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Computational Biology and Chemistry Feb 2020Tamoxifen is a prodrug and cytochrome P450 2C9 (CYP2C9) has a significant role in the formation of a therapeutically more potent metabolite (4-hydroxytamoxifen) than...
Tamoxifen is a prodrug and cytochrome P450 2C9 (CYP2C9) has a significant role in the formation of a therapeutically more potent metabolite (4-hydroxytamoxifen) than tamoxifen. Since CYP2C9 exhibits genetic polymorphism, it may contribute to different phenotypic drug response. Moreover, it may be misleading if the possibility of heterogeneous clinical observations of pharmacogenetic investigations is ignored. Above all, clinical investigation of all the polymorphic variants is beyond the scope of a pharmacogenetic study. Therefore, in order to understand the genotype-phenotype association, it is aimed to study the interatomic interactions of amino acid substitutions in CYP2C9 variants in the presence of tamoxifen. Computational structural biology approach was adopted to study the effect of amino acid substitutions of polymorphic variants of CYP2C9 R144C (*2), I359 L (*3), D360E (*5), R150H (*8), R335W (*11) and L90 P (*13) on the flexibility of the enzyme in the presence of tamoxifen. The mutations were selected based on previously determined associations on genotype and clinical outcome of drugs. Against the above plane, docking of tamoxifen was performed with the crystal structure representing the wild-type form of the enzyme. The docked conformation of tamoxifen was favourable for 4-hydroxylation with the site of metabolism within 5 Å of oxyferrylheme consistent with the drug metabolism pathway of tamoxifen. Further, the effect of amino acid substitutions CYP2C9 variants on the protein flexibility in the presence of tamoxifen in 4-hydroxy orientation was evaluated by molecular dynamics (MD) simulations. Distinct protein flexibility modulations between variants were observed in F/G segment constituting the substrate access/egress channels, helix B' involved with substrate specificity and helix I associated with the holding of substrates. Root Mean Square Fluctuation analysis of the trajectories of variants exhibited fluctuations in F/G segment, B' and I helix. Dominant motions in the structure were identified by performing Principal Component Analysis on trajectories and the porcupine plot depicted displaced F/G segment in variants. Thus, the interatomic interaction study of CYP2C9 variants in the presence of tamoxifen predicts the plausible effect of the investigated variants on the therapeutic outcome of tamoxifen. It is presumed that the observations of the study would be meaningful to understand tamoxifen pharmacogenetics.
Topics: Amino Acid Substitution; Catalytic Domain; Cytochrome P-450 CYP2C9; Humans; Molecular Docking Simulation; Molecular Dynamics Simulation; Pliability; Polymorphism, Genetic; Protein Binding; Protein Conformation, alpha-Helical; Tamoxifen
PubMed: 31785970
DOI: 10.1016/j.compbiolchem.2019.107166 -
Basic & Clinical Pharmacology &... May 2020Generic formulations of tamoxifen are commonly prescribed to oestrogen receptor-positive breast cancer patients at the Brazilian National Cancer Institute (INCA). We...
Generic formulations of tamoxifen are commonly prescribed to oestrogen receptor-positive breast cancer patients at the Brazilian National Cancer Institute (INCA). We carried out a post-marketing surveillance of the generic tamoxifen formulation in current use at INCA, by comparing plasma concentrations of the parent drug and metabolites obtained with the generic vs the reference formulation. Thirty patients participated in an open-label, bracketed protocol, comprising 3 successive phases of 30-32 days each: the generic formulation was used in phases 1 and 3 and the reference formulation in phase 2. Two blood samples were collected in the last 4 days of each phase, for LC-MS/MS quantification of tamoxifen and metabolites in plasma. The median plasma concentrations (ng/mL) for the reference formulation were as follows: tamoxifen, 135.0 (CI 95% 114.2-155.8); endoxifen, 35.3 (30.0-40.8); and 4-hydroxytamoxifen, 4.8 (4.2-5.4). The endoxifen/tamoxifen plasma concentration ratio was 0.27 (0.21-0.25). ANOVA detected no statistically significant difference in plasma concentrations of tamoxifen, metabolites or the endoxifen/tamoxifen ratio among the three phases. The genetic component (rGC) of the CYP2D6-mediated conversion of tamoxifen into endoxifen, estimated using the repeated drug administration procedure across the three phases, was 0.87, pointing to an important component of genetic variability. In conclusion, this first post-marketing surveillance trial of oncologic generic drugs carried out in Brazilian patients verified the switchability between the reference and the generic tamoxifen formulation currently used at our institution. The adopted bracketed protocol adds confidence to this conclusion and may serve as a frame for future trials of post-marketing assessment of other generic drug products.
Topics: Adult; Aged; Antineoplastic Agents, Hormonal; Brazil; Breast Neoplasms; Cytochrome P-450 CYP2D6; Drugs, Generic; Female; Genotype; Humans; Middle Aged; Product Surveillance, Postmarketing; Tamoxifen
PubMed: 31758654
DOI: 10.1111/bcpt.13368 -
Scientific Reports Sep 2019Human olfactory mucosa cells (hOMCs) have been transplanted to the damaged spinal cord both pre-clinically and clinically. To date mainly autologous cells have been...
Human olfactory mucosa cells (hOMCs) have been transplanted to the damaged spinal cord both pre-clinically and clinically. To date mainly autologous cells have been tested. However, inter-patient variability in cell recovery and quality, and the fact that the neuroprotective olfactory ensheathing cell (OEC) subset is difficult to isolate, means an allogeneic hOMC therapy would be an attractive "off-the-shelf" alternative. The aim of this study was to generate a candidate cell line from late-adherent hOMCs, thought to contain the OEC subset. Primary late-adherent hOMCs were transduced with a c-MycER gene that enables cell proliferation in the presence of 4-hydroxytamoxifen (4-OHT). Two c-MycER-derived polyclonal populations, PA5 and PA7, were generated and expanded. PA5 cells had a normal human karyotype (46, XY) and exhibited faster growth kinetics than PA7, and were therefore selected for further characterisation. PA5 hOMCs express glial markers (p75, S100ß, GFAP and oligodendrocyte marker O4), neuronal markers (nestin and ß-III-tubulin) and fibroblast-associated markers (CD90/Thy1 and fibronectin). Co-culture of PA5 cells with a neuronal cell line (NG108-15) and with primary dorsal root ganglion (DRG) neurons resulted in significant neurite outgrowth after 5 days. Therefore, c-MycER-derived PA5 hOMCs have potential as a regenerative therapy for neural cells.
Topics: Adult; Animals; Biomarkers; Cell Line; Coculture Techniques; Ganglia, Spinal; Genes, myc; Gentamicins; Humans; Karyotyping; Mice; Neuroblastoma; Olfactory Mucosa; Rats; Rats, Sprague-Dawley; Receptors, Estrogen; Recombinant Proteins; Sensory Receptor Cells; Tamoxifen; Transduction, Genetic; Transgenes
PubMed: 31519924
DOI: 10.1038/s41598-019-49315-6 -
Cell Communication and Signaling : CCS Aug 2019Ligands of the C-type lectin CLEC10A such as Tn and sialyl-Tn representing early intermediates of O-glycosylation are hallmarks of many human malignancies. A variety of...
BACKGROUND
Ligands of the C-type lectin CLEC10A such as Tn and sialyl-Tn representing early intermediates of O-glycosylation are hallmarks of many human malignancies. A variety of regulatory mechanisms underlying their expression are being discussed.
METHODS
CLEC10A ligands were detected in various tissues and cells using the recombinant glycan-binding domain of CLEC10A. In normal breast and endometrium, presence of ligands was correlated to the female cycle. Estrogen- and stress dependent induction of CLEC10A ligands was analyzed in MCF7 and T47D cells exposed to 4-hydroxy-tamoxifen (Tam), zeocin and hydrogen peroxide. The expression and localization of CLEC10A ligands was analyzed by Western blot and immunofluorescence. In breast cancer patients CLEC10A ligand expression and survival was correlated by Kaplan-Meyer analysis.
RESULT
We observed binding of CLEC10A in normal endometrial and breast tissues during the late phase of the female hormonal cycle suggesting a suppressive effect of female sex hormones on CLEC10A ligand expression. Accordingly, CLEC10A ligands were induced in MCF7- and T47D breast cancer cells after Tam treatment and accumulated on the cell surface and in the endosomal/lysosomal compartment. Phagocytosis experiments indicate that macrophages preferentially internalize CLEC10A ligands coated beads and Tam treated MCF7 cells. CLEC10A ligands were also expressed after the addition of zeocin and hydrogen-peroxide. Each substance induced the production of ROS indicating reactive oxygen species as a unifying mechanism of CLEC10A ligand induction. Mechanistically, increased expression of GalNAc-transferase 6 (GalNT6) and translocation of GalNT2 and GalNT6 from cis- towards trans-Golgi compartment was observed, while protein levels of COSMC and T-synthase remained unaffected. In breast cancer patients, positivity for CLEC10A staining in tumor tissues was associated with improved outcome and survival.
CONCLUSION
CLEC10A ligands are inducible by hormone depletion, 4-hydroxy-tamoxifen and agents inducing DNA damage and oxidative stress. Our results indicate that CLEC10A acts as a receptor for damaged and dead cells and may play an important role in the uptake of cell debris by macrophages and dendritic cells.
Topics: Antineoplastic Agents; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; DNA Damage; Drug Screening Assays, Antitumor; Female; HEK293 Cells; Humans; Lectins, C-Type; Ligands; MCF-7 Cells; Oxidative Stress; Polysaccharides; Recombinant Proteins; Signal Transduction; Tamoxifen
PubMed: 31455323
DOI: 10.1186/s12964-019-0420-9 -
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 -
Toxicology and Applied Pharmacology Sep 2019Breast cancer patients with high cholesterol biosynthesis signature had poorer therapeutic outcome. Cytochrome P450 (CYP) 2D6 is crucial in the oxidation of tamoxifen to...
Breast cancer patients with high cholesterol biosynthesis signature had poorer therapeutic outcome. Cytochrome P450 (CYP) 2D6 is crucial in the oxidation of tamoxifen to generate active metabolites, 4-hydroxytamoxifen and endoxifen. CYP2D6 variants with C100T substitution encode null or poor functional proteins. This study aims to examine the association of C100T genotypes and serum lipid levels with plasma drug levels in patients. Plasma tamoxifen concentration was positively associated with serum triglyceride concentration, adjusting for age and C100T genotype. Overweight (body mass index >24.0) patients with high serum cholesterol (≥200 mg/dL) had increased risks of ineffective endoxifen levels (<5.97 ng/mL). Compared to the low-cholesterol group, the high-cholesterol group had a lower 4-hydroxytamoxifen or endoxifen level in T/T carriers. In T/T carriers, the high-cholesterol group had an increased risk of an ineffective endoxifen level. Metastasis, hot flash/flushing, and high alanine transaminase did not relate to plasma 4-hydroxytamoxifen or endoxifen levels. Results indicate that C100T and high serum cholesterol are risk factors of ineffective endoxifen levels in Taiwanese breast cancer patients. These findings warrant further studies of a large hypercholesterolemic population to examine the outcome of increased doses of tamoxifen.
Topics: Adult; Aged; Aged, 80 and over; Antineoplastic Agents, Hormonal; Breast Neoplasms; Cholesterol; Cytochrome P-450 CYP2D6; Female; Genotype; Humans; Middle Aged; Phenotype; Tamoxifen
PubMed: 31195002
DOI: 10.1016/j.taap.2019.114619 -
Endocrine-related Cancer Aug 2019Breast cancer is the most prevalent malignancy and second leading cause of death in women worldwide, with hormone receptor-positive luminal breast cancers being the most...
Breast cancer is the most prevalent malignancy and second leading cause of death in women worldwide, with hormone receptor-positive luminal breast cancers being the most widespread subtype. While these tumors are generally amenable to endocrine therapy, cellular heterogeneity and acquired ability of tumor cells to undergo cell state switching makes these populations difficult to be fully targeted and eradicated through conventional methods. We have leveraged a quality-by-design (QbD) approach that integrates biological responses with predictive mathematical modeling to identify key combinations of commercially available drugs to induce estrogen receptor expression for therapeutic targeting. This technology utilizes a high level of automation through a custom-built platform to reduce bias as well as design-of-experiments methodology to minimize the experimental iterations required. Utilizing this approach, we identified a combination of clinical compounds, each at concentrations well below their efficacious dose, able to induce the expression of estrogen receptor alpha (ESR1) in hormone-positive breast cancer cells. Induction of ESR1 in luminal cells leads to chemosensitization. These findings provide proof of concept for the utility of the QbD strategy and identify a unique drug cocktail able to sensitize breast cancer cells to tamoxifen.
Topics: Antineoplastic Agents, Hormonal; Antineoplastic Combined Chemotherapy Protocols; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Drug Resistance, Neoplasm; Estradiol; Estrogen Receptor alpha; Everolimus; Female; Humans; Hydroxamic Acids; Indazoles; MCF-7 Cells; Paclitaxel; Sulfonamides; Tamoxifen; Tumor Cells, Cultured
PubMed: 31167163
DOI: 10.1530/ERC-19-0042