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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 -
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 -
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 -
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 -
JAMA Surgery Dec 2023Oral tamoxifen citrate benefits women with ductal carcinoma in situ (DCIS), but concern about toxic effects has limited acceptance. Previous pilot studies have suggested...
IMPORTANCE
Oral tamoxifen citrate benefits women with ductal carcinoma in situ (DCIS), but concern about toxic effects has limited acceptance. Previous pilot studies have suggested transdermal 4-hydroxytamoxifen gel has equivalent antiproliferative efficacy to oral tamoxifen, with low systemic exposure.
OBJECTIVE
To demonstrate that 4-hydroxytamoxifen gel applied to the breast skin is noninferior to oral tamoxifen in its antiproliferative effect in DCIS lesions.
DESIGN, SETTING, AND PARTICIPANTS
This randomized, double-blind, phase 2 preoperative window trial was performed at multicenter breast surgery referral practices from May 31, 2017, to January 27, 2021. Among 408 women with estrogen receptor-positive DCIS who were approached, 120 consented and 100 initiated study treatment. The most common reasons for nonparticipation were surgical delay, disinterest in research, and concerns about toxic effects. Data were analyzed from January 26, 2021, to October 5, 2022.
INTERVENTION
Random assignment to oral tamoxifen citrate, 20 mg/d, and gel placebo or 4-hydroxytamoxifen gel, 2 mg/d per breast, and oral placebo, for 4 to 10 weeks, followed by DCIS resection.
MAIN OUTCOMES AND MEASURES
The primary end point was absolute change in DCIS Ki-67 labeling index (Ki67-LI). Secondary end points included 12-gene DCIS Score, breast tissue tamoxifen metabolite concentrations, tamoxifen-responsive plasma protein levels, and patient-reported symptoms. Noninferiority of Ki67-LI reduction by 4-hydroxytamoxifen gel was tested using analysis of covariance; within- and between-arm comparisons were performed with paired t tests for mean values or the Wilcoxon rank sum test for medians.
RESULTS
Of 90 participants completing treatment (mean [SD] age, 55 [11] years; 8 [8.9%] Asian, 16 [17.8%] Black, 8 [8.9%] Latina, and 53 [58.9%] White), 15 lacked residual DCIS in the surgical sample, leaving 75 evaluable for the primary end point analysis (40 in the oral tamoxifen group and 35 in the 4-hydroxytamoxifen gel group). Posttreatment Ki67-LI was 3.3% higher (80% CI, 2.1%-4.6%) in the 4-hydroxytamoxifen gel group compared with the oral tamoxifen group, exceeding the noninferiority margin (2.6%). The DCIS Score decreased more with oral tamoxifen treatment (-16 [95% CI, -22 to -9.4]) than with 4-hydroxytamoxifen gel (-1.8 [95% CI, -5.8 to 2.3]). The median 4-hydroxytamoxifen concentrations deep in the breast were nonsignificantly higher in the oral tamoxifen group (5.7 [IQR, 4.0-7.9] vs 3.8 [IQR, 1.3-7.9] ng/g), whereas endoxifen was abundant in the oral tamoxifen group and minimal in the 4-hydroxytamoxifen gel group (median, 13.0 [IQR, 8.9-20.6] vs 0.3 [IQR, 0-0.3] ng/g; P < .001). Oral tamoxifen caused expected adverse changes in plasma protein levels and vasomotor symptoms, with minimal changes in the transdermal group.
CONCLUSIONS AND RELEVANCE
In this randomized clinical trial, antiproliferative noninferiority of 4-hydroxytamoxifen gel to oral tamoxifen was not confirmed, potentially owing to endoxifen exposure differences. New transdermal approaches must deliver higher drug quantities and/or include the most potent metabolites.
TRIAL REGISTRATION
ClinicalTrials.gov Identifier: NCT02993159.
Topics: Humans; Female; Middle Aged; Carcinoma, Intraductal, Noninfiltrating; Ki-67 Antigen; Double-Blind Method; Tamoxifen; Blood Proteins
PubMed: 37870954
DOI: 10.1001/jamasurg.2023.5113 -
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 -
Medical Oncology (Northwood, London,... Jan 2021Hormone-dependent breast cancer is the most abundant molecular subtype of the disease. Despite the availability of endocrine treatments, the use of these drugs is...
Hormone-dependent breast cancer is the most abundant molecular subtype of the disease. Despite the availability of endocrine treatments, the use of these drugs is limited by their serious adverse reactions and development of acquired resistance often mediated by growth factor receptors. The hepatocyte growth factor receptor, MET, is a receptor tyrosine kinase known for its oncogenic activity and mediating resistance to targeted therapies. Crizotinib is a small-molecule tyrosine kinase inhibitor of MET. In this study, the anticancer effects of combined crizotinib and endocrine drugs were investigated in breast cancer cells in vitro along with the molecular mechanisms associated with these effects. Results showed that crizotinib inhibited growth of MCF7 and T-47D breast cancer cells in a dose-dependent manner with IC values of 2.88 μM and 0.93 μM, respectively. Combined treatment of crizotinib and 4-hydroxytamoxifen resulted in synergistic growth inhibition of MCF7 and T-47D cells with combination index values of 0.39 and 0.8, respectively. The combined treatment significantly suppressed migration and colony formation of MCF7 and T-47D cells. Immunofluorescence showed a significant reduction of the expression of the nuclear protein Ki-67 with the combination of crizotinib and 4-hydroxytamoxifen in both cell lines. Western blotting indicated that the combination treatment reduced the levels of active and total MET, estrogen receptor α (ERα), total and active levels of AKT, ERK, c-SRC, NFĸB p65, GSK-3β, and the anti-apoptotic BCL-2 protein. Findings from this study suggest a potential role of MET inhibitors in breast cancer treatment as monotherapy or combination with endocrine drugs.
Topics: Antineoplastic Agents, Hormonal; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Cell Proliferation; Crizotinib; Drug Synergism; Estrogen Receptor alpha; Fulvestrant; Humans; Inhibitory Concentration 50; Ki-67 Antigen; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-met; Tamoxifen
PubMed: 33449292
DOI: 10.1007/s12032-021-01458-1 -
Gene May 2022The basic region leucin zipper (bZIP) protein c-Fos constitutes together with other bZIP proteins the AP-1 transcription factor complex. Expression of the c-Fos gene is...
The basic region leucin zipper (bZIP) protein c-Fos constitutes together with other bZIP proteins the AP-1 transcription factor complex. Expression of the c-Fos gene is regulated by numerous extracellular signaling molecules including mitogens, metabolites, and ligands for receptor tyrosine kinases, G protein-coupled receptors, and cytokine receptors. Here, we analyzed the effects of the stimulus-responsive MAP kinases ERK1/2 (extracellular signal-regulated protein kinase), JNK (c-Jun N-terminal protein kinase) and p38 protein kinase on transcription of the c-Fos gene. We used chromatin-integrated c-Fos promoter-luciferase reporter genes containing inactivating point mutations of DNA binding sites for distinct transcription factors. ERK1/2, JNK, and p38 protein kinases were specifically activated following expression of either a mutant of B-Raf, a truncated version of mitogen-activated/extracellular signal responsive kinase kinase kinase-1 (MEKK1), or a mutant of MAP kinase kinase-6 (MKK6), respectively. The results show that the DNA binding sites for serum response factor (SRF) and for the ternary complex factor (TCF) are of major importance for stimulating c-Fos promoter activity by MAP kinases. ERK1/2 and p38-induced stimulation of the c-Fos promoter additionally required the DNA binding site for the transcription factor AP-1. Mutation of the DNA binding site for STAT had no or only a small effect on c-Fos promoter activity. We conclude that MAP kinases do not activate distinct transcription factors involving distinct genetic elements. Rather, these kinases mainly target SRF and TCF proteins, leading to an activation of transcription of the c-Fos gene via the serum response element.
Topics: Gene Expression Regulation; HEK293 Cells; Humans; MAP Kinase Signaling System; Mitogen-Activated Protein Kinases; Mutation; Promoter Regions, Genetic; Proto-Oncogene Proteins c-fos; Serum Response Element; Serum Response Factor; TCF Transcription Factors; Tamoxifen; Transcription, Genetic
PubMed: 35143939
DOI: 10.1016/j.gene.2022.146284 -
Scientific Reports Oct 20234-hydroxytamoxifen (OHT) is an anti-cancer drug that induces apoptosis in breast cancer cells. Although changes in lipid levels and mitochondrial respiration have been...
4-hydroxytamoxifen (OHT) is an anti-cancer drug that induces apoptosis in breast cancer cells. Although changes in lipid levels and mitochondrial respiration have been observed in OHT-treated cells, the overall mechanisms underlying these metabolic alterations are poorly understood. In this study, time-series metabolomics and lipidomics were used to analyze the changes in metabolic profiles induced by OHT treatment in the MCF-7 human breast cancer cell line. Lipidomic and metabolomic analyses revealed increases in ceramide, diacylglycerol and triacylglycerol, and decreases in citrate, respectively. Gene expression analyses revealed increased expression of ATP-dependent citrate lyase (ACLY) and subsequent fatty acid biosynthetic enzymes, suggesting that OHT-treated MCF-7 cells activate citrate-to-lipid metabolism. The significance of the observed metabolic changes was evaluated by co-treating MCF-7 cells with OHT and ACLY or a diacylglycerol O-acyltransferase 1 (DGAT1) inhibitor. Co-treatment ameliorated cell death and reduced mitochondrial membrane potential compared to that in OHT treatment alone. The inhibition of cell death by co-treatment with an ACLY inhibitor has been observed in other breast cancer cell lines. These results suggest that citrate-to-lipid metabolism is critical for OHT-induced cell death in breast cancer cell lines.
Topics: Humans; Female; Lipidomics; MCF-7 Cells; Tamoxifen; Breast Neoplasms; Apoptosis; Metabolome; Citrates
PubMed: 37899460
DOI: 10.1038/s41598-023-45764-2