-
Journal of Chemical Information and... Sep 2011We provide a detailed description of the cis-trans isomerization of 4-hydroxytamoxifen/endoxifen catalyzed by several isoforms from the cytochrome P450 (CYP)...
We provide a detailed description of the cis-trans isomerization of 4-hydroxytamoxifen/endoxifen catalyzed by several isoforms from the cytochrome P450 (CYP) superfamily, including CYP1B1, CYP2B6, and CYP2C19. We show that the reactions mainly involve redox processes catalyzed by CYP. DFT calculation results strongly suggest that the isomerization occurs via a cationic intermediate. The cationic cis-isomer is more than 3 kcal/mol more stable than the trans form, resulting in an easier conversion from trans-to-cis than cis-to-trans. The cis-trans isomerization is a rarely reported CYP reaction and is ascribed to the lack of a second abstractable proton on the ethenyl group of the triarylvinyl class of substrates. The cationic intermediates thus formed instead of the stable dehydrogenation products allow for isomerization to occur. As a comparison, the reactions for the tamoxifen derivatives are compared to those of other substrates, 4-hydroxyacetanilide and raloxifene, for which the stable dehydrogenation products are formed.
Topics: Biocatalysis; Cytochrome P-450 Enzyme System; Estrogen Antagonists; Isomerism; Oxidation-Reduction; Tamoxifen
PubMed: 21870861
DOI: 10.1021/ci2001082 -
Journal of Pharmaceutical and... Mar 2013A highly sensitive HPLC-UV method for the simultaneous determination of tamoxifen, N-desmethyltamoxifen, 4-hydroxytamoxifen and endoxifen in human plasma samples was...
A highly sensitive HPLC-UV method for the simultaneous determination of tamoxifen, N-desmethyltamoxifen, 4-hydroxytamoxifen and endoxifen in human plasma samples was developed and validated. The method employs a two step liquid-liquid extraction and a reversed phase separation on a Hypersil Gold(®) C18 column (150mm×4.6mm, 5μm) with isocratic elution. Mobile phase was a mixture of triethylammonium phosphate buffer 5mM pH 3.3 and acetonitrile (57:43, v/v). Total analytical run time was 16min. Precision assays showed CV % lower than 10.53% and accuracy in the range of 93.0-104.2%. The lower limits of quantification (0.75-8.5ngml(-1)) are adequate to measure clinically relevant concentrations in plasma samples. The method was successfully applied to 110 clinical plasma samples. Median plasma levels and interquartile range were: tamoxifen 55.77ngml(-1) (38.42-83.69ngml(-1)), N-desmethyltamoxifen 124.83ngml(-1) (86.81-204.80ngml(-1)), 4-hydroxytamoxifen 1.09ngml(-1) (0.76-1.53ngml(-1)) and endoxifen 6.18ngml(-1) (4.17-8.22ngml(-1)). The procedure has adequate analytical performance and can be employed in therapeutic drug monitoring of tamoxifen or pharmacokinetics studies.
Topics: Antineoplastic Agents, Hormonal; Chromatography, High Pressure Liquid; Drug Monitoring; Female; Humans; Limit of Detection; Sensitivity and Specificity; Tamoxifen
PubMed: 23291438
DOI: 10.1016/j.jpba.2012.12.005 -
Nanoscale Aug 2019In eukaryotic cells, each process, in which DNA is involved, should take place in the context of a chromatin structure. DNA double-strand breaks (DSBs) are one of the...
In eukaryotic cells, each process, in which DNA is involved, should take place in the context of a chromatin structure. DNA double-strand breaks (DSBs) are one of the most deleterious lesions often leading to chromosomal rearrangement. In response to environmental stresses, cells have developed repair mechanisms to eliminate the DSBs. Upon DSB induction, several factors play roles in chromatin relaxation by catalysing the appropriate histone posttranslational modification (PTM) steps, therefore promoting the access of the repair factors to the DSBs. Among these PTMs, the phosphorylation of the histone variant H2AX at its Ser139 residue (also known as γH2AX) could be observed at the break sites. The structure of a DNA double-strand break induced repair focus has to be organized during the repair as it contributes to the accessibility of specific repair proteins to the damaged site. Our aim was to develop a quantitative approach to analyse the morphology of single repair foci by super-resolution dSTORM microscopy to gain insight into chromatin organization in DNA repair. We have established a specific dSTORM measurement process by developing a new analytical algorithm for gaining quantitative information about chromatin morphology and repair foci topology at an individual γH2AX enriched repair focus. Using this method we quantified single repair foci to show the distribution of γH2AX. The image of individual γH2AX referred to as the Single target Molecule response scatter Plot (SMPlot) was obtained by using high lateral resolution dSTORM images. Determination of the average localization numbers in an SMPlot was one of the key steps of quantitative dSTORM. A repair focus is made up of nanofoci. Such a substructure of repair foci can only be resolved and detected with super-resolution microscopy. Determination of the number of γH2AXs in the nanofoci was another key step of quantitative dSTORM. Additionally, based on our new analysis method, we were able to show the number of nucleosomes in each nanofocus that could allow us to define the possible chromatin structure and the nucleosome density around the break sites. This method is one of the first demonstrations of a single-cell based quantitative measurement of a discrete repair focus, which could provide new opportunities to categorize the spatial organization of nanofoci by parametric determination of topological similarity.
Topics: Algorithms; Cell Line, Tumor; Cell Nucleus; Chromatin; DNA Breaks, Double-Stranded; DNA Repair; Histones; Humans; Microscopy; Tamoxifen
PubMed: 31317161
DOI: 10.1039/c9nr03696b -
Journal of Biochemistry Jan 2018Endocrine therapy using antiestrogens and aromatase inhibitors is usually efficient to treat patients with hormone-sensitive breast cancer. Many patients with endocrine...
Endocrine therapy using antiestrogens and aromatase inhibitors is usually efficient to treat patients with hormone-sensitive breast cancer. Many patients with endocrine therapy, however, often acquire resistance. In the present study, we performed functional screening using short hairpin RNA library to dissect genes involved in antiestrogen tamoxifen resistance in MCF-7 breast cancer cells. We identified seven candidate genes that are associated with poor prognosis of breast cancer patients based on clinical dataset. The expression levels of six out of seven genes were higher in 4-hydroxytamoxifen (OHT) resistant MCF-7 (OHTR) cells compared with parental MCF-7 cells. Among the six selected genes, siRNA-mediated knockdown of PSMD1 and TSPAN12 markedly reduced the proliferation of OHTR cells. Notably, the knockdown of proteasome 26S subunit PSMD1 exhibited cell cycle arrest and the accumulation of p53 protein through inhibiting p53 protein degradation. In accordance with p53 accumulation, its target genes p21 and SFN were also upregulated by PSMD1 silencing. Taken together, PSMD1 was identified as a potential gene that plays a role in the development of tamoxifen resistance in breast cancer cells. These findings will provide a new insight for the mechanism underlying endocrine therapy resistance and a prognostic and therapeutic molecular target for advanced breast cancer.
Topics: Breast Neoplasms; Cell Proliferation; Female; Humans; MCF-7 Cells; Proteasome Endopeptidase Complex; Tamoxifen; Tumor Cells, Cultured; Tumor Suppressor Protein p53
PubMed: 28992264
DOI: 10.1093/jb/mvx053 -
Prolonged tamoxifen-enriched diet is associated with cardiomyopathy and nutritional frailty in mice.Experimental Physiology Apr 2024Tamoxifen (TAM) is required for gene recombination in the inducible Cre/lox system. The TAM-enriched diet is considered safe, with negligible impact on animal wellbeing....
Tamoxifen (TAM) is required for gene recombination in the inducible Cre/lox system. The TAM-enriched diet is considered safe, with negligible impact on animal wellbeing. However, studies reporting the long-term effects of the TAM diet and its potential impact on experimental outcomes are scarce. We conducted a longitudinal study on mice exposed to a 4-week dietary TAM citrate supplementation. Several parameters were recorded, such as body weight, body composition, mortality, and cardiac function. The collagen1a2 (Col1a2) transgenic mouse was used to assess TAM-induced recombination in vivo in cardiac fibroblasts followed by myocardial infarction (MI). The impact of TAM on the MI outcome was also evaluated. The recombination efficiency and cytotoxic effect of the TAM active metabolite, 4-hydroxy-tamoxifen (4-OHT), were assessed in vitro. Mice exposed to a TAM diet showed body weight loss and a 10% increase in mortality (P = 0.045). The TAM diet decreased cardiac function and induced cardiac remodeling, indicated by decreased fractional shortening from 32.23% to 19.23% (P = 0.001) and left ventricular (LV) wall thinning. All measured parameters were reversed to normal when mice were returned to a normal diet. Infarcted Col1a2-CreER mice on the TAM regimen showed gene recombination in fibroblasts, but it was associated with a substantial increase in mortality post-surgery (2.5-fold) compared to the controls. In vitro, 4-OHT induced gene editing in fibroblasts; however, cell growth arrest and cytotoxicity were observed at high concentrations. In conclusion, prolonged exposure to the TAM diet can be detrimental and necessitates careful model selection and interpretation of the results.
Topics: Mice; Animals; Frailty; Longitudinal Studies; Tamoxifen; Mice, Transgenic; Cardiomyopathies; Diet
PubMed: 38291801
DOI: 10.1113/EP091668 -
Pathologie-biologie Jan 1991The equilibrium binding kinetics of the interaction between the estrogen receptor and natural estrogens (estradiol, estriol and estrone), non-steroidal estrogen agonists... (Review)
Review
The equilibrium binding kinetics of the interaction between the estrogen receptor and natural estrogens (estradiol, estriol and estrone), non-steroidal estrogen agonists (11 beta-chloromethyl-estradiol-17 beta, diethyl-stilbestrol, hexestrol) and non-steroidal antiestrogens (clomiphene, tamoxifen) have been characterized. It is proposed that positive cooperative binding of ligands by the estrogen receptor reflects conformational changes in the DNA binding domain of the receptor dimer which increase its affinity to estrogen responsive elements. Weak estrogens fail to induce maximal cooperativity and are less efficient in activating the receptor complex. Antiestrogens, that inhibit the [3H]estradiol-induced cooperative binding, suppress the activation of the receptor and inhibit its nuclear interactions. Another class of antiestrogens (e.g., 4-hydroxytamoxifen) interacts with the receptor in a manner that is indistinguishable from the cooperative interaction of estradiol, and the resulting complex may also exhibit increased affinity for estrogen responsive elements. However, these complexes cannot activate transcription, presumably due to an aberrant induction of transcription-activating domain in the receptor. We suggest that the positive cooperativity of the estrogen receptor results from conformational changes in the receptor that are transmitted also to the DNA binding domain. On the other hand, conformational changes in the transcription activating domain are not revealed by equilibrium binding kinetics. Thus, compounds that block the positive cooperative binding of [3H]estradiol by the receptor act as antiestrogens. Other compounds that interact cooperatively with the receptor can activate the receptor DNA binding domain, however, they may or may not induce the full array of conformational changes required for transactivation of transcription.
Topics: Clomiphene; Drug Interactions; Estradiol; Estriol; Estrogen Antagonists; Estrone; Humans; Receptors, Estrogen; Tamoxifen
PubMed: 2011412
DOI: No ID Found -
Molecular and Cellular Endocrinology Apr 2004Estrogen-related receptor gamma (ERRgamma) is an orphan nuclear receptor lacking identified natural ligands. We have addressed the requirements for ERRgamma-mediated...
Estrogen-related receptor gamma (ERRgamma) is an orphan nuclear receptor lacking identified natural ligands. We have addressed the requirements for ERRgamma-mediated gene regulation. ERRgamma transactivates constitutively reporter genes driven by ERR response elements (ERREs) or estrogen response elements (EREs). The activation depends on an intact DNA-binding domain (DBD) and activation function-2 (AF2). ERRgamma-mediated transactivation is further enhanced by peroxisome proliferator-activated receptor coactivator-1. Interestingly, ligand-binding domain (LBD) mutations predicted to either enlarge or diminish the putative ligand-binding pocket have no effect on the transcriptional activity implying that ERRgamma activity does not depend on any ligands. Antiestrogens 4OH-tamoxifen (4OHT) and 4-hydroxytoremifene (4OHtor) inhibit the ability of ERR to transactivate ERRE and ERE reporters. In contrast, ERRgamma activates transcription at AP-1 sites in the presence of 4OHT and 4OHtor. Thus, the transcriptional activity of ERRgamma seems not to require ligand binding but is modulated by binding of certain small synthetic ligands.
Topics: Binding Sites; Cell Line; DNA-Binding Proteins; Genes, Reporter; Humans; Luciferases; Point Mutation; Protein Binding; Protein Structure, Secondary; Receptors, Cytoplasmic and Nuclear; Receptors, Estrogen; Tamoxifen; Toremifene; Transcription Factors; Transcriptional Activation
PubMed: 15149736
DOI: 10.1016/j.mce.2004.01.002 -
Science (New York, N.Y.) Jul 2002The transcription factor Pax5 is essential for initiating B cell lineage commitment, but its role in maintaining commitment is unknown. Using conditional Pax5...
The transcription factor Pax5 is essential for initiating B cell lineage commitment, but its role in maintaining commitment is unknown. Using conditional Pax5 inactivation in committed pro-B cells, we demonstrate that Pax5 is required not only to initiate its B lymphoid transcription program, but also to maintain it in early B cell development. As a consequence of Pax5 inactivation, previously committed pro-B cells regained the capacity to differentiate into macrophages in vitro and to reconstitute T cell development in vivo in RAG2-/- mice. Hence, Pax5 expression is continuously required to maintain B cell lineage commitment, because its loss converts committed pro-B cells into hematopoietic progenitors with multilineage potential.
Topics: Animals; Antigens, CD19; B-Lymphocytes; Cell Differentiation; Cell Lineage; Cells, Cultured; DNA-Binding Proteins; Gene Expression Profiling; Gene Expression Regulation; Gene Silencing; Hematopoietic Stem Cells; Macrophages; Mice; Mice, Inbred C57BL; Mice, Transgenic; PAX5 Transcription Factor; T-Lymphocytes; Tamoxifen; Transcription Factors; Transcription, Genetic
PubMed: 12098702
DOI: 10.1126/science.1067518 -
FEBS Letters Jun 2002We investigated the effect of estrogens on heart mitochondrial functions and whether estrogens can prevent calcium-induced release of cytochrome c from mitochondria. 10...
We investigated the effect of estrogens on heart mitochondrial functions and whether estrogens can prevent calcium-induced release of cytochrome c from mitochondria. 10 nM-10 microM 17beta-estradiol or 4-hydroxytamoxifen did not affect mitochondrial respiration rate and membrane potential in state 3 and state 4. Higher concentrations of both agents decreased state 3 respiration rate and membrane potential. 100 nM 17beta-estradiol and 4-hydroxytamoxifen blocked high calcium-induced cytochrome c release from mitochondria but not mitochondrial swelling. Thus, at physiological concentrations estrogens do not affect mitochondrial respiratory functions but protect heart mitochondria from high calcium-induced release of cytochrome c.
Topics: Animals; Calcium; Cytochrome c Group; Dose-Response Relationship, Drug; Estradiol; Estrogens; Female; Ion Channels; Membrane Potentials; Mitochondria, Heart; Mitochondrial Membrane Transport Proteins; Mitochondrial Permeability Transition Pore; Rats; Rats, Wistar; Receptors, Estrogen; Tamoxifen
PubMed: 12067725
DOI: 10.1016/s0014-5793(02)02820-x -
The EMBO Journal Aug 1995A conserved region in the hormone-dependent activation domain AF2 of nuclear receptors plays an important role in transcriptional activation. We have characterized a...
A conserved region in the hormone-dependent activation domain AF2 of nuclear receptors plays an important role in transcriptional activation. We have characterized a novel nuclear protein, RIP140, that specifically interacts in vitro with this domain of the estrogen receptor. This interaction was increased by estrogen, but not by anti-estrogens and the in vitro binding capacity of mutant receptors correlates with their ability to stimulate transcription. RIP140 also interacts with estrogen receptor in intact cells and modulates its transcriptional activity in the presence of estrogen, but not the anti-estrogen 4-hydroxytamoxifen. In view of its widespread expression in mammalian cells, RIP140 may interact with other members of the superfamily of nuclear receptors and thereby act as a potential co-activator of hormone-regulated gene transcription.
Topics: Adaptor Proteins, Signal Transducing; Amino Acid Sequence; Breast Neoplasms; Cell Line; Cell Nucleus; Cloning, Molecular; Cytoplasm; DNA, Complementary; Estradiol; Estrogen Antagonists; Humans; Molecular Sequence Data; Nuclear Proteins; Nuclear Receptor Interacting Protein 1; RNA, Messenger; Receptors, Estrogen; Recombinant Fusion Proteins; Sequence Analysis, DNA; Sequence Deletion; Tamoxifen; Transcription Factor TFIIB; Transcription Factors; Transcriptional Activation; Tumor Cells, Cultured
PubMed: 7641693
DOI: 10.1002/j.1460-2075.1995.tb00044.x