-
Analytical Chemistry Feb 2019Monitoring drug uptake, its metabolism, and response on the single-cell level is invaluable for sustaining drug discovery efforts. In this study, we show the possibility...
Monitoring drug uptake, its metabolism, and response on the single-cell level is invaluable for sustaining drug discovery efforts. In this study, we show the possibility of accessing the information about the aforementioned processes at the single-cell level by monitoring the anticancer drug tamoxifen using live single-cell mass spectrometry (LSC-MS) and Raman spectroscopy. First, we explored whether Raman spectroscopy could be used as a label-free and nondestructive screening technique to identify and predict the drug response at the single-cell level. Then, a subset of the screened cells was isolated and analyzed by LSC-MS to measure tamoxifen and its metabolite, 4-Hydroxytamoxifen (4-OHT) in a highly selective, sensitive, and semiquantitative manner. Our results show the Raman spectral signature changed in response to tamoxifen treatment which allowed us to identify and predict the drug response. Tamoxifen and 4-OHT abundances quantified by LSC-MS suggested some heterogeneity among single-cells. A similar phenomenon was observed in the ratio of metabolized to unmetabolized tamoxifen across single-cells. Moreover, a correlation was found between tamoxifen and its metabolite, suggesting that the drug was up taken and metabolized by the cell. Finally, we found some potential correlations between Raman spectral intensities and tamoxifen abundance, or its metabolism, suggesting a possible relationship between the two signals. This study demonstrates for the first time the potential of using Raman spectroscopy and LSC-MS to investigate pharmacokinetics at the single-cell level.
Topics: Antineoplastic Agents; Hep G2 Cells; Humans; Mass Spectrometry; Multivariate Analysis; Proof of Concept Study; Reproducibility of Results; Single-Cell Analysis; Spectrum Analysis, Raman; Tamoxifen
PubMed: 30664349
DOI: 10.1021/acs.analchem.8b04393 -
Nature Chemical Biology Nov 2016CRISPR-Cas9 has emerged as a powerful technology that enables ready modification of the mammalian genome. The ability to modulate Cas9 activity can reduce off-target...
CRISPR-Cas9 has emerged as a powerful technology that enables ready modification of the mammalian genome. The ability to modulate Cas9 activity can reduce off-target cleavage and facilitate precise genome engineering. Here we report the development of a Cas9 variant whose activity can be switched on and off in human cells with 4-hydroxytamoxifen (4-HT) by fusing the Cas9 enzyme with the hormone-binding domain of the estrogen receptor (ERT2). The final optimized variant, termed iCas, showed low endonuclease activity without 4-HT but high editing efficiency at multiple loci with the chemical. We also tuned the duration and concentration of 4-HT treatment to reduce off-target genome modification. Additionally, we benchmarked iCas against other chemical-inducible methods and found that it had the fastest on rate and that its activity could be toggled on and off repeatedly. Collectively, these results highlight the utility of iCas for rapid and reversible control of genome-editing function.
Topics: CRISPR-Cas Systems; Gene Editing; HEK293 Cells; Humans; Tamoxifen
PubMed: 27618190
DOI: 10.1038/nchembio.2179 -
Molecular Pharmacology Feb 2023G protein-coupled receptors (GPCRs) constitute the most versatile family of pharmacological target proteins. For some "orphan" GPCRs, no ligand or drug-like modulator is...
G protein-coupled receptors (GPCRs) constitute the most versatile family of pharmacological target proteins. For some "orphan" GPCRs, no ligand or drug-like modulator is known. In this study, we have established and applied a parallelized assay to coscreen 29 different human GPCRs. Three compounds, chlorhexidine, Lys-05, and 9-aminoacridine, triggered transient Ca signals linked to the expression of GPR30. GPR30, also named G protein-coupled estrogen receptor 1 (GPER1), was reported to elicit increases in cAMP in response to 17-estradiol, 4-hydroxytamoxifen, or G-1. These findings could, however, not be reproduced by other groups, and the deorphanization of GPR30 is, therefore, intensely disputed. The unbiased screen and following experiments in transiently or stably GPR30-overexpressing HEK293 cells did not show responses to 17-estradiol, 4-hydroxytamoxifen, or G-1. A thorough analysis of the activated signaling cascade revealed a canonical G-coupled pathway, including phospholipase C, protein kinase C and ERK activation, receptor internalization, and sensitivity to the G inhibitor YM-254890. When expressed in different cell lines, the localization of a fluorescent GPR30 fusion protein appeared variable. An efficient integration into the plasma membrane and stronger functional responses were found in HEK293 and in MCF-7 cells, whereas GPR30 appeared mostly retained in endomembrane compartments in Cos-7 or HeLa cells. Thus, conflicting findings may result from the use of different cell lines. The newly identified agonists and the finding that GPR30 couples to G are expected to serve as a starting point for identifying physiologic responses that are controlled by this GPCR. SIGNIFICANCE STATEMENT: This study has identified and thoroughly characterized novel and reliably acting agonists of the G protein-coupled receptor GPER1/GPR30. Applying these agonists, this study demonstrates that GPR30 couples to the canonical G-phospholipase C pathway and is rapidly internalized upon continuous exposure to the agonists.
Topics: Humans; HEK293 Cells; HeLa Cells; Receptors, G-Protein-Coupled; Estradiol
PubMed: 36400433
DOI: 10.1124/molpharm.122.000580 -
Stem Cell Research Oct 2017Induced pluripotent stem cells (iPSCs), which can be differentiated into various tissues and cell types, have been used for clinical research and disease modeling....
Induced pluripotent stem cells (iPSCs), which can be differentiated into various tissues and cell types, have been used for clinical research and disease modeling. Self-organizing three-dimensional (3D) tissue engineering has been established within the past decade and enables researchers to obtain tissues and cells that almost mimic in vivo development. However, there are no reports of practical experimental procedures that reproduce photoreceptor degeneration. In this study, we induced photoreceptor cell death in mouse iPSC-derived 3D retinal organoids (3D-retinas) by 4-hydroxytamoxifen (4-OHT), which induces photoreceptor degeneration in mouse retinal explants, and then established a live-cell imaging system to measure degeneration-related properties. Furthermore, we quantified the protective effects of representative ophthalmic supplements for treating the photoreceptor degeneration. This drug evaluation system enables us to monitor drug effects in photoreceptor cells and could be useful for drug screening.
Topics: Animals; Apoptosis; Cell Differentiation; Cytoprotection; Green Fluorescent Proteins; Induced Pluripotent Stem Cells; Mice, Inbred C57BL; Organoids; Photoreceptor Cells, Vertebrate; Retina; Retinal Degeneration; Tamoxifen
PubMed: 28886418
DOI: 10.1016/j.scr.2017.08.018 -
BioTechniques Jul 1999Existing conditional expression systems can be classified in two major categories that are based either on the induction or on the de-repression of transcription. The...
Existing conditional expression systems can be classified in two major categories that are based either on the induction or on the de-repression of transcription. The system described here combines both mechanisms, since a unique transcription factor can be shifted from a repression to a stimulation activity by simply changing its ligand. The resulting advantage of this system is the complete absence of basal expression before active induction. The principle of this method is based on the unexpected ability of the chimeric protein containing the DNA-binding domain of the yeast Gal4 transcription factor fused to the COOH half of the estradiol receptor (GalER), to act as a repressor when bound to the drug 4OH-tamoxifen, in the context of a previously described optimized Gal4-responsive promoter. The efficacy of this system has been assessed in transient expression assays using the chloramphenicol acetyl transferase (CAT), and in situ, through the activity of a Gal4 responsive beta-galactosidase gene.
Topics: Animals; CHO Cells; Chloramphenicol O-Acetyltransferase; Cricetinae; DNA-Binding Proteins; Estradiol; Eukaryotic Cells; Fungal Proteins; Gene Expression Regulation; Genes, Reporter; Genetic Techniques; Humans; Lac Operon; Ligands; Receptors, Estradiol; Recombinant Fusion Proteins; Repressor Proteins; Saccharomyces cerevisiae Proteins; Tamoxifen; Transcription Factors; Transfection; Transgenes
PubMed: 10407672
DOI: 10.2144/99271st03 -
Autophagy Sep 2013Therapy-induced autophagy is recognized as a critical determinant of treatment outcome in cancer patients, primarily as a factor underlying drug resistance. However,...
Therapy-induced autophagy is recognized as a critical determinant of treatment outcome in cancer patients, primarily as a factor underlying drug resistance. However, recent investigations point toward a context-dependent, death-inducing role for autophagy, the mechanism of which remains largely unknown. Our recent study provides evidence that autophagy can directly mediate cell killing in multiple tumor cell types by facilitating degradation of KRAS/K-Ras, a key survival protein. These findings have broad implications for strategies employing autophagy modulation to target tumor cells.
Topics: Autophagy; Cytoprotection; MAP Kinase Signaling System; Protein Kinase C; Proteolysis; Proto-Oncogene Proteins p21(ras); Tamoxifen
PubMed: 24121562
DOI: 10.4161/auto.25837 -
Biochemical and Biophysical Research... Apr 2016Triple negative breast cancer (TNBC) is the hardest breast cancer subtype to treat due to lacking therapeutic target and treatment options. In this study, we found that...
Triple negative breast cancer (TNBC) is the hardest breast cancer subtype to treat due to lacking therapeutic target and treatment options. In this study, we found that SLUG expression was much higher in TNBC MDA-MB-231 cells than estrogen receptor alpha (ERα) positive breast cancer MCF7 cells. 4-hydroxytamoxifen (4-OHT) promoted SLUG expression, which was blocked by curcumin. Further investigation showed that SLUG activated the transcription of hexokinase-2 (HK2) by binding to HK2 promoter. SLUG knockdown inhibited HK2 expression and weakened 4-OHT resistance of MDA-MB-231 cells. Conversely, SLUG overexpression elevated HK2 level and increased 4-OHT resistance of MCF7 cells. Combination of curcumin and 4-OHT suppressed SLUG and HK2 expression, leading to mitochondrion-mediated apoptosis. These results suggested SLUG as a potential target and curcumin as a promising natural agent for overcoming 4-OHT resistance of TNBC.
Topics: Antineoplastic Agents; Apoptosis; Base Sequence; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cell Survival; Chromatin Immunoprecipitation; Curcumin; Drug Resistance, Neoplasm; Estrogen Receptor alpha; Female; Flow Cytometry; Glycolysis; Hexokinase; Humans; Mitochondria; Molecular Sequence Data; Promoter Regions, Genetic; Snail Family Transcription Factors; Tamoxifen; Transcription Factors; Triple Negative Breast Neoplasms
PubMed: 27012210
DOI: 10.1016/j.bbrc.2016.03.067 -
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 -
Clinical Laboratory 2016Recent findings have revealed that abnormal expression of microRNAs (miRNA, miR) contributes to the malignancies of various cancers. Here, we report a novel miRNA that...
BACKGROUND
Recent findings have revealed that abnormal expression of microRNAs (miRNA, miR) contributes to the malignancies of various cancers. Here, we report a novel miRNA that regulates the expression of Beclin-1 in breast cancer cells.
METHODS
The expression of miR-124-3p and Beclin-1 was identified in breast cancer tissues and breast cancer cell lines. To explore whether Beclin-1 was the target gene of miR-124-3p, luciferase reporter assay was applied. MIR-124-3p was overexpressed or inhibited with the corresponding mimics or inhibitors. The expression of autophagy-related proteins including Beclin-1 and LC3II were explored by western blot and quantitative real-time PCR.
RESULTS
We first demonstrated that miR-124-3p was decreased in breast cancer tissues and breast cancer cells lines. Furthermore, we validated that miR-124-3p could negatively regulate the expression of Beclin-1. Increased miR-124-3p significantly decreased the expression of Beclin-1 and LC3I. Further study showed that overexpres- sion of miR-124-3p could partially reverse 4-hydroxytamoxifen (4-OHT)-induced autophagy in breast cancer cells.
CONCLUSIONS
Decreased miR-124-3p expression prompted breast cancer cell progression mainly by enhancing the expression of autophagy related protein, Beclin-1.
Topics: Apoptosis Regulatory Proteins; Autophagy; Beclin-1; Breast Neoplasms; Cell Survival; Down-Regulation; Female; Gene Expression Regulation, Neoplastic; Humans; MCF-7 Cells; Membrane Proteins; MicroRNAs; Selective Estrogen Receptor Modulators; Signal Transduction; Tamoxifen; Transfection
PubMed: 27468577
DOI: 10.7754/clin.lab.2015.151111 -
Development (Cambridge, England) May 2013We have generated an inducible system to control the timing of transgene expression in zebrafish and chick. An estrogen receptor variant (ERT2) fused to the GAL4...
We have generated an inducible system to control the timing of transgene expression in zebrafish and chick. An estrogen receptor variant (ERT2) fused to the GAL4 transcriptional activator rapidly and robustly activates transcription within 3 hours of treatment with the drug 4-hydroxy-tamoxifen (4-OHT) in tissue culture and transgenic zebrafish. We have generated a broadly expressed inducible ERT2-GAL4 zebrafish line using the ubiquitin (ubi) enhancer. In addition, use of ERT2-GAL4 in conjunction with tissue-specific enhancers enables the control of transgene expression in both space and time. This spatial restriction and the ability to sustain forced expression are important advantages over the currently used heat-shock promoters. Moreover, in contrast to currently available TET and LexA systems, which require separate constructs with their own unique recognition sequences, ERT2-GAL4 is compatible with the growing stock of UAS lines being generated in the community. We also applied the same inducible system to the chick embryo and find that it is fully functional, suggesting that this strategy is generally applicable.
Topics: Animals; Animals, Genetically Modified; Chick Embryo; Crosses, Genetic; Developmental Biology; Gene Expression Regulation, Developmental; HEK293 Cells; Humans; Immunohistochemistry; In Situ Hybridization; Phenotype; Receptors, Estrogen; Tamoxifen; Transgenes; Zebrafish; Zebrafish Proteins
PubMed: 23633515
DOI: 10.1242/dev.091520