-
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 -
Biochemistry. Biokhimiia Jul 2021Rat embryonic stem cells (ESCs) play an important role in the studies of genes involved in maintaining of pluripotent state and early development of this model organism....
Rat embryonic stem cells (ESCs) play an important role in the studies of genes involved in maintaining of pluripotent state and early development of this model organism. To study functions of the essential genes, as well as the processes of cell differentiation, the method of induced knockout is widely used. The CreERT2/loxP system allows obtaining an inducible knockout in cells expressing tamoxifen-inducible Cre recombinase (CreERT2) and containing loxP sites flanking the target gene by adding 4-hydroxy tamoxifen to the culture medium. However, the rat ESC lines expressing CreERT2 are absent. In this work, we tested three CRISPR/Cas systems for introduction of double-strand breaks into the Rosa26 locus in the rat ESCs and inserted tamoxifen-dependent Cre recombinase into this locus using the CRISPR/Cpf1 system. It was shown that the obtained transgenic rat ESC lines retained the characteristics of pluripotent cells. Tamoxifen-inducible Cre recombinase activity was analyzed using a reporter vector.
Topics: Animals; CRISPR-Cas Systems; Embryonic Stem Cells; Gene Editing; Gene Knockout Techniques; Integrases; Rats; Rats, Transgenic; Tamoxifen
PubMed: 34284709
DOI: 10.1134/S0006297921070051 -
Drug Design, Development and Therapy 2021In this research, we used a volumetric absorptive microsampling (VAMS) technique to collect blood samples from the patients. A rapid and simple sample preparation method...
INTRODUCTION
In this research, we used a volumetric absorptive microsampling (VAMS) technique to collect blood samples from the patients. A rapid and simple sample preparation method and LC-MS.MS assay was then developed and validated for the simultaneous analysis of tamoxifen and its three active metabolites.
METHODS
VAMS extraction was performed in methanol by sonication-assisted extraction method for 25 min after 2 hof VAMS drying. Separation was carried out using Acquity UPLC BEH C column (2.1 x 100 mm; 1.7 µm), with a flow rate of 0.2 mL/min, and the mobile phase gradient of formic acid 0.1% and formic acid 0.1% in acetonitrile for 5 min. The multiple reaction monitoring (MRM) values were set at m/z 358.31>58.27 for -desmethyltamoxifen, m/z 372.33>72.28 for tamoxifen, m/z 388.22>72.28 for 4-hydroxytamoxifen, m/z 374.25>58.25 for endoxifen, and m/z 260.26>116.12 for propranolol.
RESULTS AND DISCUSSION
The lower limit of quantification value (LLOQ) was 2.50 ng/mL for tamoxifen, 2.50 ng/mL for endoxifen, 1.50 ng/mL for 4-hydroxitamoxifen, and 2.00 ng/mL for N-desmethyltamoxifen. Accuracy (%bias) and precision (%CV) were within 20% for LLOQ and 15% for other concentrations. There were no interference responses >20% of the LLOQ and 5% of the internal standard. The level of ion suppression in all analytes was less than 7%. The preparation system developed in this study successfully extracted more than 90% of analytes from the matrix with precision below 15%. Carryover was shown to be below 6% in all analytes. Stability of analytes in VAMS was demonstrated for up to 30 days, under room temperature storage in a sealed plastic bag with desiccant. This method was successfully applied to analyze tamoxifen and the metabolites level in 30 ER+ breast cancer patients.
Topics: Antineoplastic Agents, Hormonal; Breast Neoplasms; Chromatography, High Pressure Liquid; Drug Monitoring; Female; Humans; Reproducibility of Results; Tamoxifen; Tandem Mass Spectrometry; Time Factors
PubMed: 34113081
DOI: 10.2147/DDDT.S286409 -
Journal of Medicinal Chemistry May 2021(/)-3-(4-(()-1-(4-Hydroxyphenyl)-2-phenylbut-1-enyl)phenyl)acrylic acid (GW7604) as a derivative of ()-4-hydroxytamoxifen (4-OHT) was linked by diaminoalkane spacers to...
(/)-3-(4-(()-1-(4-Hydroxyphenyl)-2-phenylbut-1-enyl)phenyl)acrylic acid (GW7604) as a derivative of ()-4-hydroxytamoxifen (4-OHT) was linked by diaminoalkane spacers to molecules that are known binders to the coactivator binding site (benzimidazole or thioxo-quinazolinone scaffolds). With this modification, an optimization of the pharmacological profile was achieved. The most active thioxo-quinazolinone derivative showed extraordinarily high affinity to the estrogen receptor (ER) β (RBA = 110%), inhibited effectively the coactivator recruitment (IC = 20.88 nM (ERα) and 28.34 nM (ERβ)), acted as a pure estradiol (E2) antagonist in a transactivation assay (IC = 18.5 nM (ERα) and 7.5 nM (ERβ)), and downregulated the ERα content in MCF-7 cells with an efficacy of 60% at 1 μM. The cytotoxicity was restricted to hormone-dependent MCF-7 (IC = 4.2 nM) and tamoxifen-resistant MCF-7TamR cells (IC = 476.6 nM). The compounds bearing a thioxo-quinazolinone moiety can therefore be assigned as pure E2-antagonistic selective ER degraders/downregulators. By contrast, the benzimidazole derivatives acted solely as pure antagonists without degradation of the ER.
Topics: Acrylates; Benzimidazoles; Binding Sites; Binding, Competitive; Dimerization; Down-Regulation; Estradiol; Estrogen Receptor alpha; Humans; Ligands; MCF-7 Cells; Molecular Docking Simulation; Quinazolinones; Structure-Activity Relationship; Tamoxifen; Transcriptional Activation
PubMed: 33904307
DOI: 10.1021/acs.jmedchem.0c02230 -
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 -
Cell Death & Disease Apr 2021DDRGK domain-containing protein 1 (DDRGK1) is an important component of the newly discovered ufmylation system and its absence has been reported to induce extensive...
DDRGK domain-containing protein 1 (DDRGK1) is an important component of the newly discovered ufmylation system and its absence has been reported to induce extensive endoplasmic reticulum (ER) stress. Recently, emerging evidence indicates that the ufmylation system is correlated with autophagy, although the exact mechanism remains largely unknown. To explore the regulation mechanism of DDRGK1 on autophagy, in this study, we established an immortalized mouse embryonic fibroblast (MEF) cell lines harvested from the DDRGK1:ROSA26-CreERT2 mice, in which DDRGK1 depletion can be induced by 4-hydroxytamoxifen (4-OHT) treatment. Here, we show that DDRGK1 deficiency in MEFs has a dual effect on autophagy, which leads to a significant accumulation of autophagosomes. On one hand, it promotes autophagy induction by impairing mTOR signaling; on the other hand, it blocks autophagy degradation by inhibiting autophagosome-lysosome fusion. This dual effect of DDRGK1 depletion on autophagy ultimately aggravates apoptosis in MEFs. Further studies reveal that DDRGK1 loss is correlated with suppressed lysosomal function, including impaired Cathepsin D (CTSD) expression, aberrant lysosomal pH, and v-ATPase accumulation, which might be a potential trigger for impairment in autophagy process. Hence, this study confirms a crucial role of DDRGK1 as an autophagy regulator by controlling lysosomal function. It may provide a theoretical basis for the treatment strategies of various physiological diseases caused by DDRGK1 deficiency.
Topics: Adaptor Proteins, Signal Transducing; Animals; Apoptosis; Autophagy; Cell Line; Endoplasmic Reticulum Stress; Fibroblasts; Kidney; Lysosomes; Mice; Tamoxifen; Transfection
PubMed: 33879777
DOI: 10.1038/s41419-021-03694-9 -
Scientific Reports Apr 2021Conditional creER-mediated gene inactivation or gene induction has emerged as a robust tool for studying gene functions in mouse models of tissue development,...
Conditional creER-mediated gene inactivation or gene induction has emerged as a robust tool for studying gene functions in mouse models of tissue development, homeostasis, and regeneration. Here, we present a method to conditionally induce cre recombination in the mouse calvarial bone while avoiding systemic recombination in distal bones. To test our method, we utilized Prx1creER-egfp;td-Tomato mice and delivered 4-hydroxytamoxifen (4-OHT) to the mouse calvaria, subperiosteally. First, we showed that two calvaria subperiosteal injections of 10 µg of 4-OHT (3.3 mg of 4-OHT/kg of body weight) can induce local recombination as efficiently as two intraperitoneal systemic injections of 200 μg of tamoxifen (70 mg of tamoxifen/kg of body weight). Then, we studied the recombination efficiency of various subperiosteal calvaria dosages and found that two subperiosteal injections of 5 µg 4-OHT (1.65 mg of 4-OHT/kg of body weight) uphold the same recombination efficiency observed with higher dosages. Importantly, the result indicated that the low dosage does not induce significant systemic recombination in remote skeletal tissues. With the proposed local low dosage protocol, the recombination efficiency at the injection site (calvarial bone) reached 94%, while the recombination efficiency at the mandible and the digits was as low as the efficiency measured in control animals.
Topics: Animals; Bone and Bones; Enzyme Activation; Female; Gene Expression Regulation; Gene Targeting; Integrases; Mice; Mice, Inbred C57BL; Mice, Transgenic; Organ Specificity; Promoter Regions, Genetic; Receptors, Estrogen; Recombination, Genetic; Skull; Tamoxifen
PubMed: 33859263
DOI: 10.1038/s41598-021-87611-2 -
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 -
Basic Research in Cardiology Feb 2021Conditional, cell-type-specific transgenic mouse lines are of high value in cardiovascular research. A standard tool for cardiomyocyte-restricted DNA editing is the...
Conditional, cell-type-specific transgenic mouse lines are of high value in cardiovascular research. A standard tool for cardiomyocyte-restricted DNA editing is the αMHC-MerCreMer/loxP system. However, there is an ongoing debate on the occurrence of cardiac side effects caused by unspecific Cre activity or related to tamoxifen/oil overload. Here, we investigated potential adverse effects of DNA editing by the αMHC-MerCreMer/loxP system in combination with a low-dose treatment protocol with the tamoxifen metabolite 4-hydroxytamoxifen (OH-Txf). αMHC-MerCreMer mice received intraperitoneally OH-Txf (20 mg/kg) for 5 or 10 days. These treatment protocols were highly efficient to induce DNA editing in adult mouse hearts. Multi-parametric magnetic resonance imaging revealed neither transient nor permanent effects on cardiac function during or up to 19 days after 5 day OH-Txf treatment. Furthermore, OH-Txf did not affect cardiac phosphocreatine/ATP ratios assessed by in vivo P MR spectroscopy, indicating no Cre-mediated side effects on cardiac energy status. No MRI-based indication for the development of cardiac fibrosis was found as mean T1 relaxation time was unchanged. Histological analysis of myocardial collagen III content after OH-Txf confirmed this result. Last, mean T2 relaxation time was not altered after Txf treatment suggesting no pronounced cardiac lipid accumulation or tissue oedema. In additional experiments, cardiac function was assessed for up to 42 days to investigate potential delayed side effects of OH-Txf treatment. Neither 5- nor 10-day treatment resulted in a depression of cardiac function. Efficient cardiomyocyte-restricted DNA editing that is free of unwanted side effects on cardiac function, energetics or fibrosis can be achieved in adult mice when the αMHC-MerCreMer/loxP system is activated by the tamoxifen metabolite OH-Txf.
Topics: Animals; Energy Metabolism; Fibrosis; Gene Editing; Gene Expression Regulation; Glycogen Synthase Kinase 3 beta; Integrases; Male; Mice, Inbred C57BL; Mice, Transgenic; Myocytes, Cardiac; Myosin Heavy Chains; Proto-Oncogene Proteins c-akt; Tamoxifen; Time Factors; Ventricular Function, Left; Ventricular Remodeling; p38 Mitogen-Activated Protein Kinases; Mice
PubMed: 33544211
DOI: 10.1007/s00395-020-00841-9 -
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