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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 -
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 -
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 -
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 -
Frontiers in Immunology 2021Cell swelling and membrane blebbing are characteristic of pyroptosis. In the present study, we explored the role of intracellular tension activity in the deformation of...
Cell swelling and membrane blebbing are characteristic of pyroptosis. In the present study, we explored the role of intracellular tension activity in the deformation of pyroptotic astrocytes. Protein nanoparticle-induced osmotic pressure (PN-OP) was found to be involved in cell swelling and membrane blebbing in pyroptotic astrocytes, and was associated closely with inflammasome production and cytoskeleton depolymerization. However, accumulation of protein nanoparticles seemed not to be absolutely required for pyroptotic permeabilization in response to cytoskeleton depolymerization. Gasdermin D activation was observed to be involved in modification of typical pyroptotic features through inflammasome-induced OP upregulation and calcium increment. Blockage of nonselective ion pores can inhibit permeabilization, but not inflammasome production and ion influx in pyroptotic astrocytes. The results suggested that the inflammasomes, as protein nanoparticles, are involved in PN-OP upregulation and control the typical features of pyroptotic astrocytes.
Topics: Animals; Astrocytes; Calcium Signaling; Caspase 1; Cell Line, Tumor; Cell Membrane; Cell Size; Cytoskeleton; Disease Models, Animal; Humans; Inflammasomes; Intracellular Signaling Peptides and Proteins; Lipopolysaccharides; Male; Mechanotransduction, Cellular; Mice, Inbred C57BL; NLR Family, Pyrin Domain-Containing 3 Protein; Nigericin; Osmotic Pressure; Phosphate-Binding Proteins; Polyethylene Glycols; Pyroptosis; Sepsis; Stress, Mechanical; Tamoxifen; Mice
PubMed: 34305921
DOI: 10.3389/fimmu.2021.688674 -
The Journal of Neuroscience : the... May 2021Memories are rarely acquired under ideal conditions, rendering them vulnerable to profound omissions, errors, and ambiguities. Consistent with this, recent work using...
Memories are rarely acquired under ideal conditions, rendering them vulnerable to profound omissions, errors, and ambiguities. Consistent with this, recent work using context fear conditioning has shown that memories formed after inadequate learning time display a variety of maladaptive properties, including overgeneralization to similar contexts. However, the neuronal basis of such poor learning and memory imprecision remains unknown. Using c-fos to track neuronal activity in male mice, we examined how these learning-dependent changes in context fear memory precision are encoded in hippocampal ensembles. We found that the total number of c-fos-encoding cells did not correspond with learning history but instead more closely reflected the length of the session immediately preceding c-fos measurement. However, using a c-fos-driven tagging method ( mouse line), we found that the degree of learning and memory specificity corresponded with neuronal activity in a subset of dentate gyrus cells that were active during both learning and recall. Comprehensive memories acquired after longer learning intervals were associated with more double-labeled cells. These were preferentially reactivated in the conditioning context compared with a similar context, paralleling behavioral discrimination. Conversely, impoverished memories acquired after shorter learning intervals were associated with fewer double-labeled cells. These were reactivated equally in both contexts, corresponding with overgeneralization. Together, these findings provide two surprising conclusions. First, engram size varies with learning. Second, larger engrams support better neuronal and behavioral discrimination. These findings are incorporated into a model that describes how neuronal activity is influenced by previous learning and present experience, thus driving behavior. Memories are not always formed under ideal circumstances. This is especially true in traumatic situations, such as car accidents, where individuals have insufficient time to process what happened around them. Such memories have the potential to overgeneralize to irrelevant situations, producing inappropriate fear and contributing to disorders, such as post-traumatic stress disorder. However, it is unknown how such poorly formed fear memories are encoded within the brain. We find that restricting learning time results in fear memories that are encoded by fewer hippocampal cells. Moreover, these fewer cells are inappropriately reactivated in both dangerous and safe contexts. These findings suggest that fear memories formed at brief periods overgeneralize because they lack the detail-rich information necessary to support neuronal discrimination.
Topics: Animals; Conditioning, Classical; Dentate Gyrus; Discrimination, Psychological; Estrogen Antagonists; Fear; Hippocampus; Learning; Male; Memory; Mice; Mice, Inbred C57BL; Models, Psychological; Neurons; Proto-Oncogene Proteins c-fos; Tamoxifen
PubMed: 33888604
DOI: 10.1523/JNEUROSCI.2786-20.2021 -
Science Advances Jun 2019Cellular senescence is a stress response program characterized by a robust cell cycle arrest and the induction of a proinflammatory senescence-associated secretory...
Cellular senescence is a stress response program characterized by a robust cell cycle arrest and the induction of a proinflammatory senescence-associated secretory phenotype (SASP) that is triggered through an unknown mechanism. Here, we show that, during oncogene-induced senescence (OIS), the Toll-like receptor 2 (TLR2) and its partner TLR10 are key mediators of senescence in vitro and in murine models. TLR2 promotes cell cycle arrest by regulating the tumor suppressors p53-p21, p16, and p15 and regulates the SASP through the induction of the acute-phase serum amyloids A1 and A2 (A-SAAs) that, in turn, function as the damage-associated molecular patterns (DAMPs) signaling through TLR2 in OIS. Last, we found evidence that the cGAS-STING cytosolic DNA sensing pathway primes TLR2 and A-SAAs expression in OIS. In summary, we report that innate immune sensing of senescence-associated DAMPs by TLR2 controls the SASP and reinforces the cell cycle arrest program in OIS.
Topics: Alarmins; Animals; Cellular Senescence; Fibroblasts; Humans; Immunity, Innate; Membrane Proteins; Mice; Mice, Inbred C57BL; Mice, Knockout; NF-kappa B; Nucleotidyltransferases; RNA Interference; RNA, Small Interfering; Serum Amyloid A Protein; Signal Transduction; Tamoxifen; Toll-Like Receptor 10; Toll-Like Receptor 2; p38 Mitogen-Activated Protein Kinases; ras Proteins
PubMed: 31183403
DOI: 10.1126/sciadv.aaw0254 -
Molecular Cancer Research : MCR Jun 2021Despite the availability of drugs that target ERα-positive breast cancer, resistance commonly occurs, resulting in relapse, metastasis, and death. Tamoxifen remains the...
Despite the availability of drugs that target ERα-positive breast cancer, resistance commonly occurs, resulting in relapse, metastasis, and death. Tamoxifen remains the most commonly-prescribed endocrine therapy worldwide, and "tamoxifen resistance" has been extensively studied. However, little consideration has been given to the role of endoxifen, the most abundant active tamoxifen metabolite detected in patients, in driving resistance mechanisms. Endoxifen functions differently from the parent drug and other primary metabolites, including 4-hydroxy-tamoxifen (4HT). Many studies have shown that patients who extensively metabolize tamoxifen into endoxifen have superior outcomes relative to patients who do not, supporting a primary role for endoxifen in driving tamoxifen responses. Therefore, "tamoxifen resistance" may be better modeled by "endoxifen resistance" for some patients. Here, we report the development of novel endoxifen-resistant breast cancer cell lines and have extensively compared these models to 4HT and fulvestrant (ICI)-resistant models. Endoxifen-resistant cells were phenotypically and molecularly distinct from 4HT-resistant cells and more closely resembled ICI-resistant cells overall. Specifically, endoxifen resistance was associated with ERα and PR loss, estrogen insensitivity, unique gene signatures, and striking resistance to most FDA-approved second- and third-line therapies. Given these findings, and the importance of endoxifen in the efficacy of tamoxifen therapy, our data indicate that endoxifen-resistant models may be more clinically relevant than existing models and suggest that a better understanding of endoxifen resistance could substantially improve patient care. IMPLICATIONS: Here we report on the development and characterization of the first endoxifen-resistant models and demonstrate that endoxifen resistance may better model tamoxifen resistance in a subset of patients.
Topics: Antineoplastic Agents, Hormonal; Blotting, Western; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Cell Proliferation; Drug Resistance, Neoplasm; Estrogen Receptor alpha; Female; Fulvestrant; Gene Expression Regulation, Neoplastic; Humans; MCF-7 Cells; Models, Biological; Reverse Transcriptase Polymerase Chain Reaction; Tamoxifen
PubMed: 33627502
DOI: 10.1158/1541-7786.MCR-20-0872