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Tissue Engineering. Part A Oct 2019Cancer invasion, metastasis, and therapy resistance are the crucial phenomena in cancer malignancy. The high expression of matrix metalloproteinase 9 (MMP9) is a...
Cancer invasion, metastasis, and therapy resistance are the crucial phenomena in cancer malignancy. The high expression of matrix metalloproteinase 9 (MMP9) is a biomarker as well as a causal factor of cancer invasiveness and metastatic activity. However, a regulatory mechanism underlying MMP9 expression in cancer is not clarified yet. In addition, a new strategy for anticancer drug discovery is becoming an important clue. In the present study, we aimed (i) to develop a novel reporter system evaluating tumorigenesis, invasiveness, metastasis, and druggability with a combination of three-dimensional tumoroid model and promoter and (ii) to examine pharmacological actions of anticancer medications using this reporter system. High expression and genetic amplification of were found in colon cancer cases. We found that proximal promoter sequences of in murine and human contained conserved binding sites for transcription factors β-catenin/TCF/LEF, glucocorticoid receptor (GR), and nuclear factor kappa-B (NF-κB). The murine promoter (-569 to +19) was markedly activated in metastatic colon cancer cells and additionally activated by tumoroid formation and by β-catenin signaling stimulator lithium chloride. The promoter-driven fluorescent reporter cells enabled the monitoring of activities of MMP9/gelatinase, tumorigenesis, invasion, and metastasis in syngeneic transplantation experiments. We also demonstrated pharmacological actions as follows: dexamethasone and hydrocortisone, steroidal medications binding to GR, inhibited the promoter but did not inhibit tumorigenesis. On the contrary, antimetabolite 5-fluorouracil, a gold standard for colon cancer chemotherapy, inhibited tumoroid formation but did not inhibit promoter activity. Notably, antimalaria medication artesunate inhibited both tumorigenesis and the promoter , potentially through inhibition of β-catenin/TCF/LEF signaling. Thus, this novel reporter system enabled monitoring tumorigenesis, invasiveness, metastasis, key regulatory signalings such as β-catenin/MMP9 axis, and druggability. Impact Statement Cancer invasion and metastasis have been shown to be driven by matrix metalloproteinase 9 (MMP9), whose expression mechanism is not clarified yet. In addition, a new strategy for anticancer drug discovery is becoming important. We established a novel reporter system evaluating tumorigenesis, invasiveness, metastasis, and druggability with a combination of three-dimensional (3D) tumoroid model and promoter. Using this reporter system, we demonstrated pharmacological actions of anticancer medications such as antimetabolite 5-fluorouracil (5-FU) and antimalaria medication artesunate (ART), which inhibited both tumorigenesis and β-catenin/MMP regulatory signaling. Our study impacts the translational fields of oncology, drug discovery, and organoid model.
Topics: Animals; Artesunate; Carcinogenesis; Cell Line, Tumor; Colonic Neoplasms; Dexamethasone; Female; Fluorescence; Gelatinases; Genes, Reporter; Humans; Matrix Metalloproteinase 9; Mice, Inbred BALB C; Neoplasm Metastasis; Promoter Regions, Genetic; Protein Stability; RNA, Messenger; Tetradecanoylphorbol Acetate; beta Catenin
PubMed: 30734664
DOI: 10.1089/ten.TEA.2018.0348 -
Frontiers in Immunology 2023The infusion of -generated regulatory B cells may represent a promising novel therapeutic approach for a variety of autoimmune and hyperinflammatory conditions including...
INTRODUCTION
The infusion of -generated regulatory B cells may represent a promising novel therapeutic approach for a variety of autoimmune and hyperinflammatory conditions including graft-versus-host disease.
METHODS
Previously, we developed a protocol for the generation of a novel population of regulatory B cells, which are characterized by secretion of enzymatically active granzyme B (). This protocol uses recombinant interleukin 21 (IL-21) and goat-derived F(ab)'2 fragments against the human B cell receptor (anti-BCR). Generally, the use of xenogeneic material for the manufacturing of advanced therapy medicinal products should be avoided to prevent adverse immune reactions as well as potential transmission of so far unknown diseases.
RESULTS
In the present work we demonstrated that phorbol-12-myristate-13-acetate (PMA/TPA), a phorbol ester with a particular analogy to the second messenger diacylglycerol (DAG), is a potent enhancer of IL-21-induced differentiation of pre-activated B cells into . The percentage of after stimulation of pre-activated B cells with IL-21 and PMA/TPA was not significantly lower compared to stimulation with IL-21 and anti-BCR.
DISCUSSION
Given that PMA/TPA has already undergone encouraging clinical testing in patients with certain haematological diseases, our results suggest that PMA/TPA may be a safe and feasible alternative for manufacturing of .
Topics: Humans; B-Lymphocytes, Regulatory; Granzymes; Tetradecanoylphorbol Acetate
PubMed: 37588597
DOI: 10.3389/fimmu.2023.1194880 -
Alcoholism, Clinical and Experimental... Jul 2020EtOH has a significant effect on synaptic plasticity. Munc13-1 is an essential presynaptic active zone protein involved in priming the synaptic vesicle and releasing...
BACKGROUND
EtOH has a significant effect on synaptic plasticity. Munc13-1 is an essential presynaptic active zone protein involved in priming the synaptic vesicle and releasing neurotransmitter in the brain. It is a peripheral membrane protein and binds to the activator, diacylglycerol (DAG)/phorbol ester at its membrane-targeting C1 domain. Our previous studies identified Glu-582 of C1 domain as the alcohol-binding residue (Das, J. et al, J. Neurochem., 126, 715-726, 2013).
METHODS
Here, we describe a 250 ns molecular dynamics (MD) simulation study on the interaction of EtOH and the activator-bound Munc13-1 C1 in the presence of varying concentrations of phosphatidylserine (PS).
RESULTS
In this study, Munc13-1 C1 shows higher conformational stability in EtOH than in water. It forms fewer hydrogen bonds with phorbol 13-acetate in the presence of EtOH than in water. EtOH also affected the interaction between the protein and the membrane and between the activator and the membrane. Similar studies in a E582A mutant suggest that these effects of EtOH are mostly mediated through Glu-582.
CONCLUSIONS
EtOH forms hydrogen bonds with Glu-582. While occupancy of the EtOH molecules at the vicinity (4Å) of Glu-582 is 34.4%, the occupancy in the E582A mutant is 26.5% of the simulation time. In addition, the amount of PS in the membrane influences the conformational stability of the C1 domain and interactions in the ternary complex. This study is important in providing the structural basis of EtOH's effects on synaptic plasticity.
Topics: Central Nervous System Depressants; Ethanol; Humans; Molecular Dynamics Simulation; Mutation; Nerve Tissue Proteins; Phorbol Esters; Presynaptic Terminals; Protein Conformation; Protein Domains; Synaptic Membranes
PubMed: 32424866
DOI: 10.1111/acer.14363 -
Scientific Reports Jul 2023Jatropha curcas is an oilseed crop with biorefinery applications. Whilst cake generated following oil extraction offers potential as a protein source for animal feed,...
Jatropha curcas is an oilseed crop with biorefinery applications. Whilst cake generated following oil extraction offers potential as a protein source for animal feed, inactivation of toxic phorbol esters present in the material is necessary. Pleurotus pulmonarius is a detoxifying agent for jatropha cake with additional potential as animal feed, edible mushroom and for enzyme production. For the characterization of fungal genes involved in phorbol ester degradation, together with other industrial applications, reverse transcription-quantitative PCR (RT-qPCR) is a tool that enables accurate quantification of gene expression. For this, reliable analysis requires reference genes for normalization of mRNA levels validated under conditions employed for target genes. The stability of potential reference genes β-TUB, ACTIN, GAPDH, PHOS, EF1α, TRPHO, LAC, MNP3, MYP and VP were evaluated following growth of P. pulmonarius on toxic, non-toxic jatropha cake and a combined treatment, respectively. NormFinder and geNorm algorithms for expression stability analysis identified PHOS, EF1α and MNP3 as appropriate for normalizing gene expression. Reference gene combinations contrasting in ranking were compared following normalization of relative expression of the CHU_2040 gene, encoding an esterase enzyme potentially involved in phorbol ester degradation. The reference genes for P. pulmonarius will facilitate the elucidation of mechanisms involved in detoxification of phorbol esters as well as analysis of target genes for application in biorefinery models.
Topics: Animals; Reverse Transcription; Pleurotus; Agaricales; Animal Feed; Jatropha
PubMed: 37516784
DOI: 10.1038/s41598-023-39115-4 -
Frontiers in Immunology 2023Neutrophil extracellular traps (NET) formation is one important host innate defense mechanism elicited by polymorphonuclear neutrophils (PMN). NETs are composed by...
Neutrophil extracellular traps (NET) formation is one important host innate defense mechanism elicited by polymorphonuclear neutrophils (PMN). NETs are composed by chromatin and proteins with microbicidal and signaling activity. So far, there is one report on triggered NETs in cattle, however, exact mechanisms, including signalling pathways and dynamics governing this reaction remain largely unknown. Recently, involvement of cell cycle proteins was demonstrated for phorbol myristate acetate (PMA)-triggered human PMN-derived NETs. Here, we studied the involvement of cell cycle proteins in -induced NETs in exposed bovine PMN. Through confocal and transmission electron microscopy we discovered that Ki-67 and lamin B1 signals are upregulated and relocated during -induced NETosis. Nuclear membrane disruption was also observed as a hallmark of NET formation in bovine PMN confronted with viable tachyzoites, mimicking some steps of mitosis. However, we did not observe centrosome duplication as previously described for human PMN-derived NET formation stimulated with PMA.
Topics: Humans; Cattle; Animals; Extracellular Traps; Cell Cycle Proteins; Toxoplasma; Neutrophils; Cell Cycle; Receptor Protein-Tyrosine Kinases; Tetradecanoylphorbol Acetate
PubMed: 36875070
DOI: 10.3389/fimmu.2023.1125667 -
Microorganisms Aug 2022This work focused on obtaining fermented oil cake (cotton or ) via macrofungi growth with potential characteristics for animal feed formulations, such as the presence of...
This work focused on obtaining fermented oil cake (cotton or ) via macrofungi growth with potential characteristics for animal feed formulations, such as the presence of extracellular enzymes, bioactive (ergosterol and antioxidants), and detoxification of antinutritional compounds. The concentration of phorbol esters was reduced by four macrofungi in seed cake (JSC) to non-toxic levels. At least two macrofungi efficiently degraded free gossypol in cottonseed cake (CSC). Fermentation with sp. INPA1646 and sp. INPA1696 resulted in increased ergosterol concentrations, antioxidant activity reduction, and high activity of laccases and proteases. Bromatological analysis indicated high crude protein concentrations, with partial solubilization by fungal proteases. Fermented products from sp. and sp. in JSC or CSC can be considered important biological inputs for monogastric and polygastric animal feed.
PubMed: 36014089
DOI: 10.3390/microorganisms10081670 -
ELife Aug 2020Previously, we showed that modulation of the energy barrier for synaptic vesicle fusion boosts release rates supralinearly (Schotten, 2015). Here we show that mouse...
Previously, we showed that modulation of the energy barrier for synaptic vesicle fusion boosts release rates supralinearly (Schotten, 2015). Here we show that mouse hippocampal synapses employ this principle to trigger Ca-dependent vesicle release and post-tetanic potentiation (PTP). We assess energy barrier changes by fitting release kinetics in response to hypertonic sucrose. Mimicking activation of the C2A domain of the Ca-sensor Synaptotagmin-1 (Syt1), by adding a positive charge (Syt1) or increasing its hydrophobicity (Syt1), lowers the energy barrier. Removing Syt1 or impairing its release inhibitory function (Syt1) increases spontaneous release without affecting the fusion barrier. Both phorbol esters and tetanic stimulation potentiate synaptic strength, and lower the energy barrier equally well in the presence and absence of Syt1. We propose a model where tetanic stimulation activates Syt1-independent mechanisms that lower the energy barrier and act additively with Syt1-dependent mechanisms to produce PTP by exerting multiplicative effects on release rates.
Topics: Animals; Calcium; Cells, Cultured; Female; Hippocampus; Male; Membrane Fusion; Mice; Mice, Inbred C57BL; Neuronal Plasticity; Rats; Rats, Wistar; Synaptic Vesicles; Synaptotagmin I
PubMed: 32831174
DOI: 10.7554/eLife.55713 -
Genetics Jul 2022Activated Gαq signals through phospholipase-Cβ and Trio, a Rho GTPase exchange factor (RhoGEF), but how these distinct effector pathways promote cellular responses to...
Activated Gαq signals through phospholipase-Cβ and Trio, a Rho GTPase exchange factor (RhoGEF), but how these distinct effector pathways promote cellular responses to neurotransmitters like serotonin remains poorly understood. We used the egg-laying behavior circuit of Caenorhabditis elegans to determine whether phospholipase-Cβ and Trio mediate serotonin and Gαq signaling through independent or related biochemical pathways. Our genetic rescue experiments suggest that phospholipase-Cβ functions in neurons while Trio Rho GTPase exchange factor functions in both neurons and the postsynaptic vulval muscles. While Gαq, phospholipase-Cβ, and Trio Rho GTPase exchange factor mutants fail to lay eggs in response to serotonin, optogenetic stimulation of the serotonin-releasing HSN neurons restores egg laying only in phospholipase-Cβ mutants. Phospholipase-Cβ mutants showed vulval muscle Ca2+ transients while strong Gαq and Trio Rho GTPase exchange factor mutants had little or no vulval muscle Ca2+ activity. Treatment with phorbol 12-myristate 13-acetate that mimics 1,2-diacylglycerol, a product of PIP2 hydrolysis, rescued egg-laying circuit activity and behavior defects of Gαq signaling mutants, suggesting both phospholipase-C and Rho signaling promote synaptic transmission and egg laying via modulation of 1,2-diacylglycerol levels. 1,2-Diacylglycerol activates effectors including UNC-13; however, we find that phorbol esters, but not serotonin, stimulate egg laying in unc-13 and phospholipase-Cβ mutants. These results support a model where serotonin signaling through Gαq, phospholipase-Cβ, and UNC-13 promotes neurotransmitter release, and that serotonin also signals through Gαq, Trio Rho GTPase exchange factor, and an unidentified, phorbol 12-myristate 13-acetate-responsive effector to promote postsynaptic muscle excitability. Thus, the same neuromodulator serotonin can signal in distinct cells and effector pathways to coordinate activation of a motor behavior circuit.
Topics: Animals; Caenorhabditis elegans; Calcium; Diglycerides; GTP-Binding Proteins; Myristates; Neurotransmitter Agents; Phorbols; Phospholipases; Rho Guanine Nucleotide Exchange Factors; Serotonin; rho GTP-Binding Proteins
PubMed: 35579369
DOI: 10.1093/genetics/iyac084 -
International Journal of Molecular... Feb 2022A phenyl ethanoid, salidroside (SAL), and two secoiridoids, 8()-nuezhenide (NZD) and ligustroside (LIG), were isolated from fruits of used as traditional folk medicine,...
A phenyl ethanoid, salidroside (SAL), and two secoiridoids, 8()-nuezhenide (NZD) and ligustroside (LIG), were isolated from fruits of used as traditional folk medicine, and their chemical structures were elucidated by the comparison of spectral data with published literature. Matrix metalloproteinases (MMPs) are major enzymes that play crucial roles in the metastasis and invasive behavior of tumors. In particular, MMP-2 and MMP-9, regulated by the MAPK signaling pathways, including p38, ERK and JNK, are known to play a key role in the degradation of the basement membrane. In the present study, the effects of SAL, NZD and LIG on the expression of MMP-2 and -9 were examined in phorbol 12-myristate 13-acetate (PMA)-induced HT 1080 cells. All the compounds significantly lowered the amount of MMP-2 and MMP-9 released, as determined by gelatin zymography and ELISA. In addition, the mRNA and protein expression levels of MMP-2 and MMP-9 were significantly suppressed, as measured by RT-PCR and Western blotting. According to the Western blotting assay, SAL and LIG effectively reduced the expression of MMP-2 in a dose-dependent manner. NZD lowered the expression of MMP-9 in a similar way. The phosphorylation of p38, ERK and JNK was also significantly suppressed by these compounds. These findings suggest that all the compounds regulate the release and expression of MMP-2 and MMP-9 via MAPK signaling pathways.
Topics: Fibrosarcoma; Fruit; Glucosides; Humans; Ligustrum; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Phenols; Pyrans; Tetradecanoylphorbol Acetate
PubMed: 35269801
DOI: 10.3390/ijms23052660 -
Cells Sep 2020Podoplanin and CD44 are transmembrane glycoproteins involved in inflammation and cancer. In this paper, we report that podoplanin is coordinately expressed with the CD44...
Podoplanin and CD44 are transmembrane glycoproteins involved in inflammation and cancer. In this paper, we report that podoplanin is coordinately expressed with the CD44 standard (CD44s) and variant (CD44v) isoforms in vivo-in hyperplastic skin after a pro-inflammatory stimulus with 12--tetradecanoylphorbol-13-acetate (TPA)-and in vitro-in cell lines representative of different stages of mouse-skin chemical carcinogenesis, as well as in human squamous carcinoma cell (SCC) lines. Moreover, we identify CD44v10 in the mouse-skin carcinogenesis model as the only CD44 variant isoform expressed in highly aggressive spindle carcinoma cell lines together with CD44s and podoplanin. We also characterized CD44v3-10, CD44v6-10 and CD44v8-10 as the major variant isoforms co-expressed with CD44s and podoplanin in human SCC cell lines. Immunofluorescence confocal microscopy experiments show that these CD44v isoforms colocalize with podoplanin at plasma membrane protrusions and cell-cell contacts of SCC cells, as previously reported for CD44s. Furthermore, CD44v isoforms colocalize with podoplanin in chemically induced mouse-skin SCCs in vivo. Co-immunoprecipitation experiments indicate that podoplanin physically binds to CD44v3-10, CD44v6-10 and CD44v8-10 isoforms, as well as to CD44s. Podoplanin-CD44 interaction is mediated by the transmembrane and cytosolic regions and is negatively modulated by glycosylation of the extracellular domain. These results point to a functional interplay of podoplanin with both CD44v and CD44s isoforms in SCCs and give insight into the regulation of the podoplanin-CD44 association.
Topics: Animals; Carcinogenesis; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Membrane; Cell Surface Extensions; Humans; Hyaluronan Receptors; Membrane Glycoproteins; Mice; Phorbol Esters; Protein Binding; Protein Domains; Protein Isoforms
PubMed: 33003440
DOI: 10.3390/cells9102200