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Medicina (Kaunas, Lithuania) Jun 2024: As modulators of the tumor microenvironment, macrophages have been extensively studied for their potential in developing anticancer strategies, particularly in...
: As modulators of the tumor microenvironment, macrophages have been extensively studied for their potential in developing anticancer strategies, particularly in regulating macrophage polarization towards an antitumorigenic (M1) phenotype rather than a protumorigenic (M2) one in various experimental models. Here, we evaluated the effect of PD98059, a mitogen-activated protein kinase kinase MAPKK MEK1-linked pathway inhibitor, on the differentiation and polarization of THP-1 monocytes in response to phorbol-12-myristate-13-acetate (PMA) under various culture conditions for tumor microenvironmental application. : Differentiation and polarization of THP-1 were analyzed by flow cytometry and RT-PCR. Polarized THP-1 subsets with different treatment were compared by motility, phagocytosis, and so on. : Clearly, PMA induced THP-1 differentiation occurs in adherent culture conditions more than nonadherent culture conditions by increasing CD11b expression up to 90%, which was not affected by PD98059 when cells were exposed to PMA first (post-PD) but inhibited when PD98059 was treated prior to PMA treatment (pre-PD). CD11b THP-1 cells treated with PMA and PMA-post-PD were categorized into M0 (HLA-DR and CD206), M1 (HLA-DR and CD206), and M2 (HLA-DR and CD206), resulting in an increased population of M1 macrophages. The transcription levels of markers of macrophage differentiation and polarization confirmed the increased M1 polarization of THP-1 cells with post-PD treatment rather than with PMA-only treatment. The motility and cytotoxicity of THP-1 cells with post-PD treatment were higher than THP-1 cells with PMA, suggesting that post-PD treatment enhanced the anti-tumorigenicity of THP-1 cells. Confocal microscopy and flow cytometry showed the effect of post-PD treatment on phagocytosis by THP-1 cells. : We have developed an experimental model of macrophage polarization with THP-1 cells which will be useful for further studies related to the tumor microenvironment.
Topics: Humans; Macrophages; Tetradecanoylphorbol Acetate; Flavonoids; THP-1 Cells; Cell Differentiation; Monocytes; Flow Cytometry; Phagocytosis
PubMed: 38929626
DOI: 10.3390/medicina60061009 -
International Journal of Molecular... Jun 2024LPA receptors were expressed in TREx HEK 293 cells, and their signaling and phosphorylation were studied. The agonist, lysophosphatidic acid (LPA), increased...
LPA receptors were expressed in TREx HEK 293 cells, and their signaling and phosphorylation were studied. The agonist, lysophosphatidic acid (LPA), increased intracellular calcium and ERK phosphorylation through pertussis toxin-insensitive processes. Phorbol myristate acetate, but not LPA, desensitizes LPA-mediated calcium signaling, the agonists, and the phorbol ester-induced LPA internalization. Pitstop 2 (clathrin heavy chain inhibitor) markedly reduced LPA-induced receptor internalization; in contrast, phorbol ester-induced internalization was only delayed. LPA induced rapid β-arrestin-LPA receptor association. The agonist and the phorbol ester-induced marked LPA receptor phosphorylation, and phosphorylation sites were detected using mass spectrometry. Phosphorylated residues were detected in the intracellular loop 3 (S221, T224, S225, and S229) and in the carboxyl terminus (S321, S325, S331, T333, S335, Y337, and S343). Interestingly, phosphorylation sites are within sequences predicted to constitute β-arrestin binding sites. These data provide insight into LPA receptor signaling and regulation.
Topics: Humans; Receptors, Lysophosphatidic Acid; Phosphorylation; HEK293 Cells; Signal Transduction; Lysophospholipids; beta-Arrestins; Binding Sites; Calcium Signaling
PubMed: 38928196
DOI: 10.3390/ijms25126491 -
Biomolecules May 2024Nuclear hormone receptors exist in dynamic equilibrium between transcriptionally active and inactive complexes dependent on interactions with ligands, proteins, and...
Nuclear hormone receptors exist in dynamic equilibrium between transcriptionally active and inactive complexes dependent on interactions with ligands, proteins, and chromatin. The present studies examined the hypothesis that endogenous ligands activate peroxisome proliferator-activated receptor-β/δ (PPARβ/δ) in keratinocytes. The phorbol ester treatment or HRAS infection of primary keratinocytes increased fatty acids that were associated with enhanced PPARβ/δ activity. Fatty acids caused PPARβ/δ-dependent increases in chromatin occupancy and the expression of angiopoietin-like protein 4 () mRNA. Analyses demonstrated that stearoyl Co-A desaturase 1 () mediates an increase in intracellular monounsaturated fatty acids in keratinocytes that act as PPARβ/δ ligands. The activation of PPARβ/δ with palmitoleic or oleic acid causes arrest at the G2/M phase of the cell cycle of HRAS-expressing keratinocytes that is not found in similarly treated HRAS-expressing -null keratinocytes. HRAS-expressing -null mouse keratinocytes exhibit enhanced cell proliferation, an effect that is mitigated by treatment with palmitoleic or oleic acid. Consistent with these findings, the ligand activation of PPARβ/δ with GW0742 or oleic acid prevented UVB-induced non-melanoma skin carcinogenesis, an effect that required PPARβ/δ. The results from these studies demonstrate that PPARβ/δ has endogenous roles in keratinocytes and can be activated by lipids found in diet and cellular components.
Topics: Keratinocytes; PPAR-beta; Animals; Mice; Stearoyl-CoA Desaturase; PPAR delta; Fatty Acids; Angiopoietin-Like Protein 4; Humans; Oleic Acid; Proto-Oncogene Proteins p21(ras); Fatty Acids, Monounsaturated; Skin Neoplasms
PubMed: 38927010
DOI: 10.3390/biom14060606 -
Chemical Research in Toxicology Jun 2024Peroxymonocarbonate (HCO/HOOCO) is produced by the reversible reaction of CO/HCO with HO ( = 0.33 M, pH 7.0). Although produced in low yields at physiological pHs and HO...
Peroxymonocarbonate (HCO/HOOCO) is produced by the reversible reaction of CO/HCO with HO ( = 0.33 M, pH 7.0). Although produced in low yields at physiological pHs and HO and CO/HCO concentrations, HCO oxidizes most nucleophiles with rate constants 10 to 100 times higher than those of HO. Boronate probes are known examples because HCO reacts with coumarin-7-boronic acid pinacolate ester (CBE) with a rate constant that is approximately 100 times higher than that of HO and the same holds for fluorescein-boronate (Fl-B) as reported here. Therefore, we tested whether boronate probes could provide evidence for HCO formation under biologically relevant conditions. Glucose/glucose oxidase/catalase were adjusted to produce low steady-state HO concentrations (2-18 μM) in Pi buffer at pH 7.4 and 37 °C. Then, CBE (100 μM) was added and fluorescence increase was monitored with time. The results showed that each steady-state HO concentration reacted more rapidly (∼30%) in the presence of CO/HCO (25 mM) than in its absence, and the data permitted the calculation of consistent rate constants. Also, RAW 264.7 macrophages were activated with phorbol 12-myristate 13-acetate (PMA) (1 μg/mL) at pH 7.4 and 37 °C to produce a time-dependent HO concentration (8.0 ± 2.5 μM after 60 min). The media contained 0, 21.6, or 42.2 mM HCO equilibrated with 0, 5, or 10% CO, respectively. In the presence of CBE or Fl-B (30 μM), a time-dependent increase in the fluorescence of the bulk solution was observed, which was higher in the presence of CO/HCO in a concentration-dependent manner. The Fl-B samples were also examined by fluorescence microscopy. Our results demonstrated that mammalian cells produce HCO and boronate probes can evidence and distinguish it from HO under biologically relevant concentrations of HO and CO/HCO.
PubMed: 38916595
DOI: 10.1021/acs.chemrestox.4c00059 -
Natural Product Research Jun 2024Two unusual phorbol esters, namely 20-deoxyphorbol-3,4,12-triacetate-13-phenylacetate () and phorbol-3,4,12,13-tetraacetate-20-phenylacetate () plus...
Two unusual phorbol esters, namely 20-deoxyphorbol-3,4,12-triacetate-13-phenylacetate () and phorbol-3,4,12,13-tetraacetate-20-phenylacetate () plus ingol-3,8,12-triacetate-7-phenylacetate () were isolated from the latex of and identified by HRESIMS and 2D NMR. Compound is herein described for the first time. Assignment of the phenylacetyl group at C-7 in compound was suggested by the HMBC and NOESY spectra obtained in pyridine-. In addition to the latex and its distinct terpenoid fractions, the isolated compounds were tested as latent reversal agents against HIV-1-infected J-Lat cells, with reference to phorbol-12-myristate-13-acetate and ingenol-B. Compound reverted 75-80% the viral latency on the GFP-positive cells, resulting EC 3.70 μg/mL (SI 6.7), while induced 34-40% reactivation at the same concentration range (4-20 µg/mL). The ingol derivative was ineffective. Phorbol esters were confirmed as effective constituents in the latex since the fraction containing them was 2.4-fold more active than the lyophilised latex at the lowest concentration assayed.
PubMed: 38902957
DOI: 10.1080/14786419.2024.2364261 -
The Journal of Endocrinology Jun 2024Cells actively engaged in de novo steroidogenesis rely on an expansive intracellular network to efficiently transport cholesterol. The final link in the transport chain...
Cells actively engaged in de novo steroidogenesis rely on an expansive intracellular network to efficiently transport cholesterol. The final link in the transport chain is STARD1, which transfers cholesterol to the enzyme complex that initiates steroidogenesis. However, the regulation of ovarian STARD1 is not fully characterized and even less is known for upstream cytosolic cholesterol transporters STARD4 and STARD6. Here, we identified both STARD4 and STARD6 mRNAs in the human ovary but only detected STARD4 protein since the primary STARD6 transcript turned out to be a splice variant. Corpora lutea contained the highest levels of STARD4 and STARD1 mRNA and STARD1 protein, while STARD4 protein was uniformly distributed across ovarian tissues. Cyclic AMP analog (8Br-cAMP) and phorbol ester (PMA) individually increased STARD1 and STARD4 mRNA along with STARD1 protein and its phosphoform in cultured primary human luteinized granulosa cells (hGC). STARD6 transcripts and STARD4 protein were unresponsive to these stimuli. Combining lower doses of PMA and 8Br-cAMP blunted the 8Br-cAMP stimulation of STARD1 protein. Increasing cholesterol levels by blocking its conversion to steroid with aminoglutethimide or by adding LDL reduced the STARD4 mRNA response to stimuli. Sterol depletion reduced the STARD1 mRNA and protein response to PMA. These data support a possible role for STARD4, but not STARD6, in supplying cholesterol for steroidogenesis in the ovary. We demonstrate for the first time how cAMP, PMA and sterol pathways separately and combined differentially regulate STARD4, STARD6 and STARD1 mRNA levels, and STARD1 and STARD4 protein in human primary ovarian cells.
PubMed: 38829257
DOI: 10.1530/JOE-23-0385 -
Ecotoxicology and Environmental Safety Jul 2024The correlation between formaldehyde (FA) exposure and prevalence of asthma has been widely reported. However, the underlying mechanism is still not fully understood. FA...
The correlation between formaldehyde (FA) exposure and prevalence of asthma has been widely reported. However, the underlying mechanism is still not fully understood. FA exposure at 2.0 mg/m was found to exacerbate asthma in OVA-induced murine models. IFN-γ, the cytokine produced by T helper 1 (Th1) cells, was significantly induced by FA in serum and bronchoalveolar lavage fluid (BALF) of asthmatic mice, which was different from cytokines secreted by other Th cells. The observation was also confirmed by mRNA levels of Th marker genes in CD4+ T cells isolated from BALF. In addition, increased production of IFN-γ and expression of T-bet in Jurkat T cells primed with phorbol ester and phytohaemagglutinin were also observed with 100 μM FA treatment in vitro. Upregulated STAT1 phosphorylation, T-bet expression and IFN-γ production induced by FA was found to be restrained by STAT1 inhibitor fludarabine, indicating that FA promoted Th1 commitment through the autocrine IFN-γ/STAT1/T-bet pathway in asthma. This work not only revealed that FA could bias Th lineage commitment to exacerbate allergic asthma, but also identified the signaling mechanism of FA-induced Th1 differentiation, which may be utilized as the target for development of interfering strategies against FA-induced immune disorders.
Topics: Asthma; Animals; STAT1 Transcription Factor; Interferon-gamma; Mice; T-Box Domain Proteins; Formaldehyde; Inflammation; Mice, Inbred BALB C; Humans; Female; Bronchoalveolar Lavage Fluid; T-Lymphocytes, Helper-Inducer; Signal Transduction; Th1 Cells; Jurkat Cells
PubMed: 38823345
DOI: 10.1016/j.ecoenv.2024.116534 -
Zhongguo Zhong Yao Za Zhi = Zhongguo... May 2024This study aims to optimize the conditions for the formation of neutrophil extracellular traps(NETs) in vitro, so as to establish a relatively stable experimental...
This study aims to optimize the conditions for the formation of neutrophil extracellular traps(NETs) in vitro, so as to establish a relatively stable experimental research platform. Different conditions were compared, including commonly used laboratory animals(rats and mice) and a variety of cell sources(bone marrow neutrophils and peripheral blood neutrophils separated by percoll density gradient centrifugation). Different inducers like lipopolysaccharide(LPS) and phorbol 12-myristate 13-acetate(PMA) were used for induction in vitro. Myeloperoxidase(MPO)/citrullinated histone H3(CitH3)/DAPI immunofluorescence and cell free DNA(cf-DNA) content determination were used for comprehensive evaluation to screen the optimal conditions for the formation of NETs induced in vitro. Furthermore, the stability of the selected conditions for inducing the formation of NETs in vitro was evaluated by tetramethylpyrazine(TMP), an active component in Chinese herbal medicines. The results showed that coated poly-D-lysine(PDL) induced the formation of NETs in bone marrow neutrophils of mice to a certain extent. Both LPS and PMA significantly up-regulated the protein levels of MPO and CitH3 in mouse bone marrow neutrophils and elevated the cfDNA level in the supernatant of rat peripheral blood neutrophils. The cfDNA level in the PMA-induced group increased more significantly than that in the LPS-induced group(P<0.05). The results of immunofluorescence staining showed that the expression of MPO and CitH3 in mouse bone marrow neutrophils, rat bone marrow neutrophils, and rat peripheral blood neutrophils were significantly increased after PMA induction, especially in rat peripheral blood neutrophils. TMP significantly down-regulated the protein levels of MPO, CitH3, and neutrophil elastase(NE) in rat peripheral blood neutrophils induced by PMA. In conclusion, treating the peripheral blood neutrophils of rats with PMA is the optimal condition for inducing the formation of NETs in vitro. This study provides an optimal platform for in vitro studies based on NETs and a basis for studying the effects of traditional Chinese medicines targeting NETs.
Topics: Extracellular Traps; Animals; Neutrophils; Mice; Rats; Peroxidase; Tetradecanoylphorbol Acetate; Male; Lipopolysaccharides; Rats, Sprague-Dawley; Histones; Humans
PubMed: 38812134
DOI: 10.19540/j.cnki.cjcmm.20240124.403 -
Microorganisms Apr 2024Chagas disease is caused by the single-flagellated protozoan , which affects several million people worldwide. Understanding the signal transduction pathways involved in...
In Vitro Identification of Phosphorylation Sites on TcPolβ by Protein Kinases TcCK1, TcCK2, TcAUK1, and TcPKC1 and Effect of Phorbol Ester on Activation by TcPKC of TcPolβ in Epimastigotes.
Chagas disease is caused by the single-flagellated protozoan , which affects several million people worldwide. Understanding the signal transduction pathways involved in this parasite's growth, adaptation, and differentiation is crucial. Understanding the basic mechanisms of signal transduction in could help to develop new drugs to treat the disease caused by these protozoa. In the present work, we have demonstrated that Fetal Calf Serum (FCS) can quickly increase the levels of both phosphorylated and unphosphorylated forms of DNA polymerase beta (TcPolβ) in tissue-cultured trypomastigotes. The in vitro phosphorylation sites on TcPolβ by protein kinases TcCK1, TcCK2, TcAUK1, and TcPKC1 have been identified by Mass Spectrometry (MS) analysis and with antibodies against phosphor Ser-Thr-Tyr. MS analysis indicated that these protein kinases can phosphorylate Ser and Thr residues on several sites on TcPolβ. Unexpectedly, it was found that TcCK1 and TcPKC1 can phosphorylate a different Tyr residue on TcPolβ. By using a specific anti-phosphor Tyr monoclonal antibody, it was determined that TcCK1 can be in vitro autophosphorylated on Tyr residues. In vitro and in vivo studies showed that phorbol 12-myristate 13-acetate (PMA) can activate the PKC to stimulate the TcPolβ phosphorylation and enzymatic activity in epimastigotes.
PubMed: 38792752
DOI: 10.3390/microorganisms12050907 -
Journal of Natural Products Jun 2024Natural products represent a rich source of bioactive compounds, covering a large chemical space. Even if challenging, this diversity can be extended by applying...
Natural products represent a rich source of bioactive compounds, covering a large chemical space. Even if challenging, this diversity can be extended by applying chemical modifications. However, these studies generally require multigram amounts of isolated natural products and face frequent testing failures. To overcome this limitation, we propose a rapid and efficient approach that uses molecular networking (MN) to visualize the new chemical diversity generated by simple chemical modifications of natural extracts. Moreover, the strategy deployed enables the most appropriate reagents to be defined quickly upstream of a reaction on a pure compound, in order to maximize chemical diversity. This methodology was applied to the latex extract of to follow the reactivity toward a series of Brønsted and Lewis acids of three class of diterpene esters identified in this species: jatrophane, terracinolide, and phorbol. Through the molecular networking interpretation, with the aim to illustrate our approach, BF·OEt was selected for chemical modification on isolated jatrophane esters. Three rearranged compounds (-) were obtained, showing that the most appropriate reagents can be selected by MN interpretation.
Topics: Euphorbia; Diterpenes; Biological Products; Plant Extracts; Esters; Molecular Structure
PubMed: 38789921
DOI: 10.1021/acs.jnatprod.4c00190