoprotein (P-gp) in neoplastic cells. In the current study, we used three L1210 cell variants: S cells - parental drug-sensitive cells; R cells - drug-resistant cells with P-gp overexpression due to selection with vincristine; T cells - drug-resistant cells with P-gp overexpression due to stable transfection with the pHaMDRwt plasmid, which encodes human full-length P-gp. Several authors have described the induction of P-gp expression/activity in malignant cell lines after treatment with all-trans retinoic acid (AtRA; ligand of retinoic acid nuclear receptors, RARs). An isomer of AtRA also exists, 9-cis retinoic acid, which is a ligand of both RARs and nuclear retinoid X receptors (RXRs). In a previous work, we described that the combined treatment of R cells with verapamil and AtRA induces the downregulation of P-gp expression/activity. In the current study, we studied the expression of RARs and RXRs in S, R and T cells and the effects of treatment with AtRA, 9cRA and verapamil on P-gp expression, cellular localization and efflux activity in R and T cells. We found that the overexpression of P-gp in L1210 cells is associated with several changes in the specific transcription of both subgroups of nuclear receptors, RARs and RXRs. We also demonstrated that treatment with AtRA, 9cRA and verapamil induces alterations in P-gp expression in R and T cells. Particularly, combined treatment of R cells with verapamil and AtRA induced downregulation of P-gp content/activity. In contrast, similar treatment of T cells induced slight increase of P-gp content without any changes in efflux activity of this protein. These findings indicate that active crosstalk between the RAR and RXR regulatory pathways and P-gp-mediated MDR could take place.

Effects of retinoic acid isomers on proteomic pattern in human breast cancer MCF-7 cell line.

Retinoids, acting through their cognate nuclear receptors, are crucial transcriptional regulators of many cellular processes such as differentiation, development, apoptosis, carbohydrate and lipid metabolism, homeostasis, etc. The aim of this study was the exploration of molecular mechanisms in relation to therapy of human breast cancer. One of the efficient strategies is identification of biomarkers as important tools in early cancer diagnosis and advisable treatment. Retinoids have been regarded as important therapeutic agents for many types of cancers, including human breast cancer. The effects of all-trans retinoic acid and 9-cis retinoic acid or their combination on proteomic pattern in human MCF-7 breast cancer line were investigated. The total cell proteins were extracted utilizing a commercially Radio-Immunoprecipitation Assay (RIPA) buffer and separated on 1D sodium dodecyl sulfate polyacrylamide gel electrophoresis (1D SDS-PAGE). The proteins were subsequently digested in-gel by trypsin and identified by matrix assisted laser desorption ionization technique with time of flight mass analyzer (MALDI-TOF/TOF). Our data offer novel information on the proteomic pattern of proteins evaluated after treatment of MCF-7 cells with retinoic acid isomers.

Morphology of 1-methyl-1-nitrosourea induced rat mammary tumours after treatment with precursor of phytanic acid or its combination with vitamin D analogue.

OBJECTIVE: The proposed therapeutical effect of phytol (PHY), a precursor of the phytanic acid (PHYA), on mammary tumours induced with 1-methyl-1-nitrosourea (MNU), was investigated in Sprague-Dawley rats in combination with vitamin D analogue, Seocalcitol (SEO). METHODS: Female Sprague-Dawley rats were administered intraperitoneally with MNU (50 mg/kg of body weight) at the 46th and 52th days of age. Controls and MNU animals received propyleneglycol appropriate to their body weight. PHY (MNU + PHY) (500 mg/kg) was administered after tumour detection (approximately in 100th day of the life) three times/week. Combination of PHY with SEO (7 µg/kg per week) was administered to rats after tumour detection (approximately in 100th day of the life) until the 181st day of age. Then the animals were sacrificed, the tumours removed, and fixed in 10% formalin. Haematoxylin and eosine stained sections were evaluated under microscope. RESULTS: Tumour invasiveness observed in all groups of animals was ranging from 80 to 90%. Treatment with PHY alone did not inhibit the progression of the MNU induced tumours in the rat breast but it decreased the tumour burden and volume in comparison with MNU treated controls. Decreased tumour burden and volume were induced by combined treatment of PHY with SEO. Malignity and invasivity of carcinomas were not affected. CONCLUSION: No redifferentiating effect on mammary tumour cells induced by NMU after treatment with PHY alone or in combination with SEO was observed in rats. SEO alone or in combination with PHY inhibited the progression of MNU induced mammary tumours and also inhibited the increase of tumour burden and volume in comparison with MNU treated control group. However, none of the compounds, either alone or in mutual combination, reduced the malignity or the number of invasive tumours in this experimental study.

Detection of circulating tumor cells in metastatic breast cancer patients.

OBJECTIVE: The objective of this study was the detection of circulating tumor cells (CTCs) in metastatic breast cancer patients. METHODS: Since only small numbers of circulating tumor cells (CTCs) are found in peripheral blood, at first we performed immunomagnetic separation as a concentration method suitable for selecting circulating tumor cells in peripheral blood. This was followed by analysis of isolated cells with the aid of laser scanning cytometry (LSC). Twenty eight patients with metastatic breast cancer were enrolled in the study and the control group consisted of 19 clinically healthy women. Six milliliters of peripheral blood was drawn for the analyses, but only in two patients the blood has been drawn twice. Blood samples were taken when no chemotherapy was administered, but hormonal therapy has been allowed. RESULTS: The positivity for CTCs was found in 20 (50.0 %) patients with metastatic breast cancer patients, while in 6 (31.6 %) healthy controls false positive circulating epithelial-like cells were detected. Because we did not use CD45 staining, we could not distinguish these circulating epithelial-like cells from CTCs. In a majority of metastatic breast cancer patients we found a mixed population of HER-2 gene expressing CTCs. We found that HER2+ CTCs in high numbers are CK19 + CTCs, while almost all HER2-CTCs are CK19- CTCs. CONCLUSION: The described method was found promising for estimating HER2 status on CTCs from peripheral blood in metastatic breast cancer patients.

Short term 13-cis-retinoic acid treatment at therapeutic doses elevates expression of leptin, GLUT4, PPARgamma and aP2 in rat adipose tissue.

Temporary defects in the plasma lipid and glucose homeostasis are frequent complication accompanying chronic treatment with 13-cis-retinoic acid (13cRA). White adipose tissue acts as an endocrine organ producing a variety of hormones (adipocytokines) including leptin, adiponectin, tumor-necrosis factor alpha (TNFalpha) and angiotensin II (Ang II), which influence lipid metabolism, systemic insulin sensitivity and inflammation. To study the effect of a short-term 13cRA administration on metabolism of epididymal fat tissue, we treated Wistar rats with five identical therapeutic doses of 13cRA (0.8 mg/kg b.w.) by gavage during a period of 10 days. Expression of adiponectin, leptin, TNFalpha and selected proteins such as adipocyte fatty acid binding protein (aP2), insulin-dependent glucose transporter GLUT4, peroxisome proliferator-activated receptor gamma (PPARgamma) and retinoid X receptors (RXRs) was investigated using RT-PCR. Short-term treatment with therapeutic doses of 13cRA caused significant increase of the aP2, PPARgamma and moderately RXRalpha gene expression. Similarly, the relative amount of mRNA for leptin and GLUT4 was increased, while the TNFa transcript was decreased after treatment with 13cRA. The gene expression and plasma concentration of adiponectin were without any significant changes. Since local adipose renin-angiotensin system (RAS) has been presumed to be involved in the regulation of fat tissue metabolism, we also investigated the gene expression of RAS components in epididymal fat depot. Our data has shown that 13cRA elevated Ang II receptor type 1 (AT(1) receptor)--at both, mRNA and protein level. Thus, our results demonstrate that short-term 13cRA treatment is inducing alterations in fat tissue metabolism in relation to stimulated adipogenesis.

The effect of all-trans retinoic acid and/or colchicine on expression of rexinoid and thyroid hormone nuclear receptors and their coregulators in primary rat hepatocytes.

In the present work, the effects of colchicine (COL) and/or all-trans retinoic acid (ATRA) on expression of rexinoid receptors (RXRs) (alpha, beta, gamma), thyroid hormone receptor alpha and coregulators N-CoR, SMRT and SRC-1 mRNA in primary rat hepatocytes as a model of no-proliferating cells were investigated. Treatment with these components, either alone or in combination, induced differences of the expression profiles between distinct treatment groups.

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.

[Mechanisms of the intracellular activities of thyroid gland hormones]. / Mechanizmus vnútrobunkového pôsobenia hormónov stitnej zl'azy.

Function of thyroid hormones affecting broad spectrum of various biochemical and molecular biology reactions in organisms is unimaginable without fully functional nuclear receptors. On account of this fact, research on the role and function of thyroid hormone receptors that play a role as thyroid hormone inducible transcription factors, belongs to dynamically developing branches of molecular endocrinology. In organism, full functionality of thyroid hormone receptors in the form of heterodimer with nuclear 9-cis retinoic acid receptor is essential for biological effects of 3,5,3'-triiodothyronine.

Role of dihydroxyvitamin D(3) and its nuclear receptor in novel directed therapies for cancer.

Dihydroxyvitamin D(3) is known to affect broad spectrum of various biochemical and molecular biological reactions in organisms. Research on the role and function of nuclear vitamin D(3) receptors (VDR) playing a role as dihydroxyvitamin D(3) inducible transcription factor belongs to dynamically developing branches of molecular endocrinology. In higher organisms, full functionality of VDR in the form of heterodimer with nuclear 9-cis retinoic acid receptor is essential for biological effects of dihydroxyvitamin D(3). This article summarizes selected effects of biologically active vitamin D(3) acting through their cognate nuclear receptors, and also its potential use in therapy and prevention of various types of cancer.

Distinct modulation of a gene expression of the type 1 and 2 IP(3) receptors by retinoic acid in brain areas.

Inositol 1,4,5-trisphosphate (IP(3)) receptors belong to the intracellular calcium channels that release calcium from the intracellular stores after binding IP(3). Three types of IP(3) receptors occurred in a tissue specific manner and different promoters direct their gene expression. Thus, understanding of the transcriptional regulation is the first step towards comprehension of the function of these receptors. Since the retinoic acid activates RARE and AP2 transcription factors, the present study focuses on determination of whether or not expression of type 1 and 2 IP(3) receptors is modulated by retinoic acid in selected brain areas. We have found that mRNA levels of the type 1 IP(3) receptors were decreased significantly in cerebellum and hypothalamus, but not in the brain stem of rats treated with retinoic acid, compared to untreated littermates. The mRNA levels of the type 2 IP(3) receptor were significantly decreased in all tested tissues, cerebellum, hypothalamus, and also in brain stem after the treatment with retinoic acid. These results show that gene expression of both type 1 and 2 IP(3) receptors is regulated by retinoic acid, although the effect of retinoic acid on mRNA levels of the type 1 IP(3) receptors is dependent on brain area.

Thyroid tumors: histological classification and genetic factors involved in the development of thyroid cancer.

Classification of thyroid tumours and their variants is described with special respect to some recent findings on somatic mutations characteristics which are associated with individual types of malignity. Special attention is paid to the interrelations between thyroid nodules and malignity and predictive risk factors are listed.

1alpha,25-dihydroxyvitamin D3 inducible transcription factor and its role in the vitamin D action.

Vitamin D is considered multifunctional steroid hormone that modulates calcium homeostasis through actions predominantly in kidney, bone and the intestinal tract. Nuclear vitamin D receptor (VDR) is a specific nuclear protein, a member of steroid hormone receptor superfamily. The amino acid sequence of the VDR shows a significant homology with other members of the nuclear hormone receptor superfamily, including receptors for glucocorticoids (GR), oestrogen (ER), androgen (AR), progesteron (PR), thyroid hormone (T3R), retinoic acid (RAR), retinoid X (RXR) and over 150 orphan receptors. VDR is known to mediate the pleiotropic biological actions of 1a,25-dihydroxyvitamin D3 through its ability to modulate the expression of target genes. VDR upon binding 1a,25-dihydroxyvitamin D3 regulates specific gene transcription predominantly by binding as a heterodimer with the retinoid X receptor (RXR) to DNA enhancer sequence, termed the vitamin D-responsive element (VDRE) that is present within the promoter region of vitamin D-controlled genes. The VDR has been shown to associate with several additional molecules to form the active transcriptional complex required for gene regulation. The regulation of this ligand-activated cellular transcription factor occurs at both transcriptional and posttranslational levels. This article summarizes a variety of effects of 1a,25-dihydroxyvitamin D3, acting through its cognate nuclear receptor, and its use in chemotherapy and chemoprevention of cancer.

Kojic acid and its derivatives: history and present state of art.

Kojic acid (5-hydroxy-2-hydroxymethyl-4-pyranone) represents an attractive polyfunctional skeleton for development of biologically active compounds. The authors prepared a great variety of kojic acid derivatives and selected biological properties have been studied. Thus, kojic acid derivatives are promising compounds that might advantageously be used in human and/or veterinary medicine and also in preparation of new, even more biologically active preparations.

Effects of kojic acid on oxidative damage and on iron and trace element level in iron-overloaded mice and rats.

Since members of hydroxypyrone series posses iron chelating properties, kojic acid (KA), 5-hydroxy-2-(hydroxymethyl)-4H-pyran-one, a fungal metabolite of natural origin, has been suggested to might play a role in iron-overload diseases and in oxidative stress conditions involving transition metal. In our experiments in vivo models of iron-overload were used to study iron-chelating properties of KA and its effect on oxidative damage in mice and rats. The treatment of iron-preloaded rats (25 mg Fe x kg(-1) b.w., i.p., daily for five days) with 0.5% KA in drinking water for four weeks did not lower the iron concentration accumulated in the liver, neither diminished the induced hepatic lipid peroxidation in iron-loaded rats. The GSH level decreased in KA-treated group. Similarly, in iron-loaded mice model experiment, the following oral treatment with KA (100 mg x kg(-1)) daily for 7 days did not decrease the level of Fe accumulated in the liver and the lipid peroxidation even enhanced after KA treatment. Though in our experiments in vivo the ability of kojic acid to affect iron kinetics in the organism could not be proved, kojic acid as a molecule of natural origin may serve as a template for the preparation of new biologically active derivatives possessing capability of chelating iron.