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Tissue & Cell Aug 2022Adipose-derived stromal cells (ASCs) are a promising cell source for novel tissue engineering approaches to breast reconstruction following cancer resection. However...
BACKGROUND AND AIM
Adipose-derived stromal cells (ASCs) are a promising cell source for novel tissue engineering approaches to breast reconstruction following cancer resection. However there is limited knowledge on the effect of adjuvant therapies such as hormonal therapy on ASCs, which may affect their efficacy in regenerative strategies. The present study aims to investigate the effects of Tamoxifen and its metabolites Afimoxifene (4-Hydroxy-Tamoxifen) and Endoxifen (N-desmethyl-4-hydroxytamoxifen) on patient-derived ASC viability, apoptosis, adipogenic differentiation and angiogenic potential.
METHODS
ASCs were isolated from fat harvested from female breast cancer patients undergoing breast reconstruction surgery or cosmetic procedures. Oestrogen receptor (ER α, β) expression was analysed using immunofluorescence. ASCs were then treated with various concentrations of Afimoxifene, Endoxifen and Tamoxifen (combination), and the impact on ASC viability and apoptosis determined. ASCs were cultured in adipogenic-differentiation media with or without tamoxifen and derivatives, and adipogenesis was measured using quantitative Real-time Polymerase chain reaction (qRT-PCR) and histological staining (Oil Red O). The effect on secreted VEGF levels was also quantified in ASC conditioned media RESULTS: ASCs were successfully isolated and characterised from human abdominal lipoaspirates or fat tissues (n = 8). ASCs subjected to varying doses of Tamoxifen and metabolites (up to 1000 nM) showed no decline in cell viability or increase in apoptosis, at physiological doses (upto 100 nM). Functional decline in adipogenic differentiation or gene expression was observed at supraphysiological concentrations of Tamoxifen (1000 nM). VEGF protein secretion in ASC-cell conditioned media was not significantly impacted irrespective of dosage.
CONCLUSION
At physiologically relevant doses, Tamoxifen treatment did not result in any deleterious effect on ASC survival and functionality and is unlikely to negatively impact ASC based breast reconstruction strategies for breast cancer patients receiving this adjuvant hormonal therapy.
Topics: Adipose Tissue; Breast Neoplasms; Cell Differentiation; Cells, Cultured; Culture Media, Conditioned; Female; Humans; Stromal Cells; Tamoxifen; Vascular Endothelial Growth Factor A
PubMed: 35777289
DOI: 10.1016/j.tice.2022.101858 -
ChemMedChem Aug 2020In the search for new and effective treatments of breast and prostate cancer, a series of hybrid compounds based on tamoxifen, estrogens, and artemisinin were...
In the search for new and effective treatments of breast and prostate cancer, a series of hybrid compounds based on tamoxifen, estrogens, and artemisinin were successfully synthesized and analyzed for their in vitro activities against human prostate (PC-3) and breast cancer (MCF-7) cell lines. Most of the hybrid compounds exhibit a strong anticancer activity against both cancer cell lines - for example, EC (PC-3) down to 1.07 μM, and EC (MCF-7) down to 2.08 μM - thus showing higher activities than their parent compounds 4-hydroxytamoxifen (afimoxifene, 7; EC =75.1 (PC-3) and 19.3 μM (MCF-7)), dihydroartemisinin (2; EC =263.6 (PC-3) and 49.3 μM (MCF-7)), and artesunic acid (3; EC =195.1 (PC-3) and 32.0 μM (MCF-7)). The most potent compounds were the estrogen-artemisinin hybrids 27 and 28 (EC =1.18 and 1.07 μM, respectively) against prostate cancer, and hybrid 23 (EC =2.08 μM) against breast cancer. These findings demonstrate the high potential of hybridization of artemisinin and estrogens to further improve their anticancer activities and to produce synergistic effects between linked pharmacophores.
Topics: Antineoplastic Agents; Artemisinins; Breast Neoplasms; Cell Proliferation; Dose-Response Relationship, Drug; Drug Screening Assays, Antitumor; Estrogens; Female; Humans; MCF-7 Cells; Male; Molecular Structure; PC-3 Cells; Prostatic Neoplasms; Structure-Activity Relationship; Tamoxifen
PubMed: 32374071
DOI: 10.1002/cmdc.202000174 -
Nucleic Acids Research Nov 1999Conditional DNA excision between two LoxP sites can be achieved in the mouse using Cre-ER(T), a fusion protein between a mutated ligand binding domain of the human... (Comparative Study)
Comparative Study
Temporally-controlled site-specific mutagenesis in the basal layer of the epidermis: comparison of the recombinase activity of the tamoxifen-inducible Cre-ER(T) and Cre-ER(T2) recombinases.
Conditional DNA excision between two LoxP sites can be achieved in the mouse using Cre-ER(T), a fusion protein between a mutated ligand binding domain of the human estrogen receptor (ER) and the Cre recombinase, the activity of which can be induced by 4-hydroxy-tamoxifen (OHT), but not natural ER ligands. We have recently characterized a new ligand-dependent recombinase, Cre-ER(T2), which was approximately 4-fold more efficiently induced by OHT than Cre-ER(T) in cultured cells. In order to compare the in vivo efficiency of these two ligand-inducible recombinases to generate temporally-controlled somatic mutations, we have engineered transgenic mice expressing a LoxP-flanked (floxed) transgene reporter and either Cre-ER(T) or Cre-ER(T2) under the control of the bovine keratin 5 promoter that is specifically active in the epidermis basal cell layer. No background recombinase activity could be detected, while recombination was induced in basal keratinocytes upon OHT administration. Interestingly, a dose-response study showed that Cre-ER(T2) was approximately 10-fold more sensitive to OHT induction than Cre-ER(T).
Topics: Animals; Enzyme Induction; Epidermis; Estrogen Receptor Modulators; Genes, Reporter; Humans; Integrases; Keratinocytes; Mice; Mice, Transgenic; Mutagenesis, Site-Directed; Receptors, Estrogen; Recombinant Fusion Proteins; Tamoxifen; Viral Proteins
PubMed: 10536138
DOI: 10.1093/nar/27.22.4324 -
Journal of Pharmacological Sciences Jul 2015Previous studies demonstrated that sulfate conjugation is involved in the metabolism of three commonly used breast cancer drugs, tamoxifen, raloxifene and fulvestrant....
Previous studies demonstrated that sulfate conjugation is involved in the metabolism of three commonly used breast cancer drugs, tamoxifen, raloxifene and fulvestrant. The current study was designed to systematically identify the human cytosolic sulfotransferases (SULTs) that are capable of sulfating raloxifene, fulvestrant, and two active metabolites of tamoxifen, afimoxifene and endoxifen. A systematic analysis using 13 known human SULTs revealed SULT1A1 and SULT1C4 as the major SULTs responsible for the sulfation of afimoxifene, endoxifen, raloxifene and fulvestrant. Kinetic parameters of these two human SULTs in catalyzing the sulfation of these drug compounds were determined. Sulfation of afimoxifene, endoxifen, raloxifene and fulvestrant under metabolic conditions was examined using HepG2 human hepatoma cells and MCF-7 breast cancer cells. Moreover, human intestine, kidney, liver, and lung cytosols were examined to verify the presence of afimoxifene/endoxifen/raloxifene/fulvestrant-sulfating activity.
Topics: Catalysis; Cytosol; Estradiol; Fulvestrant; Hep G2 Cells; Humans; MCF-7 Cells; Raloxifene Hydrochloride; Sulfates; Sulfotransferases; Tamoxifen
PubMed: 26169578
DOI: 10.1016/j.jphs.2015.06.004 -
Biochimica Et Biophysica Acta Sep 2015Tamoxifen, a triphenylethylene antiestrogen and one of the first-line endocrine therapies used to treat estrogen receptor-positive breast cancer, has a number of... (Review)
Review
Tamoxifen, a triphenylethylene antiestrogen and one of the first-line endocrine therapies used to treat estrogen receptor-positive breast cancer, has a number of interesting, off-target effects, and among these is the inhibition of sphingolipid metabolism. More specifically, tamoxifen inhibits ceramide glycosylation, and enzymatic step that can adventitiously support the influential tumor-suppressor properties of ceramide, the aliphatic backbone of sphingolipids. Additionally, tamoxifen and metabolites N-desmethyltamoxifen and 4-hydroxytamoxifen, have been shown to inhibit ceramide hydrolysis by the enzyme acid ceramidase. This particular intervention slows ceramide destruction and thereby depresses formation of sphingosine 1-phosphate, a mitogenic sphingolipid with cancer growth-promoting properties. As ceramide-centric therapies are becoming appealing clinical interventions in the treatment of cancer, agents like tamoxifen that can retard the generation of mitogenic sphingolipids and buffer ceramide clearance via inhibition of glycosylation, take on new importance. In this review, we present an abridged, lay introduction to sphingolipid metabolism, briefly chronicle tamoxifen's history in the clinic, examine studies that demonstrate the impact of triphenylethylenes on sphingolipid metabolism in cancer cells, and canvass works relevant to the use of tamoxifen as adjuvant to drive ceramide-centric therapies in cancer treatment. The objective is to inform the readership of what could be a novel, off-label indication of tamoxifen and structurally-related triphenylethylenes, an indication divorced from estrogen receptor status and one with application in drug resistance.
Topics: Acid Ceramidase; Antineoplastic Agents, Hormonal; Biotransformation; Breast Neoplasms; Ceramides; Drug Resistance, Neoplasm; Female; Humans; Hydrolysis; Lipid Metabolism; Lysophospholipids; Sphingosine; Tamoxifen
PubMed: 25964209
DOI: 10.1016/j.bbalip.2015.05.001 -
International Journal of Molecular... Jan 2020Alpha-fetoprotein (AFP) is a major embryo- and tumor-associated protein capable of binding and transporting a variety of hydrophobic ligands, including estrogens. AFP...
Alpha-fetoprotein (AFP) is a major embryo- and tumor-associated protein capable of binding and transporting a variety of hydrophobic ligands, including estrogens. AFP has been shown to inhibit estrogen receptor (ER)-positive tumor growth, which can be attributed to its estrogen-binding ability. Despite AFP having long been investigated, its three-dimensional (3D) structure has not been experimentally resolved and molecular mechanisms underlying AFP-ligand interaction remains obscure. In our study, we constructed a homology-based 3D model of human AFP (HAFP) with the purpose of molecular docking of ERα ligands, three agonists (17β-estradiol, estrone and diethylstilbestrol), and three antagonists (tamoxifen, afimoxifene and endoxifen) into the obtained structure. Based on the ligand-docked scoring functions, we identified three putative estrogen- and antiestrogen-binding sites with different ligand binding affinities. Two high-affinity binding sites were located (i) in a tunnel formed within HAFP subdomains IB and IIA and (ii) on the opposite side of the molecule in a groove originating from a cavity formed between domains I and III, while (iii) the third low-affinity binding site was found at the bottom of the cavity. Here, 100 ns molecular dynamics (MD) simulation allowed us to study their geometries and showed that HAFP-estrogen interactions were caused by van der Waals forces, while both hydrophobic and electrostatic interactions were almost equally involved in HAFP-antiestrogen binding. Molecular mechanics/Generalized Born surface area (MM/GBSA) rescoring method exploited for estimation of binding free energies (ΔG) showed that antiestrogens have higher affinities to HAFP as compared to estrogens. We performed in silico point substitutions of amino acid residues to confirm their roles in HAFP-ligand interactions and showed that Thr132, Leu138, His170, Phe172, Ser217, Gln221, His266, His316, Lys453, and Asp478 residues, along with two disulfide bonds (Cys224-Cys270 and Cys269-Cys277), have key roles in both HAFP-estrogen and HAFP-antiestrogen binding. Data obtained in our study contribute to understanding mechanisms underlying protein-ligand interactions and anticancer therapy strategies based on ERα-binding ligands.
Topics: Amino Acid Sequence; Amino Acid Substitution; Binding Sites; Estradiol; Estrogen Receptor Modulators; Estrogen Receptor alpha; Estrogens; Female; Humans; Ligands; Models, Molecular; Molecular Docking Simulation; Molecular Dynamics Simulation; Mutagenesis; Sequence Alignment; alpha-Fetoproteins
PubMed: 32019136
DOI: 10.3390/ijms21030893 -
Biomedicine & Pharmacotherapy =... Dec 2021Sex differences in immune-mediated diseases are linked to the activity of estrogens on innate immunity cells, including macrophages. Tamoxifen (TAM) is a selective...
Sex differences in immune-mediated diseases are linked to the activity of estrogens on innate immunity cells, including macrophages. Tamoxifen (TAM) is a selective estrogen receptor modulator (SERM) used in estrogen receptor-alpha (ERα)-dependent breast cancers and off-target indications such as infections, although the immune activity of TAM and its active metabolite, 4-OH tamoxifen (4HT), is poorly characterized. Here, we aimed at investigating the endocrine and immune activity of these SERMs in macrophages. Using primary cultures of female mouse macrophages, we analyzed the expression of immune mediators and activation of effector functions in competition experiments with SERMs and 17β-estradiol (E2) or the bacterial endotoxin LPS. We observed that 4HT and TAM induce estrogen antagonist effects when used at nanomolar concentrations, while pharmacological concentrations that are reached by TAM in clinical settings regulate the expression of VEGFα and other immune activation genes by ERα- and G protein-coupled receptor 1 (GPER1)-independent mechanisms that involve NRF2 through PI3K/Akt-dependent mechanisms. Importantly, we observed that SERMs potentiate cell phagocytosis and modify the effects of LPS on the expression of inflammatory cytokines, such as TNFα and IL1β, with an overall increase in cell inflammatory phenotype, further sustained by potentiation of IL1β secretion through caspase-1 activation. Altogether, our data unravel a novel molecular mechanism and immune functions for TAM and 4HT, sustaining their repurposing in infective and other estrogen receptors-unrelated pathologies.
Topics: Animals; Cells, Cultured; Estrogen Receptor alpha; Female; Immunomodulating Agents; Inflammation Mediators; Lipopolysaccharides; Macrophages, Peritoneal; Mice, Inbred C57BL; Mice, Knockout; NF-E2-Related Factor 2; Phagocytosis; Phenotype; Receptors, Estrogen; Receptors, G-Protein-Coupled; Selective Estrogen Receptor Modulators; Signal Transduction; Tamoxifen; Mice
PubMed: 34653752
DOI: 10.1016/j.biopha.2021.112274 -
Journal of Biomedical Informatics Aug 2015Preparing a systematic review can take hundreds of hours to complete, but the process of reconciling different results from multiple studies is the bedrock of...
Preparing a systematic review can take hundreds of hours to complete, but the process of reconciling different results from multiple studies is the bedrock of evidence-based medicine. We introduce a two-step approach to automatically extract three facets - two entities (the agent and object) and the way in which the entities are compared (the endpoint) - from direct comparative sentences in full-text articles. The system does not require a user to predefine entities in advance and thus can be used in domains where entity recognition is difficult or unavailable. As with a systematic review, the tabular summary produced using the automatically extracted facets shows how experimental results differ between studies. Experiments were conducted using a collection of more than 2million sentences from three journals Diabetes, Carcinogenesis and Endocrinology and two machine learning algorithms, support vector machines (SVM) and a general linear model (GLM). F1 and accuracy measures for the SVM and GLM differed by only 0.01 across all three comparison facets in a randomly selected set of test sentences. The system achieved the best performance of 92% for objects, whereas the accuracy for both agent and endpoints was 73%. F1 scores were higher for objects (0.77) than for endpoints (0.51) or agents (0.47). A situated evaluation of Metformin, a drug to treat diabetes, showed system accuracy of 95%, 83% and 79% for the object, endpoint and agent respectively. The situated evaluation had higher F1 scores of 0.88, 0.64 and 0.62 for object, endpoint, and agent respectively. On average, only 5.31% of the sentences in a full-text article are direct comparisons, but the tabular summaries suggest that these sentences provide a rich source of currently underutilized information that can be used to accelerate the systematic review process and identify gaps where future research should be focused.
Topics: Female; Humans; Algorithms; Blood Glucose; Comparative Effectiveness Research; Data Mining; Electronic Data Processing; Evidence-Based Medicine; Insulin; Language; Linear Models; Linguistics; Machine Learning; Metformin; Natural Language Processing; Raloxifene Hydrochloride; Randomized Controlled Trials as Topic; Reproducibility of Results; Support Vector Machine; Tamoxifen; Uterus; Systematic Reviews as Topic
PubMed: 26003938
DOI: 10.1016/j.jbi.2015.05.004 -
European Journal of Medicinal Chemistry Dec 2022In the last four decades, treatment of oestrogen receptor positive (ER+) breast cancer (BCa), has focused on targeting the estrogenic receptor signaling pathway. This...
In the last four decades, treatment of oestrogen receptor positive (ER+) breast cancer (BCa), has focused on targeting the estrogenic receptor signaling pathway. This signaling function is pivotal to sustain cell proliferation. Tamoxifen, a competitive inhibitor of oestrogen, has played a major role in therapeutics. However, primary and acquired resistance to hormone blockade occurs in a large subset of these cancers, and new approaches are urgently needed. Aromatase inhibitors and receptor degraders were approved and alternatively used. Yet, resistance appears in the metastatic setting. Here we report the design and synthesis of a series of proteolysis targeting chimeras (PROTACs) that induce the degradation of estrogen receptor alpha in breast cancer MCF-7 (ER+) cells at nanomolar concentration. Using a warhead based on 4-hydroxytamoxifen, bifunctional degraders recruiting either cereblon or the Von Hippel Lindau E3 ligases were synthesized. Our efforts resulted in the discovery of TVHL-1, a potent ERα degrader (DC: 4.5 nM) that we envisage as a useful tool for biological study and a platform for potential therapeutics.
Topics: Humans; Female; Receptors, Estrogen; Proteolysis; Von Hippel-Lindau Tumor Suppressor Protein; Chimera; Tamoxifen; Ubiquitin-Protein Ligases; Estrogen Receptor alpha; Breast Neoplasms
PubMed: 36148710
DOI: 10.1016/j.ejmech.2022.114770 -
Angiogenesis Nov 2023Longitudinal mouse models of brain arteriovenous malformations (AVMs) are crucial for developing novel therapeutics and pathobiological mechanism discovery underlying...
BACKGROUND
Longitudinal mouse models of brain arteriovenous malformations (AVMs) are crucial for developing novel therapeutics and pathobiological mechanism discovery underlying brain AVM progression and rupture. The sustainability of existing mouse models is limited by ubiquitous Cre activation, which is associated with lethal hemorrhages resulting from AVM formation in visceral organs. To overcome this condition, we developed a novel experimental mouse model of hereditary hemorrhagic telangiectasia (HHT) with CreER-mediated specific, localized induction of brain AVMs.
METHODS
Hydroxytamoxifen (4-OHT) was stereotactically delivered into the striatum, parietal cortex, or cerebellum of R26; Alk1 (Alk1-iKO) littermates. Mice were evaluated for vascular malformations with latex dye perfusion and 3D time-of-flight magnetic resonance angiography (MRA). Immunofluorescence and Prussian blue staining were performed for vascular lesion characterization.
RESULTS
Our model produced two types of brain vascular malformations, including nidal AVMs (88%, 38/43) and arteriovenous fistulas (12%, 5/43), with an overall frequency of 73% (43/59). By performing stereotaxic injection of 4-OHT targeting different brain regions, Alk1-iKO mice developed vascular malformations in the striatum (73%, 22/30), in the parietal cortex (76%, 13/17), and in the cerebellum (67%, 8/12). Identical application of the stereotaxic injection protocol in reporter mice confirmed localized Cre activity near the injection site. The 4-week mortality was 3% (2/61). Seven mice were studied longitudinally for a mean (SD; range) duration of 7.2 (3; 2.3-9.5) months and demonstrated nidal stability on sequential MRA. The brain AVMs displayed microhemorrhages and diffuse immune cell invasion.
CONCLUSIONS
We present the first HHT mouse model of brain AVMs that produces localized AVMs in the brain. The mouse lesions closely resemble the human lesions for complex nidal angioarchitecture, arteriovenous shunts, microhemorrhages, and inflammation. The model's longitudinal robustness is a powerful discovery resource to advance our pathomechanistic understanding of brain AVMs and identify novel therapeutic targets.
Topics: Animals; Mice; Humans; Telangiectasia, Hereditary Hemorrhagic; Arteriovenous Malformations; Arteriovenous Fistula; Brain
PubMed: 37219736
DOI: 10.1007/s10456-023-09881-w