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Journal of Natural Products May 2023Three new phenanthrene derivatives (, , ), one new fluorenone (), and four known compounds (-) were isolated from the ethyl acetate extract of Sw. stems using column...
Three new phenanthrene derivatives (, , ), one new fluorenone (), and four known compounds (-) were isolated from the ethyl acetate extract of Sw. stems using column chromatography. The chemical structures were elucidated by analysis of spectroscopic data. The absolute configuration of was determined by electronic circular dichroism calculation. We also evaluated the immunomodulatory effects of compounds isolated from in human peripheral blood mononuclear cells from healthy individuals and those from patients with multiple sclerosis in vitro. Dendrocrumenol B () and dendrocrumenol D () showed strong immunomodulatory effects on both CD3 T cells and CD14 monocytes. Compounds and could reduce IL-2 and TNF production in T cells and monocytes that were treated with phorbol-12-myristate-13-acetate and ionomycin (PMA/Iono). Deep immune profiling using high-dimensional single-cell mass cytometry could confirm immunomodulatory effects of , quantified by the reduction of activated T cell population under PMA/Iono stimulation, in comparison to the stimulated T cells without treatment.
Topics: Humans; Dendrobium; Leukocytes, Mononuclear; Monocytes; Phenanthrenes; T-Lymphocytes; Tetradecanoylphorbol Acetate; Fluorenes
PubMed: 37140218
DOI: 10.1021/acs.jnatprod.3c00107 -
Carcinogenesis Aug 2023Non-melanoma skin cancer (NMSC) is the most common cancer in the world. Environmental exposure to carcinogens is one of the major causes of NMSC initiation and...
Non-melanoma skin cancer (NMSC) is the most common cancer in the world. Environmental exposure to carcinogens is one of the major causes of NMSC initiation and progression. In the current study, we utilized a two-stage skin carcinogenesis mouse model generated by sequential exposure to cancer-initiating agent benzo[a]pyrene (BaP) and promoting agent 12-O-tetradecanoylphorbol-13-acetate (TPA), to study epigenetic, transcriptomic and metabolic changes at different stages during the development of NMSC. BaP/TPA caused significant alterations in DNA methylation and gene expression profiles in skin carcinogenesis, as evidenced by DNA-seq and RNA-seq analysis. Correlation analysis between differentially expressed genes and differentially methylated regions found that the mRNA expression of oncogenes leucine rich repeat LGI family member 2 (Lgi2), kallikrein-related peptidase 13 (Klk13) and SRY-Box transcription factor (Sox5) are correlated with the promoter CpG methylation status, indicating BaP/TPA regulates these oncogenes through regulating their promoter methylation at different stages of NMSC. Pathway analysis identified that the modulation of macrophage-stimulating protein-recepteur d'origine nantais and high-mobility group box 1 signaling pathways, superpathway of melatonin degradation, melatonin degradation 1, sirtuin signaling and actin cytoskeleton signaling pathways are associated with the development of NMSC. The metabolomic study showed BaP/TPA regulated cancer-associated metabolisms like pyrimidine and amino acid metabolisms/metabolites and epigenetic-associated metabolites, such as S-adenosylmethionine, methionine and 5-methylcytosine, indicating a critical role in carcinogen-mediated metabolic reprogramming and its consequences on cancer development. Altogether, this study provides novel insights integrating methylomic, transcriptomic and metabolic-signaling pathways that could benefit future skin cancer treatment and interception studies.
Topics: Mice; Animals; Benzo(a)pyrene; Carcinogens, Environmental; Melatonin; Carcinogenesis; Skin Neoplasms; Tetradecanoylphorbol Acetate; Epigenesis, Genetic
PubMed: 37100755
DOI: 10.1093/carcin/bgad024 -
Molecular and Cellular Endocrinology Jun 2023LPA internalization to endosomes was studied employing Förster Resonance Energy Transfer (FRET) in cells coexpressing the mCherry-lysophosphatidic acid LPA receptors...
LPA internalization to endosomes was studied employing Förster Resonance Energy Transfer (FRET) in cells coexpressing the mCherry-lysophosphatidic acid LPA receptors and distinct eGFP-tagged Rab proteins. Lysophosphatidic acid (LPA)-induced internalization was rapid and decreased afterward: phorbol myristate acetate (PMA) action was slower and sustained. LPA stimulated LPA-Rab5 interaction rapidly but transiently, whereas PMA action was rapid but sustained. Expression of a Rab5 dominant-negative mutant blocked LPA-Rab5 interaction and receptor internalization. LPA-induced LPA-Rab9 interaction was only observed at 60 min, and LPA-Rab7 interaction after 5 min with LPA and after 60 min with PMA. LPA triggered immediate but transient rapid recycling (i.e., LPA-Rab4 interaction), whereas PMA action was slower but sustained. Agonist-induced slow recycling (LPA-Rab11 interaction) increased at 15 min and remained at this level, whereas PMA action showed early and late peaks. Our results indicate that LPA receptor internalization varies with the stimuli.
Topics: Receptors, Lysophosphatidic Acid; Fluorescence Resonance Energy Transfer; Phosphorylation; Tetradecanoylphorbol Acetate; Endosomes; Lysophospholipids
PubMed: 37054840
DOI: 10.1016/j.mce.2023.111930 -
ACS Biomaterials Science & Engineering May 2023As one imaging method to evaluate monocyte-macrophage differentiation, cationized gelatin nanospheres incorporating a molecular beacon (MB) (cGNS) were designed....
As one imaging method to evaluate monocyte-macrophage differentiation, cationized gelatin nanospheres incorporating a molecular beacon (MB) (cGNS) were designed. Cationized gelatin nanospheres (cGNS) of different apparent sizes were prepared by the conventional coacervation method, and then the MB of CD204 was incorporated into cGNS to prepare cGNS. When three types of cGNS were cultured with human monocytoma (THP-1) cells, the cGNS with a 110 nm diameter showed the highest MB delivery efficiency. In addition, no influence on the monocyte-macrophage differentiation was observed in terms of CD204 gene expression and cell viability. After incubation with cGNS incorporating CD204 MB (cGNS), THP-1 cells were stimulated by phorbol 12-myristate 13-acetate (PMA) for monocyte differentiation into macrophages. The fluorescence intensity of macrophages increased with the incubation time. In contrast, the fluorescence intensity of macrophages incubated with MB alone was not changed. On the other hand, there was no change in the fluorescence intensity of original THP-1 cells cultured with cGNS. It is concluded that the cGNS are promising to trace the differentiation of THP-1 cells into macrophages in their live condition.
Topics: Humans; Monocytes; Gelatin; Nanospheres; Macrophages; Cell Differentiation; Tetradecanoylphorbol Acetate
PubMed: 37014020
DOI: 10.1021/acsbiomaterials.2c01420 -
Frontiers in Bioscience (Landmark... Mar 2023To investigate the effects of extract (GBE) on autophagy in human macrophages stimulated by cigarette smoke extract (CSE).
OBJECTIVE
To investigate the effects of extract (GBE) on autophagy in human macrophages stimulated by cigarette smoke extract (CSE).
METHODS
The human monocyte cell line U937 was cultured , and phorbol ester (PMA) was added to the cell culture medium to induce differentiation into human macrophages. CSE was prepared by traditional methods for experiments. The cells were divided into four groups: the blank group, the CSE model group, the GBE + CSE group, and the rapamycin + CSE group. Immunofluorescence was used to identify human macrophages, transmission electron microscopy was used to observe the ultrastructure of human macrophages in each group, ELISA was used to measure the amount of IL-6 and IL-10 in the supernatant from each group of cells, the mRNA levels of p62, ATG5, ATG7, and Rab7 were measured by real-time qPCR, and the protein expression levels of p62, ATG5, ATG7, and Rab7 were measured by Western blotting.
RESULTS
U937 cells were successfully differentiated into human macrophages after induction with PMA. The CSE model group had many more autophagosomes than the blank group. Compared with the CSE model group, the GBE + CSE group and the rapamycin + CSE group had significantly more autophagolysosomal. Compared with the other groups, the CSE model group had a higher level of IL-6 but a lower level of IL-10 in the supernatant ( 0.05). Compared with the blank group, the mRNA and protein expression levels of p62 in the CSE model group were significantly decreased, while the mRNA and protein expression levels of ATG5 and ATG7 were significantly increased in the CSE model group ( 0.05). No difference was found in the mRNA and protein expression levels of Rab7 between the blank group and the CSE model group. Compared with the CSE model group, the IL-6 level in the GBE + CSE group and the rapamycin + CSE group cell culture supernatant decreased significantly, p62 mRNA and protein expression significantly decreased, while ATG5, ATG7, and Rab7 mRNA and protein expression levels were significantly increased ( 0.05). Moreover, increased LC3-II/LC3-I ratio were also found in the GBE + CSE group and the rapamycin + CSE group compared with the CSE model group.
CONCLUSIONS
GBE could promote the fusion of autophagosomes and lysosomes in human macrophages, enhance the autophagy function of human macrophages, and reduce the damaging effect of CSE on the autophagy function of macrophages.
Topics: Humans; Interleukin-10; Cigarette Smoking; Interleukin-6; RNA, Messenger; Autophagy; Macrophages
PubMed: 37005757
DOI: 10.31083/j.fbl2803050 -
Life Sciences May 2023Overproduction of pro-inflammatory cytokines and its-mediated immune cell infiltration play a crucial role in asthma progression. In this study, we investigated the role...
AIMS
Overproduction of pro-inflammatory cytokines and its-mediated immune cell infiltration play a crucial role in asthma progression. In this study, we investigated the role of ginsenoside Rh1 (Rh1) in ovalbumin (OVA)/lipopolysaccharide (LPS)-induced allergic asthma both in vitro and in vivo.
MATERIALS AND MAIN METHODS
The phorbol ester (PMA) and LPS were used to induce inflammation in lung airway cells and macrophage activation, respectively. Western blotting, quantitative reverse transcription-PCR, and immunofluorescence (IF) assays were performed to elucidate the underlying molecular mechanisms. To evaluating the effects of Rh1 in vivo, OVA and LPS were used to establish allergic asthma models.
KEY FINDINGS
Rh1 significantly suppressed PMA-induced lung inflammation and macrophage activation by suppressing pro-inflammatory cytokines (TNF-α, IL-1β, MCP-1), ICMA-1, and matrix metallopeptidase 9 (MMP9) in A549 cells. Rh1 abolished the PMA-induced inflammation by suppressing MAPK, Akt, and NF-κB p65. Pretreatment with Rh1 blocked PMA-mediated translocation of NF-κB, a key marker of pro-inflammatory cytokine release, into the nucleus. Similar to PMA-induced lung inflammation, Rh1 suppressed LPS-induced macrophage activation by suppressing NF-κB p65 activation and inducible nitric oxide synthase protein and mRNA expression. Consistent with in vitro data, LPS injection enhanced the number of immune cells induced by OVA in bronchoalveolar lavage fluid, whereas 20 mg/kg Rh1 significantly decreased OVA/LPS-mediated immune cell induction. In addition, Rh1 inhibited eosinophil, macrophage, and neutrophil maturation through by IL-4 and OVA-specific IgE production.
SIGNIFICANCE
Rh1 protects against OVA/LPS-induced allergic asthma by suppressing immune cell infiltration by blocking the activation of MAPK, Akt, and NF-κB signaling pathways.
Topics: Humans; NF-kappa B; Proto-Oncogene Proteins c-akt; Lipopolysaccharides; Asthma; Signal Transduction; Inflammation; Lung; Cytokines; Pneumonia; Bronchoalveolar Lavage Fluid; Ovalbumin
PubMed: 36958436
DOI: 10.1016/j.lfs.2023.121607 -
Cell Biology International Jul 2023Ormeloxifene (ORM) (3,4-trans-2,2-dimethyl-3-phenyl-4-p-(β-pyrrolidinoethoxy) phenyl-7-methoxychroman), world's first nonsteroidal selective estrogen receptor...
Ormeloxifene (ORM) (3,4-trans-2,2-dimethyl-3-phenyl-4-p-(β-pyrrolidinoethoxy) phenyl-7-methoxychroman), world's first nonsteroidal selective estrogen receptor modulator approved for contraception in India has been shown to have potential anticancer activities. Here, we show that ORM can induce megakaryocyte and myeloid (granulocytic) but not erythroid differentiation in multipotent human myeloid leukemia cell line K562. We show that ORM at an IC50 of 7.5 µM can induce morphological changes similar to megakaryocytes in K562 cells. ORM led to increase in levels of megakaryocytic differentiation markers (CD41 and CD61) as well as key transcription factors GATA1 and AML1. We further show that ORM induces megakaryocytic differentiation in K562 cells through ERK activation and induction of autophagy in a fashion similar to other known inducers of megakaryocytic differentiation such as phorbol esters. In addition, as shown earlier, we yet again observed that ORM led to activation of caspases since their inhibition through pan-caspase inhibitor mitigated megakaryocytic differentiation as they led to significant decrease in CD41 and CD61. Because induction of megakaryocytic differentiation in K562 involves growth arrest and exit from cell cycle, we also observed an increase in levels of p21 and p27 with decrease in c-Myc protein levels in K562 cells treated with 7.5 µM ORM for 24 and 48 h, respectively. Taken together, these findings indicate that ORM can markedly induce megakaryocytic differentiation in K562 cells.
Topics: Humans; Megakaryocytes; K562 Cells; Cell Differentiation; Leukemia
PubMed: 36950830
DOI: 10.1002/cbin.12017 -
Frontiers in Immunology 2023Alpha-fetoprotein(AFP) is a cancer biomarker for the diagnosis of hepatocellular carcinoma(HCC); however, its role in macrophage polarization and phagocytosis remains...
Alpha-fetoprotein(AFP) is a cancer biomarker for the diagnosis of hepatocellular carcinoma(HCC); however, its role in macrophage polarization and phagocytosis remains unclear. In the present study, we explored the correlation between AFP regulation of macrophage function and the possible regulatory mechanisms. Human mononuclear leukemia cells (THP-1) and monocytes from healthy donors were used to analyze the effect of AFP on the macrophages' phenotype and phagocytosis. THP-1 cells and healthy human donor-derived monocytes were polarized into M0 macrophages induced by phorbol ester (PMA), and M0 macrophages were polarized into M1 macrophages induced by lipopolysaccharide(LPS) and interferon-γ(IFN-γ). Interleukin-4(IL-4) and interleukin-13(IL-13) were used to induce M0 macrophage polarization into M2 macrophages. Tumor-derived AFP(tAFP) stimulated M0 macrophage polarization into M2 macrophages and inhibited M1 macrophages to phagocytize HCC cells. The role of AFP in promoting macrophage polarization into M2 macrophages and inhibiting the M1 macrophages to phagocytize HCC cells may be involved in activating the PI3K/Akt signaling pathway. AFP could also enhanced the migration ability of macrophages and inhibited the apoptosis of HCC cells when co-cultured with M1-like macrophages. AFP is a pivotal cytokine that inhibits macrophages to phagocytize HCC cells.
Topics: Humans; alpha-Fetoproteins; Carcinoma, Hepatocellular; Phosphatidylinositol 3-Kinases; Liver Neoplasms; Macrophages; Interferon-gamma; Phenotype
PubMed: 36911723
DOI: 10.3389/fimmu.2023.1081572 -
International Journal of Molecular... Feb 2023Protein kinase C delta (PKC-δ) is an important signaling molecule in human cells that has both proapoptotic as well as antiapoptotic functions. These conflicting...
Protein kinase C delta (PKC-δ) is an important signaling molecule in human cells that has both proapoptotic as well as antiapoptotic functions. These conflicting activities can be modulated by two classes of ligands, phorbol esters and bryostatins. Phorbol esters are known tumor promoters, while bryostatins have anti-cancer properties. This is despite both ligands binding to the C1b domain of PKC-δ (δC1b) with a similar affinity. The molecular mechanism behind this discrepancy in cellular effects remains unknown. Here, we have used molecular dynamics simulations to investigate the structure and intermolecular interactions of these ligands bound to δC1b with heterogeneous membranes. We observed clear interactions between the δC1b-phorbol complex and membrane cholesterol, primarily through the backbone amide of L250 and through the K256 side-chain amine. In contrast, the δC1b-bryostatin complex did not exhibit interactions with cholesterol. Topological maps of the membrane insertion depth of the δC1b-ligand complexes suggest that insertion depth can modulate δC1b interactions with cholesterol. The lack of cholesterol interactions suggests that bryostatin-bound δC1b may not readily translocate to cholesterol-rich domains within the plasma membrane, which could significantly alter the substrate specificity of PKC-δ compared to δC1b-phorbol complexes.
Topics: Humans; Protein Kinase C-delta; Bryostatins; Isoenzymes; Phorbol Esters; Phorbols; Lactones
PubMed: 36902029
DOI: 10.3390/ijms24054598 -
Journal of Leukocyte Biology Jul 2023CR3 (CD11b/CD18; αmβ2 integrin) is a conserved phagocytic receptor. The active conformation of CR3 binds the iC3b fragment of complement C3 as well as many host and...
CR3 (CD11b/CD18; αmβ2 integrin) is a conserved phagocytic receptor. The active conformation of CR3 binds the iC3b fragment of complement C3 as well as many host and microbial ligands, leading to actin-dependent phagocytosis. There are conflicting reports about how CR3 engagement affects the fate of phagocytosed substrates. Using imaging flow cytometry, we confirmed that binding and internalization of iC3b-opsonized polystyrene beads by primary human neutrophils was CR3-dependent. iC3b-opsonized beads did not stimulate neutrophil reactive oxygen species, and most beads were found in primary granule-negative phagosomes. Similarly, Neisseria gonorrhoeae that does not express phase-variable Opa proteins suppresses neutrophil reactive oxygen species and delays phagolysosome formation. Here, binding and internalization of Opa-deleted (Δopa) N. gonorrhoeae by adherent human neutrophils was inhibited using blocking antibodies against CR3 and by adding neutrophil inhibitory factor, which targets the CD11b I-domain. No detectable C3 was deposited on N. gonorrhoeae in the presence of neutrophils alone. Conversely, overexpressing CD11b in HL-60 promyelocytes enhanced Δopa N. gonorrhoeae phagocytosis, which required the CD11b I-domain. Phagocytosis of N. gonorrhoeae was also inhibited in mouse neutrophils that were CD11b-deficient or treated with anti-CD11b. Phorbol ester treatment upregulated surface CR3 on neutrophils in suspension, enabling CR3-dependent phagocytosis of Δopa N. gonorrhoeae. Neutrophils exposed to Δopa N. gonorrhoeae had limited phosphorylation of Erk1/2, p38, and JNK. Neutrophil phagocytosis of unopsonized Mycobacterium smegmatis, which also resides in immature phagosomes, was CR3-dependent and did not elicit reactive oxygen species. We suggest that CR3-mediated phagocytosis is a silent mode of entry into neutrophils, which is appropriated by diverse pathogens to subvert phagocytic killing.
Topics: Mice; Animals; Humans; Neutrophils; Reactive Oxygen Species; Phagocytosis; Macrophage-1 Antigen; Complement C3b; Receptors, Complement
PubMed: 36882066
DOI: 10.1093/jleuko/qiad028