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International Journal of Molecular... Jan 2022Tamoxifen, a therapeutic agent for breast cancer, has been associated with genetic polymorphisms in the metabolism of ,-dialkylaminoethyl substituent, which plays an...
Tamoxifen, a therapeutic agent for breast cancer, has been associated with genetic polymorphisms in the metabolism of ,-dialkylaminoethyl substituent, which plays an important role in the expression of selective estrogen receptor modulator (SERM) activity. To solve this problem, we developed a novel estrogen receptor (ER) modulator, Az-01, on the basis of the aromaticity, dipole moment, and isopropyl group of guaiazulene. Az-01 showed four-fold lower binding affinity for ER than E2 but had similar ER-binding affinity to that of 4-hydroxytamoxifen (4-HOtam). Unlike tamoxifen, Az-01 acted as a partial agonist with very weak estrogenic activity at high concentrations when used alone, and it showed potent anti-estrogenic activity in the presence of E2. The cell proliferation and inhibition activities of Az-01 were specific to ER-expressing MCF-7 cells, and no effect of Az-01 on other cell proliferation signals was observed. These findings are important for the development of new types of SERMs without the ,-dialkylaminoethyl substituent as a privileged functional group for SERMs.
Topics: Azulenes; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cell Survival; Drug Development; Drug Synergism; Estradiol; Estrogen Receptor Modulators; Female; Humans; MCF-7 Cells; Models, Molecular; Molecular Structure; Protein Binding; Protein Conformation; Receptors, Estrogen; Sesquiterpenes, Guaiane; Tamoxifen
PubMed: 35163039
DOI: 10.3390/ijms23031113 -
The Journal of Biological Chemistry Jul 2023Therapeutic strategies targeting nuclear receptors (NRs) beyond their endogenous ligand binding pocket have gained significant scientific interest driven by a need to...
Therapeutic strategies targeting nuclear receptors (NRs) beyond their endogenous ligand binding pocket have gained significant scientific interest driven by a need to circumvent problems associated with drug resistance and pharmacological profile. The hub protein 14-3-3 is an endogenous regulator of various NRs, providing a novel entry point for small molecule modulation of NR activity. Exemplified, 14-3-3 binding to the C-terminal F-domain of the estrogen receptor alpha (ERα), and small molecule stabilization of the ERα/14-3-3ζ protein complex by the natural product Fusicoccin A (FC-A), was demonstrated to downregulate ERα-mediated breast cancer proliferation. This presents a novel drug discovery approach to target ERα; however, structural and mechanistic insights into ERα/14-3-3 complex formation are lacking. Here, we provide an in-depth molecular understanding of the ERα/14-3-3ζ complex by isolating 14-3-3ζ in complex with an ERα protein construct comprising its ligand-binding domain (LBD) and phosphorylated F-domain. Bacterial co-expression and co-purification of the ERα/14-3-3ζ complex, followed by extensive biophysical and structural characterization, revealed a tetrameric complex between the ERα homodimer and the 14-3-3ζ homodimer. 14-3-3ζ binding to ERα, and ERα/14-3-3ζ complex stabilization by FC-A, appeared to be orthogonal to ERα endogenous agonist (E2) binding, E2-induced conformational changes, and cofactor recruitment. Similarly, the ERα antagonist 4-hydroxytamoxifen inhibited cofactor recruitment to the ERα LBD while ERα was bound to 14-3-3ζ. Furthermore, stabilization of the ERα/14-3-3ζ protein complex by FC-A was not influenced by the disease-associated and 4-hydroxytamoxifen resistant ERα-Y537S mutant. Together, these molecular and mechanistic insights provide direction for targeting ERα via the ERα/14-3-3 complex as an alternative drug discovery approach.
Topics: Humans; 14-3-3 Proteins; Estrogen Receptor alpha; Ligands; Tamoxifen; Protein Binding; Drug Discovery; Estrogen Antagonists
PubMed: 37224961
DOI: 10.1016/j.jbc.2023.104855 -
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 -
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 -
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 -
Journal of Pharmaceutical and... Jan 2024A high rate of interindividual variability in response to tamoxifen (TAM) in breast cancer patients with CYP2D6 polymorphism has been reported, which affects the...
Evaluation of the effect of CYP2D6*3, *4,*10, and *17 polymorphisms on the pharmacokinetic of tamoxifen and its metabolites in patients with hormone-positive breast cancer.
BACKGROUND AND OBJECTIVE
A high rate of interindividual variability in response to tamoxifen (TAM) in breast cancer patients with CYP2D6 polymorphism has been reported, which affects the patient's therapeutic outcome. The objective of this study was to investigate the pharmacogenomics of CYP2D6 genotyping in Iranian patients with breast cancer treated with adjuvant TAM.
METHODS
A peripheral blood sample was obtained to determine the steady-state plasma concentrations of TAM and its metabolites (Endoxifen (EN) and 4-Hydroxytamoxifen (4-OHT)) using high-performance liquid chromatography with fluorescence detection (HPLC-FLU) assay. We detected CYP2D6 * 3, * 4, * 10, and * 17 single nucleotide polymorphisms via polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) method.
RESULTS
A total of 84 Iranian estrogen receptor‑positive breast cancer patients receiving the daily dose of 20 mg tamoxifen were recruited. Although a consequent decrease in the median EN and 4-OHT concentrations was observed by comparing poor or intermediate metabolizer patients with an extensive metabolizer population, this difference did not reach a significant level. The mean plasma EN concentrations in poor and intermediate metabolizers were 46.1% (95% CI, 7.4-27.8%) and 59.4% (95% CI, 11.9-37.3%) of extensive metabolizer subjects, respectively. Poor and intermediate metabolizers had the mean plasma 4-OHT concentrations that were 46.6% (95% CI, 0.9-61.7%) and 73.2% (95% CI, 2.7-93.1%) of those of subjects who were extensive metabolizer, respectively.
CONCLUSIONS
The possible role of genotyping in Iranian patients' response to treatment may explain inter-individual differences in the plasma concentrations of active metabolites of TAM.
Topics: Humans; Female; Breast Neoplasms; Cytochrome P-450 CYP2D6; Iran; Antineoplastic Agents, Hormonal; Tamoxifen; Polymorphism, Single Nucleotide; Hormones; Genotype
PubMed: 37976989
DOI: 10.1016/j.jpba.2023.115839 -
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 -
Cells Sep 2021Cancer cells have an increased need for glucose and, despite aerobic conditions, obtain their energy through aerobic oxidation and lactate fermentation, instead of...
Cancer cells have an increased need for glucose and, despite aerobic conditions, obtain their energy through aerobic oxidation and lactate fermentation, instead of aerobic oxidation alone. Glutamine is an essential amino acid in the human body. Glutaminolysis and glycolysis are crucial for cancer cell survival. In the therapy of estrogen receptor α (ERα)-positive breast cancer (BC), the focus lies on hormone sensitivity targeting therapy with selective estrogen receptor modulators (SERMs) such as 4-hydroxytamoxifen (4-OHT), although this therapy is partially limited by the development of resistance. Therefore, further targets for therapy improvement of ERα-positive BC with secondary 4-OHT resistance are needed. Hence, increased glucose requirement and upregulated glutaminolysis in BC cells could be used. We have established sublines of ERα-positive MCF7 and T47D BC cells, which were developed to be resistant to 4-OHT. Further, glycolysis inhibitor 2-Deoxy-D-Glucose (2-DG) and glutaminase inhibitor CB-839 were analyzed. Co-treatments using 4-OHT and CB-839, 2-DG and CB-839, or 4-OHT, 2-DG and CB-839, respectively, showed significantly stronger inhibitory effects on viability compared to single treatments. It could be shown that tamoxifen-resistant BC cell lines, compared to the non-resistant cell lines, exhibited a stronger reducing effect on cell viability under co-treatments. In addition, the tamoxifen-resistant BC cell lines showed increased expression of proto-oncogene c-Myc compared to the parental cell lines. This could be reduced depending on the treatment. Suppression of c-Myc expression using specific siRNA completely abolished resistance to 4OH-tamoxifen. In summary, our data suggest that combined treatments affecting the metabolism of BC are suitable depending on the cellularity and resistance status. In addition, the anti-metabolic treatments affected the expression of the proto-oncogene c-Myc, a key player in the regulation of cancer cell metabolism.
Topics: Antimetabolites; Apoptosis; Benzeneacetamides; Breast Neoplasms; Cell Proliferation; Deoxyglucose; Drug Resistance, Neoplasm; Drug Therapy, Combination; Estrogen Antagonists; Female; Glutaminase; Glycolysis; Humans; Proto-Oncogene Mas; Proto-Oncogene Proteins c-myc; Tamoxifen; Thiadiazoles; Tumor Cells, Cultured
PubMed: 34572047
DOI: 10.3390/cells10092398 -
Environmental Science & Technology Oct 2023Wastewater treatment plants (WWTPs) are regarded as the main sources of estrogens that reach the aquatic environment. Hence, continuous monitoring of potential...
Wastewater treatment plants (WWTPs) are regarded as the main sources of estrogens that reach the aquatic environment. Hence, continuous monitoring of potential estrogenic-active compounds by a biosensor is an appealing approach. However, existing biosensors cannot simultaneously distinguish and quantify estrogenic agonists and antagonists. To overcome the challenge, we developed an estrogen receptor-based biosensor that selectively screened estrogenic agonists and antagonists by introducing rationally designed agonist/antagonist conformation-specific reporters. The double functional conformation-specific reporters consist of a Cy5.5-labeled streptavidin moiety and a peptide moiety, serving as signal recognition and signal transduction elements. In addition, the conformation recognition mechanism was further validated at the molecular level through molecular docking. Based on the two-step "turn-off" strategy, the biosensor exhibited remarkable sensitivity, detecting 17β-estradiol-binding activity equivalent (E-BAE) at 7 ng/L and 4-hydroxytamoxifen-binding activity equivalent (4-OHT-BAE) at 91 ng/L. To validate its practicality, the biosensor was employed in a case study involving wastewater samples from two full-scale WWTPs across different treatment stages to map their estrogenic agonist and antagonist binding activities. Comparison with the yeast two-hybrid bioassay showed a strong liner relationship ( = 0.991, < 0.0001), indicating the excellent accuracy and reliability of this technology in real applications.
Topics: Wastewater; Molecular Docking Simulation; Reproducibility of Results; Estrogens; Estrone; Biosensing Techniques; Water Pollutants, Chemical
PubMed: 37802504
DOI: 10.1021/acs.est.3c03223 -
Biophysical Chemistry Nov 2021The anticancer drug tamoxifen and its primary metabolite 4-hydroxytamoxifen tend to accumulate in membranes due to its strong hydrophobic character. Thus, in this work...
The anticancer drug tamoxifen and its primary metabolite 4-hydroxytamoxifen tend to accumulate in membranes due to its strong hydrophobic character. Thus, in this work we have carried out a systematic study to investigate their effects on model phosphatidylcholine membranes. Tamoxifen and 4-hydroxytamoxifen affect the phase behaviour of phosphatidylcholine model membranes, giving rise to formation of drug/dipalmitoylphosphatidylcholine domains, which is more evident in the case of 4-hydroxytamoxifen. These drugs have differential effects on the polar and apolar regions of the phospholipid supporting a different location of both compounds within the bilayer. Both compounds induce contents leakage in fluid phosphatidylcholine unilamellar liposomes, the effect of 4-hydroxytamoxifen being negligible as compared to that of tamoxifen. Molecular dynamics confirmed the tendency of both drugs to form clusters, tamoxifen locating all along the bilayer, whereas 4-hydroxytamoxifen mostly locates near the lipid/water interface, which can explain the different effects of both drugs in fluid phosphatidylcholine membranes.
Topics: Phosphatidylcholines; Tamoxifen
PubMed: 34530285
DOI: 10.1016/j.bpc.2021.106681