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.

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.

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.

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.

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.

Retinoic acid increased expression of the Na+/Ca2+ exchanger in the heart and brain.

The Na+/Ca2+ exchanger (NCX) is an important calcium transport system, which regulates intracellular calcium homeostasis. In particular, NCX is highly expressed in the plasma membrane of excitable neuronal and cardiac cells. We report here that binding of retinoic acid enhances expression of the NCX1 in the rat cardiac atria, brain stem and hypothalamus, but not in cerebellum. Differences in the regulation of the NCX1 by retinoic acid might suggest that GATA4-retinoic acid inducible transcription factor is activated in the hypothalamus and brain stem, but not in the cerebellum. Our results support the idea that inducible transcription factors play an important role in the fine-tuning of local tissue calcium homeostasis.

Analysis of altered gene expression profiles in retinoic acid or CpG-treated Sprague-Dawley rats with MNU-induced mammary adenocarcinoma by cDNA macro array.

In the present work the effects of 13-cis retinoic acid (RA) and CpG-containing oligodeoxynucleotides (CpG-ODN) on the gene expression profile of spleen and tumor tissue in a MNU-induced mammary gland carcinoma ratmodel were investigated by the use of a commercial cDNA macro array (Atlas rat toxicology array 1.2, Clontech). Treatment with these components, either alone or in combination, induced differences of the expression profiles between the distinct treatment groups in both tissues. The large number of genes with altered expression (> 200) points to a highly complex process in vivo.

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.

Reduction of 1-methyl-1-nitrosourea-induced tumor burden with DNA vaccines encoding mutated ras epitopes and the costimulatory molecule B7.1.

1-methyl-1-nitrosourea (MNU), a well characterized carcinogen, was used to induce adenocarcinomas in rat mammary gland. 150 days after the first injection of MNU, the animals were treated with DNA minigene vaccines encoding ras T cell epitopes together with the co-stimulatory molecule B7.1 (CD 80). Five injections with a biolistic device (gene gun) in monthly intervals significantly reduced the tumor burden. A therapeutic effect could be measured with both, DNA vaccines encoding ras epitopes and B7.1, as well as with a DNA vaccine expressing solely the B7.1 molecule thus indicating the potential of genetic vaccination for turnor treatment.

Transglutamines and endocrine system (minireview).

Transglutaminases catalyze the posttranslation modification of proteins by catalyzing Ca2+ dependent acyl-transfer reaction resulting in the formation of new g-amide bonds between g-carboxamide groups of peptide-bound glutamine residues and various primary amines. Such glutamine residue serves as acyl-donor and the most common acyl-acceptors are e-amino groups of peptide-bound lysine residues or primary amino groups of some naturally occurring polyamines, like putrescine or spermidine. The active site of cysteine reacts first with the g-carboxamide group of glutamine residue to form the acyl-enzyme intermediate under the release of ammonia. In the second step, the complex reacts with a primary amine to form an isopeptide bond and liberate the reactivated enzyme. The presence of transglutaminases has been observed in various endocrine glands such as human pituitary, which was investigated by immunohistochemical methods using specific antibodies. A significant increase in the expression and activity of tissue transglutaminase was observed during involution of thymus. In the genital tract of the male rat two different forms of the enzyme transglutaminase could be identified and characterized. the presence of p53 and tissues transglutaminase gene expressions in human normal and pathologic adrenal tissues. The Ca2+-responsive enzyme transglutaminase, which catalyzes the cross-bridging of proteins, was found in pancreatic islet cells.

Effect of cadmium and mercury on the nuclear retinoic acid receptors.

The actions of retinoic acids (RA) are mediated by their cognate nuclear receptors--ligand inducible transcription factors (retinoic acid receptors (RAR)). Possible interactions of toxic heavy metals on the RAR system are of interest due to involvement of the RAR system in multiple systemic processes. We assayed cadmium chloride and mercury chloride for their influence on the RAR system in rat and in cell culture. Mercury chloride was observed to decrease the maximal binding capacity in vitro of RARs for all-trans RA in liver nuclear fraction containing sets of nuclear receptors by seventy percent at a concentration of 0.1 mmol/l, though not cadmium chloride. Neither mercury chloride nor cadmium chloride induced any changes with respect to mRNA levels of RAR and binding properties of nuclear receptor fraction for RA or retinoic acid responsive elements (RARE) in male Wistar rats receiving tap water with cadmium chloride (9.7 mg/l) or mercury chloride (11.5 mg/l) for six weeks. In rat pituitary GH4C1 cells, neither mRNA levels nor binding properties for RARE in cell culture were affected by non-toxic concentrations of these heavy metals. From the data obtained it is suggested that, in vivo, cadmium or mercury have no significant impact on RA nuclear receptor system.

Chemically induced carcinogenesis: a comparison of 1-methyl-1-nitrosourea, 7,12-dimethylbenzanthracene,diethylnitroso-amine and azoxymethan models (minireview).

The induction of mammary gland and other organ tumours by selected chemical carcinogens, 1-methyl-1-nitrosourea, 7,12-dimethylbenzanthracene, diethylnitrosoamine and azoxymethane is described and their application in experimental carcinogenesis research is discussed.

Mammary gland carcinoma-related increase of type I iodothyronine 5'-deiodinase activity in Sprague-Dawley rats.

Type I, iodothyronine 5'-deiodinase (5'-DI) catalyses deiodination of the prohormone thyroxine (T4) to the metabolically active 3,5,3'-triiodo-L-thyronine (T3). The present study was undertaken to investigate the activity of 5'-DI in rat mammary gland tumours representing various combinations of histologically defined papillary, cribriform or comedo patterns of ductal carcinomas. Female Sprague-Dawley rats were given two doses 50 mg x kg(-1) 1-methyl-1-nitrosourea (MNU) in abdominal parts on the 52nd day and 113th day of age. We have found that in comparison with non-lactating mammary gland, the activity of 5'-DI in all mammary gland tumours studied was significantly (p < 0.0001) increased and that the 5'-DI activity, expressed as pmol of 125I- released per min and per mg of protein, in malignant mammary gland tumours was found to be at least two order higher than that of intact mammary non-lactating gland. From our data, we suggest that thyroid hormone in mammary gland tumours might play a significant role to support high energetic expenditure of neoplastic tissues.