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1.
Immune Network ; : e26-2018.
Article in English | WPRIM | ID: wpr-716243

ABSTRACT

Thapsigargin (TGN) is a potent and selective inhibitor of sarco-endoplasmic Ca²⁺-ATPase, leading to rapid elevation of cytoplasmic Ca2+ concentration. Previous reports have shown that TGN increases the production of various cytokines from macrophages and dendritic cells. Here, we examine the effects of TGN on murine T cells. Nanomolar concentrations of TGN are a significant inducer of IL-2 production with full activity at 50 nM. Micromolar concentrations of TGN, however, are inhibitory to IL-2 production and T cell proliferation. The IL-2 production-inducing activity of TGN is much more prominent when T cells are primed with concanavalin A or anti-CD3 mAb, and is due to the increase of cytoplasmic Ca²⁺ concentration. TGN at 50 nM does not affect interferon-gamma or IL-4 production from T cells. Thus, the present study shows that low nanomolar concentrations of TGN could be useful in potentiating IL-2 production from antigen-primed T cells.


Subject(s)
Cell Proliferation , Concanavalin A , Cytokines , Cytoplasm , Dendritic Cells , Interferon-gamma , Interleukin-2 , Interleukin-4 , Macrophages , T-Lymphocytes , Tetradecanoylphorbol Acetate , Thapsigargin
2.
Article in English | WPRIM | ID: wpr-285260

ABSTRACT

Infection of schistosomiasis japonica may eventually lead to liver fibrosis, and no effective antifibrotic therapies are available but liver transplantation. Hedgehog (HH) signaling pathway has been involved in the process and is a promising target for treating liver fibrosis. This study aimed to explore the effects of pentoxifylline (PTX) on liver fibrosis induced by schistosoma japonicum infection by inhibiting the HH signaling pathway. Phorbol12-myristate13-acetate (PMA) was used to induce human acute mononuclear leukemia cells THP-1 to differentiate into macrophages. The THP-1-derived macrophages were stimulated by soluble egg antigen (SEA), and the culture supernatants were collected for detection of activation of macrophages. Cell Counting Kit-8 (CCK-8) was used to detect the cytotoxicity of the culture supernatant and PTX on the LX-2 cells. The LX-2 cells were administered with activated culture supernatant from macrophages and(or) PTX to detect the transforming growth factor-β gene expression. The mRNA expression of shh and gli-1, key parts in HH signaling pathway, was detected. The mRNA expression of shh and gli-1 was increased in LX-2 cells treated with activated macrophages-derived culture supernatant, suggesting HH signaling pathway may play a key role in the activation process of hepatic stellate cells (HSCs). The expression of these genes decreased in LX-2 cells co-cultured with both activated macrophages-derived culture supernatant and PTX, indicating PTX could suppress the activation process of HSCs. In conclusion, these data provide evidence that PTX prevents liver fibrogenesis in vitro by the suppression of HH signaling pathway.


Subject(s)
Animals , Antigens, Helminth , Pharmacology , Cell Culture Techniques , Cell Differentiation , Cell Line , Culture Media, Conditioned , Chemistry , Pharmacology , Gene Expression Regulation , Hedgehog Proteins , Genetics , Allergy and Immunology , Hepatic Stellate Cells , Cell Biology , Metabolism , Humans , Liver Cirrhosis , Metabolism , Parasitology , Macrophage Activation , Macrophages , Cell Biology , Allergy and Immunology , Models, Biological , Monocytes , Cell Biology , Metabolism , Pentoxifylline , Pharmacology , Phosphodiesterase Inhibitors , Pharmacology , RNA, Messenger , Genetics , Allergy and Immunology , Schistosoma japonicum , Chemistry , Signal Transduction , Tetradecanoylphorbol Acetate , Pharmacology , Zinc Finger Protein GLI1 , Genetics , Allergy and Immunology , Zygote , Chemistry
3.
Article in English | WPRIM | ID: wpr-812176

ABSTRACT

The present study was designed to target fish for potential bioactive components contained in a Huang Lian Jie Du decoction (HLJDD) and identify the underlying mechanisms of action for the treatment of sepsis at the molecular level. he bioactive components database of HLJDD was constructed and the sepsis-associated targets were comprehensively investigated. The 3D structures of the PAFR and TXA2R proteins were established using the homology modelling (HM) method, and the molecular effects for sepsis treatment were analysed by comparing the bioactive components database and the sepsis targets using computational biology methods. The results of the screening were validated with biological testing against the human oral epidermal carcinoma cell line KB in vitro. We found that multiple bioactive compounds contained in the HLJDD interacted with multiple targets. We also predicted the promising compound leads for sepsis treatment, and the first 28 compounds were characterized. Several compounds, such as berberine, berberrubine and epiberberine, dose-dependently inhibited PGE2 production in human KB cells, and the effects were similar in the presence or absence of TPA. This study demonstrates a novel approach to identifying natural chemical compounds as new leads for the treatment of sepsis.


Subject(s)
Anti-Inflammatory Agents, Non-Steroidal , Pharmacokinetics , Berberine , Pharmacokinetics , Dinoprostone , Drugs, Chinese Herbal , Chemistry , Pharmacokinetics , Humans , KB Cells , Platelet Membrane Glycoproteins , Protein Transport , Receptors, G-Protein-Coupled , Receptors, Thromboxane A2, Prostaglandin H2 , Sepsis , Drug Therapy , Metabolism , Tetradecanoylphorbol Acetate , Pharmacokinetics
4.
Acta Pharmaceutica Sinica ; (12): 482-489, 2014.
Article in English | WPRIM | ID: wpr-245058

ABSTRACT

This study investigated the effect of arctigenin (Arc) on the cell activation, cytokines expression, proliferation, and cell-cycle distribution of mouse T lymphocytes. Mouse lymphocytes were prepared from lymph node and treated with Phorbol-12-myristate-13-acetate (PMA)/Ionimycin (Ion) and/or Arc. CD69, CD25, cytokines, proliferation and cell cycle were assayed by flow cytometry. The results showed that, at concentrations of less than 1.00 micromol x L(-1), Arc expressed non-obvious cell damage to cultured lymphocytes, however, it could significantly down-regulate the expression of CD69 and CD25, as well as TNF-alpha, IFN-gamma, IL-2, IL-4, IL-6 and IL-10 on PMA/Ion stimulated lymphocytes. At the same time, Arc could also inhibit the proliferation of PMA/Ion-activated lymphocytes and exhibited lymphocyte G 0/G1 phase cycle arrest. These results suggest that Arc possesses significant anti-inflammatory effects that may be mediated through the regulation of cell activation, cytokines expression and cell proliferation.


Subject(s)
Animals , Anti-Inflammatory Agents , Pharmacology , Antigens, CD , Metabolism , Antigens, Differentiation, T-Lymphocyte , Metabolism , Arctium , Chemistry , Cell Cycle Checkpoints , Cell Proliferation , Cytokines , Metabolism , Female , Furans , Pharmacology , Interferon-gamma , Metabolism , Interleukin-10 , Metabolism , Interleukin-2 , Metabolism , Interleukin-2 Receptor alpha Subunit , Metabolism , Interleukin-4 , Metabolism , Interleukin-6 , Metabolism , Ionomycin , Pharmacology , Lectins, C-Type , Metabolism , Lignans , Pharmacology , Lymphocyte Activation , Mice , Mice, Inbred BALB C , Plants, Medicinal , Chemistry , T-Lymphocytes , Cell Biology , Allergy and Immunology , Tetradecanoylphorbol Acetate , Pharmacology , Tumor Necrosis Factor-alpha , Metabolism
5.
Article in English | WPRIM | ID: wpr-247071

ABSTRACT

Relaxin is known to inhibit cardiac fibrosis. However, it is unclear whether relaxin could regulate the effects of Phorbol 12-myristate 13-acetate (PMA, PKC activator) on cardiac fibrosis. So the influence of relaxin on the cell proliferation and collagen expression induced by PMA in cultured cardiac fibroblasts was studied. It showed that PMA significantly increased cardiac fibroblasts proliferation, Type I pro-collagen protein expression, Type I pro-collagen mRNA expression, and rhRLX absolutely significantly decreased PMA induced effects on cardiac fibroblasts proliferation and Type I pro-collagen expressions, indicating that relaxin could inhibit cardiac fibrosis induced by PMA.


Subject(s)
Animals , Animals, Newborn , Cells, Cultured , Fibroblasts , Pathology , Fibrosis , Heart Diseases , Pathology , Rats , Rats, Sprague-Dawley , Relaxin , Therapeutic Uses , Tetradecanoylphorbol Acetate
6.
Article in Chinese | WPRIM | ID: wpr-302368

ABSTRACT

This study was aimed to investigate the effects of arsenic trioxide (As2O3) combined with TPA on cell cycle, cell differentiation and apoptosis of K562 cell line, and their possible mechanisms. K562 cells were treated with 200 nmol/L TPA, 2 µmol/L As2O3 alone and 200 nmol/L TPA combined with 2 µmol/L As2O3. The proliferative inhibition rates were determined with CCK-8. Annexin V and agarose gel electrophoresis were adopted to detect apoptosis. Colony formation test was used to determine the colony-formation efficiency. Flow cytometry was used to detect the cell differentiation and cell cycle changes. Western blot was employed to detect the expression of P38 and p-P38 proteins. The results showed that combination treatment had synergistic effects on the proliferative inhibition and apoptosis, which were much higher than those treated alone. As2O3 could decrease the colony formation ability of K562 cells. The cells treated with both TPA and As2O3 expressed far more CD11b antigens compared with cells exposed to As2O3 alone. K562 cells treated with TPA were arrested in G1 phase compared with the control group, As2O3 increased the percentage of K562 cells in the G2 phase. The combination treatment increased the expression of p-P38 of K562 cells compared with the cells exposed As2O3 alone. It is concluded that TPA can enhance the effect of As2O3 on inducing apoptosis and adjusting cell cycle , which will expect to provide a new therapeutic program.


Subject(s)
Apoptosis , Arsenicals , Pharmacology , Cell Cycle , Drug Synergism , Humans , K562 Cells , Oxides , Pharmacology , Tetradecanoylphorbol Acetate , Pharmacology
7.
Article in Korean | WPRIM | ID: wpr-648117

ABSTRACT

BACKGROUND AND OBJECTIVES: MUC5AC and MUC5B are representative secretory mucin genes in the human airway, whose expressions are increased by a variety of inflammatory mediators. Betulinic acid, a naturally occurring pentacyclic triterpenoid, is known to have an anti-inflammatory property. However, the effects of betulinic acid on mucin secretion of airway epithelial cells still have not been reported. Therefore, in this study, the effect of betulinic acid on inflammatory mediators-induced MUC5AC and MUC5B expressions was investigated in human airway epithelial cells. SUBJECTS AND METHOD: In the mucin-producing human NCI-H292 airway epithelial cells, the effects of betulinic acid on interleukin-1beta (IL-1beta)-, lipopolysaccharide (LPS)-, and phorbol myristate acetate (PMA)-induced MUC5AC and MUC5B expressions were analyzed by reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay. RESULTS: Betulinic acid attenuated IL-1beta-, LPS-, and PMA-induced MUC5B mRNA and glycoprotein expression in NCI-H292 cells. On the other hand, betulinic acid did not attenuate IL-1beta-, and LPS-, but induced PMA-induced MUC5AC mRNA and glycoprotein expressions in NCI-H292 cells. CONCLUSION: These results suggest that betulinic acid attenuates IL-1beta-, LPS-, and PMA-induced MUC5B expression in the airway epithelial cells. Therefore, betulinic acid may modulate a control of mucus-hypersecretion in airway inflammatory diseases.


Subject(s)
Enzyme-Linked Immunosorbent Assay , Epithelial Cells , Glycoproteins , Hand , Humans , Interleukin-1beta , Mucins , RNA, Messenger , Tetradecanoylphorbol Acetate
8.
Acta sci., Health sci ; 35(1): 105-109, jan.-jun. 2013. tab
Article in Portuguese | LILACS | ID: biblio-1904

ABSTRACT

Indivíduos fumantes apresentam aumento na proporção de leucócitos polimorfonucleares (LPMN), como por exemplo, no tecido pulmonar, resultando em aumento nos níveis de enzimas proteolíticas e espécies reativas de oxigênio, que ocasionam efeito destrutivo na matriz celular e contribuem para a progressão da doença pulmonar, além de favorecer o aparecimento de infecções microbianas, e determinam que as células fagocíticas estejam em frequente estado de ativação (fagocitose). Neste trabalho, avaliou-se a influência da nicotina (NIC) sobre a viabilidade de LPMN e macrófagos ativados ou não, pelos estímulos zymosan e acetato de forbol miristato. Os resultados indicaram que a NIC promove aumento na viabilidade de LPMN e macrófagos ativados em relação a essas células ativadas sem a presença de NIC, avaliada 'ex vivo' pelo teste de exclusão do azul de trypan. Esse efeito foi significativamente mais pronunciado sobre LPMN que sobre os macrófagos. Essa redução na citotoxicidade favorece a sobrevida da célula, podendo exacerbar os seus efeitos deletérios, especialmente em no seu estado ativado, pela maior produção de espécies reativas de oxigênio.


Smokers show increased rates of polymorphonuclear leukocytes (PMNL), including in pulmonary tissue, resulting increased levels of proteolytic enzymes and reactive oxygen species, which have a destructive effect on the cellular matrix and contribute to the progression of pulmonary disease, in addition to promoting the onset of microbial infections which cause phagocytic cells to be in a state of frequent activation (phagocytosis). This work evaluated the influence of nicotine (NIC) on the viability of PMNL and macrophages (activated or not), by stimuli zymosan and phorbol myristate acetate. The results indicated that NIC led to increased viability of PMNL and activated macrophages compared to these cells activated without NIC, measured ex vivo by trypan blue exclusion test. This effect was significantly higher on PMNL than on macrophages. This reduction in cytotoxicity favors cell survival, and may exacerbate its deleterious effect, especially in the active state, due to increased production of reactive oxygen species.


Subject(s)
Lung Diseases , Microbial Viability , Neutrophils , Nicotine , Peptide Hydrolases , Reactive Oxygen Species , Tetradecanoylphorbol Acetate , Zymosan
9.
Indian J Biochem Biophys ; 2013 Apr; 50(2): 105-113
Article in English | IMSEAR | ID: sea-147293

ABSTRACT

The modulation in biochemical status of skin and hepatic tissue at the time point of commencement of promotion stage of skin carcinogenesis in mice and its intervention with aqueous Azadirachta indica leaf extract (AAILE) were investigated. 7,12-Dimethylbenz(a)anthracene (DMBA, 500 nmol/100 ul of acetone) was applied topically for 2 weeks (twice weekly), followed by phorbol-12-myristate-13-acetate (TPA, 1.7 nmol/100 ul) twice weekly for 6 weeks on the depilated skin of mice and AAILE was administered orally at a dose level of 300 mg/kg body wt thrice a week for 10 weeks. DMBA/TPA treatment upregulated the phase I enzymes in skin and hepatic tissue, as revealed by the increased cytochrome P450 (CYP) and cytochrome b5 (cyt b5) levels and aryl hydrocarbon hydroxylase (AHH) activity when compared to the control group and differentially modulated the activities of phase II enzymes like glutathione-s-transferase (GST), DT-diaphorase (DTD) and uridine diphosphate glucuronosyltransferase (UDP-GT). AAILE treatment decreased the DMBA/TPA-induced increase in cutaneous CYP level and enhanced the DTD and UDP-GT activities when compared with DMBA/TPA group. In the hepatic tissue of AAILE + DMBA/TPA group, an increase in UDP-GT activity was observed when compared to DMBA/TPA group. DMBA/TPA treatment did not alter the skin lipid peroxidation (LPO) level when compared to control group, however, in the animals that received AAILE treatment along with DMBA/TPA, a significant increase in LPO was observed when compared to control group. This was associated with a decrease in cutaneous reduced glutathione (GSH) level of AAILE + DMBA/TPA group. Enhanced LPO level was observed in the hepatic tissue of DMBA/TPA and AAILE + DMBA/TPA groups when compared to control group. However, no alteration was observed in their hepatic GSH levels. The micronuclei score in hepatic tissue did not exhibit significant inter-group differences. The results of the present study suggest that apart from skin, liver may be affected during DMBA/TPA-induced skin tumorigenesis. AAILE treatment has the ability to modulate these changes potentially influencing the process of tumor formation. These findings seem to be important to carcinogenesis and its intervention with anti-cancer agents.


Subject(s)
9,10-Dimethyl-1,2-benzanthracene/pharmacology , Animals , Antineoplastic Agents/pharmacology , Antioxidants/metabolism , Azadirachta/chemistry , Cell Transformation, Neoplastic , Cytochrome P-450 Enzyme System/metabolism , Cytochromes b5/metabolism , Gene Expression Regulation, Neoplastic , Glutathione Transferase/metabolism , Lipid Peroxidation , Liver/drug effects , Liver/metabolism , Male , Mice , Micronucleus Tests , Neoplasms, Experimental/chemically induced , Phytotherapy/methods , Plant Extracts/pharmacology , Plant Leaves , Skin/drug effects , Skin/metabolism , Skin Neoplasms/chemically induced , Skin Neoplasms/drug therapy , Tetradecanoylphorbol Acetate/pharmacology , Xenobiotics/chemistry
10.
Article in English | WPRIM | ID: wpr-293292

ABSTRACT

<p><b>OBJECTIVE</b>Cheongseoikki-tang (CIT, Korean), also called Qingshu Yiqi decoction () and Seisho-ekki-to (Japanese), is well known as an effective traditional combination of herbs for treating cardiovascular diseases. This study was to research its effects on bone marrow-derived mast cell (BMMC)-mediated allergy and inflammation mechanisms.</p><p><b>METHODS</b>In this study, the biological effect of Cheongseoikki-tang ethanol extract (CITE) was evaluated, focusing on its effects on the production of allergic mediators by phorbol 12-myristate 13-acetate (PMA) plus calcium ionophore A23187 (A23187)-stimulated BMMCs. These allergic mediators included interleukin-6 (IL-6), prostaglandin D2 (PGD2), leukotriene C4 (LTC4), and β-hexosaminidase (β-hex).</p><p><b>RESULTS</b>Our data revealed that CITE inhibited the production of IL-6, PGD2, LTC4, and β-hex induced by PMA plus A23187 (P<0.05).</p><p><b>CONCLUSION</b>These findings indicate that CITE has the potential for use in the treatment of allergy.</p>


Subject(s)
Animals , Anti-Inflammatory Agents , Pharmacology , Therapeutic Uses , Bone Marrow Cells , Pathology , Calcimycin , Pharmacology , Cell Degranulation , Cell Survival , Drugs, Chinese Herbal , Pharmacology , Therapeutic Uses , Hypersensitivity , Drug Therapy , Pathology , Interleukin-6 , Bodily Secretions , Leukotriene C4 , Pharmacology , Male , Mast Cells , Pathology , Physiology , Mice , Mice, Inbred BALB C , Prostaglandin D2 , Tetradecanoylphorbol Acetate , Pharmacology , beta-N-Acetylhexosaminidases , Metabolism
11.
Chinese Journal of Oncology ; (12): 331-336, 2013.
Article in Chinese | WPRIM | ID: wpr-284181

ABSTRACT

<p><b>OBJECTIVE</b>To investigate the effects of sphingosine kinase 1 (SphK1) on the proliferation, migration and invasion of human colon cancer LoVo cells, and to explore the related mechanisms.</p><p><b>METHODS</b>Human colon cancer LoVo cells were divided into three groups: phorbol 12-myristate 13-acetate (PMA) was used to induce the activation of SphK1 in the PMA group, N,N-dimethylsphingosine (DMS) used to suppress the activity of SphK1 in DMS group, and the cells treated with equal amount of 0.9 % NaCl instead of drugs served as the control group. The activity of SphK1 was assayed by autoradiography, the cell proliferation was assessed by MTT assay, cell migration and invasion were examined by Boyden chamber assay, concentrations of sICAM-1 and sVCAM-1 were assayed by ELISA, and RT-PCR and Western blot were used to evaluate the mRNA and protein expression in the cells.</p><p><b>RESULTS</b>The activity of SphK1 was efficiently induced by PMA and significantly suppressed by DMS. PMA induced cell proliferation in a time- and dose-dependent manner. On the contrast, DMS suppressed cell proliferation in a time- and dose-dependent manner. After treating with PMA, the number of migrating and invasing cells were increased to 143.36 ± 8.73 and 118.46 ± 6.25, significantly higher than those of the control group (75.48 ± 6.12 and 64.19 ± 5.36). After treating with DMS, the number of migrating and invasing cells were decreased to 38.57 ± 3.24 and 32.48 ± 4.27, significantly lower than those of the control group (P < 0.01). The relative expression levels of FAK, ICAM-1 and VCAM-1 mRNA in the PMA group were 0.82 ± 0.06, 0.74 ± 0.05 and 0.89 ± 0.09, and those in the DMS group were 0.23 ± 0.02, 0.26 ± 0.03 and 0.37 ± 0.04, with significant differences between the PMA, DMS and control groups (P < 0.01). Compared with the control group, the relative expression levels of FAK and p-FAK proteins in the PMA group (0.52 ± 0.06 and 0.51 ± 0.06) were significantly elevated, and those of the DMS group (0.20 ± 0.03 and 0.09 ± 0.02) were significantly decreased. In addition, the concentrations of sICAM-1 and sVCAM-1 were significantly elevated with the activation of SphK1. On the contrary, those of the DMS group were significantly reduced with the suppression of SphK1 (Both P < 0.01).</p><p><b>CONCLUSIONS</b>SphK1 may enhance the proliferation, migration and invasion of colon cancer LoVo cells through activating FAK pathway and up-regulating the expression of ICAM-1 and VCAM-1.</p>


Subject(s)
Cell Line, Tumor , Cell Movement , Cell Proliferation , Colonic Neoplasms , Metabolism , Pathology , Dose-Response Relationship, Drug , Enzyme Inhibitors , Pharmacology , Focal Adhesion Kinase 1 , Genetics , Metabolism , Humans , Intercellular Adhesion Molecule-1 , Genetics , Metabolism , Neoplasm Invasiveness , Phosphorylation , Phosphotransferases (Alcohol Group Acceptor) , Metabolism , RNA, Messenger , Metabolism , Signal Transduction , Sphingosine , Pharmacology , Tetradecanoylphorbol Acetate , Pharmacology , Vascular Cell Adhesion Molecule-1 , Genetics , Metabolism
12.
Article in English | WPRIM | ID: wpr-289745

ABSTRACT

<p><b>OBJECTIVE</b>To investigate the effects of ursolic acid (UA) on T-cell proliferation and activation, as well as to examine its effect on nuclear factor-κB (NF-κB) signaling pathway in T cells.</p><p><b>METHODS</b>T-cells isolated from BALB/c mice were incubated with UA at concentrations ranging from 5-30 μmol/L in the presence of phorbol 12-myristate 13-acetate (PMA) or PMA plus ionomycin. The proliferation of T cells was measured by the MTT assay. The expressions of CD69, CD25, and CD71 on T-cell surface were analyzed using flow cytometry. The level of interleukin-2 (IL-2) in the culture supernatant of activated T cells was quantified by enzyme-linked immunosorbent assay (ELISA). The level of phosphorylated IκB-α (p-IκB-α) in total protein and p65, a subunit of NF-κB, nuclear translocation were measured by Western blot analysis.</p><p><b>RESULTS</b>UA in a dose-dependent manner significantly decreased the proliferation and inhibited the surface expressions of CD69, CD25, and CD71 in murine T lymphocytes upon in vitro activation (P<0.01). Significant reduction of IL-2 production was found in activated T cells treated with UA (P<0.01). The PMA-induced increase in p-IκB-α protein was inhibited, and nuclear translocation of p65 from the cytoplasm was blocked by UA.</p><p><b>CONCLUSION</b>UA is a potent inhibitor for T cell activation and proliferation; these effects are associated with the inhibition of NF-κB signaling pathway.</p>


Subject(s)
Animals , Cell Nucleus , Metabolism , Cell Proliferation , I-kappa B Proteins , Metabolism , Interleukin-2 , Bodily Secretions , Ionomycin , Pharmacology , Lymphocyte Activation , Mice , Mice, Inbred BALB C , NF-KappaB Inhibitor alpha , NF-kappa B , Metabolism , Phosphorylation , Protein Transport , Signal Transduction , T-Lymphocytes , Cell Biology , Allergy and Immunology , Bodily Secretions , Tetradecanoylphorbol Acetate , Pharmacology , Triterpenes , Pharmacology
13.
Article in English | WPRIM | ID: wpr-243262

ABSTRACT

<p><b>OBJECTIVE</b>To investigate the main proteinases responsible for CD16b shedding under different stimulators.</p><p><b>METHODS</b>HEK293 cell line stably expressing CD16b was constructed by lentivirus system. The cell line was then overexpressed with a disintegrin and metalloproteinase 10 (ADAM10) or ADAM17, suppressed with short hairpin RNA of ADAM10 or ADAM17, and reconstituted with ADAM10 or ADAM17, respectively. After each treatment, the cell line was stimulated with ionomycin or phorbol 12-myristate- 13-acetate (PMA) for 12 hours. The soluble CD16b released from cell membrane was detected by immunoprecipition and immunoblot. Quantitation was then implemented to compare the amount of soluble CD16b in cell supernatant after stimulation.</p><p><b>RESULTS</b>HEK293 cell line stably expressing CD16b was successfully established. When CD16b expressing cell line was overexpressed with ADAM10, shedding of CD16b was increased after stimulation with ionomycin but not PMA; when the cell line overexpressed with ADAM17, shedding of CD16b was increased after stimulation with PMA but not ionomycin. Similarly, when ADAM10 was suppressed by short hairpin RNA, CD16b shedding was decreased after stimulation with ionomycin; when ADAM17 was suppressed by short hairpin RNA, CD16b shedding was decreased after stimulation with PMA. The shedding of CD16b was increased again when CD16b expressing cell line was reconstituted with ADAM10 and stimulated by ionomycin or reconstituted with ADAM17 and stimulated by PMA.</p><p><b>CONCLUSIONS</b>Both ADAM10 and ADAM17 could shed CD16b, but they possess differed preferences. ADAM10 is the main sheddase under stimulation of ionomycin, while ADAM17 is the main sheddase under stimulation of PMA.</p>


Subject(s)
ADAM Proteins , Genetics , Metabolism , Physiology , ADAM10 Protein , ADAM17 Protein , Amyloid Precursor Protein Secretases , Genetics , Metabolism , Physiology , Calcium Ionophores , Pharmacology , Carcinogens , Pharmacology , Cells, Cultured , Drug Evaluation, Preclinical , GPI-Linked Proteins , Metabolism , Gene Knockdown Techniques , HEK293 Cells , Humans , Ionomycin , Pharmacology , Membrane Proteins , Genetics , Metabolism , Physiology , Protein Processing, Post-Translational , Protein Transport , Proteolysis , Receptors, IgG , Metabolism , Tetradecanoylphorbol Acetate , Pharmacology , Transfection
14.
Chinese Journal of Oncology ; (12): 425-429, 2012.
Article in Chinese | WPRIM | ID: wpr-307370

ABSTRACT

<p><b>OBJECTIVE</b>To investigate the effect of DADLE, a δ-opioid receptor agonist, on the proliferation of human liver cancer HepG2 cells and explore the mechanism involving PKC pathway.</p><p><b>METHODS</b>HepG2 cells were treated with DADLE at different doses (0.01, 0.1, 1.0 and 10 µmol/L). Cell viability was determined using methyl thiazolyl terazolium (MTT) assay. The expression of PKC mRNA and p-PKC protein were examined by RT-PCR and Western blot assay. After treated separately with DADLE plusing NAL or PMA, the cell cycle of HepG2 cells was analyzed by flow cytometer. MTT was used to detect their proliferation capacity and Western blot was used to examine the p-PKC expression. The growth inhibitory rate of HepG2 cells treated with DADLE and cis-diammine dichloridoplatinum (CDDP) was analyzed.</p><p><b>RESULTS</b>DADLE at different concentrations showed an inhibitory effect on the proliferation of HepG2 cells though inhibiting the expression of PKC mRNA and p-PKC protein. The results of flow cytometry showed that compared with the control group, the percentage of S + G(2)/M cells in DADLE-treated group was lowered by 3.94% (P < 0.01). Meanwhile, after treated with NAL and PMA, the percentage was elevated by 3.22% and 3.63%, respectively (P < 0.01). The MTT and Western blot assays showed that compared with the control group, the values of A570 and p-PKC protein levels in the HepG2 cells of DADLE-treated group were significantly decreased (P < 0.01). After treatment with NAL and PMA, the values of A570 and p-PKC protein levels were elevated significantly (P < 0.01). The growth inhibitory rate of DADLE + CDDP group was 79.9%, significantly lower than 25.2% and 43.2% of the DADLE and CDDP groups, respectively.</p><p><b>CONCLUSIONS</b>Activation of δ-opioid receptor by DADLE inhibits the apoptosis of human liver cancer HepG2 cells. The underlying mechanism may be correlated with PKC pathway. DADLE can enhance the chemosensitivity of HepG2 cells to CDDP.</p>


Subject(s)
Antineoplastic Agents , Pharmacology , Cell Cycle , Cell Proliferation , Cisplatin , Pharmacology , Dose-Response Relationship, Drug , Drug Resistance, Neoplasm , Enkephalin, Leucine-2-Alanine , Pharmacology , Hep G2 Cells , Humans , Naltrexone , Pharmacology , Phosphorylation , Protein Kinase C , Genetics , Metabolism , RNA, Messenger , Metabolism , Receptors, Opioid, delta , Signal Transduction , Tetradecanoylphorbol Acetate , Pharmacology
15.
Chinese Journal of Cancer ; (12): 153-162, 2011.
Article in English | WPRIM | ID: wpr-296301

ABSTRACT

The increasing incidence and mortality associated with advanced stages of melanoma are cause for concern. Few treatment options are available for advanced melanoma and the 5-year survival rate is less than 15%. Targeted therapies may revolutionize melanoma treatment by providing less toxic and more effective strategies. However, maximizing effectiveness requires further understanding of the molecular alterations that drive tumor formation, progression, and maintenance, as well as elucidating the mechanisms of resistance. Several different genetic alterations identified in human melanoma have been recapitulated in mice. This review outlines recent progress made in the development of mouse models of melanoma and summarizes what these findings reveal about the human disease. We begin with a discussion of traditional models and conclude with the recently developed RCAS/TVA somatic cell gene delivery mouse model of melanoma.


Subject(s)
9,10-Dimethyl-1,2-benzanthracene , Animals , Avian Leukosis Virus , Genetics , Avian Proteins , Genetics , Metabolism , Cell Line, Tumor , Disease Models, Animal , Gene Transfer Techniques , Genetic Vectors , Genetics , Humans , Melanocytes , Metabolism , Melanoma , Genetics , Pathology , Melanoma, Experimental , Genetics , Mice , Mice, Transgenic , Neoplasm Transplantation , Receptors, Virus , Genetics , Metabolism , Skin Neoplasms , Genetics , Pathology , Tetradecanoylphorbol Acetate , Transgenes
16.
Chinese Journal of Oncology ; (12): 178-182, 2011.
Article in Chinese | WPRIM | ID: wpr-303319

ABSTRACT

<p><b>OBJECTIVE</b>To investigate the effect of sphingosine kinase 1 (SphK1) on the proliferation, apoptosis, migration and invasion of colon cancer TH-29 cells and to explore its molecular mechanisms.</p><p><b>METHODS</b>Phorbol 12-myristate 13-acetate (PMA) was used to induce the activity of SphK1 and N, N-dimethylsphingosine (DMS) was used to suppress the activity of SphK1. Cell prolieration and apoptosis were detected by MTT assay and flow cytometry, respectively. The migration and invasion capabilities of the cells were assessed in Transwell chambers. The activity of SphK1 was assayed by autoradiography. Western blot was used to evaluate the protein expression of SphK1, p38, phosphorylated p38 (p-p38) and SAPK/JNK.</p><p><b>RESULTS</b>PMA and DMS were able to induce and suppress the activity and protein expression of SphK1 in a time-dependent manner, respectively. PMA enhanced and DMS suppressed the cell viability in a time- and dose-dependent manner. Being treated with 100 nmol/L PMA or 50 µmol/L DMS for 0, 6, 12, 24 h, the cell apoptosis rates of PMA group were (9.35 ± 0.84)%, (7.61 ± 0.48)%, (5.53 ± 0.76)% and (0.56 ± 0.33)%, contrastly, that of DMS group were (9.18 ± 0.94)%, (12.06 ± 1.41)%, (19.80 ± 2.36)% and (31.85 ± 3.60)%, respectively. Compared with the control group, the cell migration and invasion capabilities of the PMA group were significantly enhanced, and that of the DMS group were significantly suppressed. The migration cell number of control, PMA and DMS groups were 68.75 ± 6.15, 109.33 ± 11.63 and 10.83 ± 2.48, the invasion cell number of control, PMA and DMS groups were 55.42 ± 4.50, 90.58 ± 7.06 and 9.58 ± 2.39, respectively. With the elevating activity and expression of SphK1, the protein expressions of p38, p-p38 and SAPK/JNK were strikingly suppressed. On the contrary, after treating with DMS the protein expressions of p38, p-p38 and SAPK/JNK were enhanced.</p><p><b>CONCLUSIONS</b>SphK1 potently enhances the prolieration, migration and invasion of colon cancer HT-29 cells, meanwhile suppresses the cell apoptosis. The suppressing of the p38 and SAPK/JNK signalling pathways may be one of its molecular mechanisms.</p>


Subject(s)
Apoptosis , Carcinogens , Pharmacology , Cell Movement , Cell Proliferation , Dose-Response Relationship, Drug , Enzyme Inhibitors , Pharmacology , HT29 Cells , Humans , MAP Kinase Kinase 4 , Metabolism , Neoplasm Invasiveness , Phosphorylation , Phosphotransferases (Alcohol Group Acceptor) , Metabolism , Physiology , Sphingosine , Pharmacology , Tetradecanoylphorbol Acetate , Pharmacology , Time Factors , p38 Mitogen-Activated Protein Kinases , Metabolism
17.
Article in English | WPRIM | ID: wpr-11467

ABSTRACT

OBJECTIVES: Doxycycline is commonly used in medicine for its bacteriostatic antimicrobial properties. Recent studies have reported that doxycycline also has anti-inflammatory effects. Matrix metalloproteinase (MMP)-9 has been found to be involved in the physiological and pathological process of inflammatory airway disease. Phorbol 12-myristate 13-acetate (PMA), a protein kinase C activator, is known to stimulate the expression of MMP and mucin genes in the airway and intestinal epithelial cells. Therefore, the effects and signal pathways of doxycycline on PMA-induced MUC5B expression dependent MMP-9 in human airway epithelial cells were investigated. METHODS: In human NCI-H292 airway epithelial cells, MUC5B and MMP-9 mRNA expression, MUC5B protein expression, and MMP-9 protein activity after the treatment with PMA, MMP-9 or doxycycline were determined by reverse transcriptase-polymerase chain reaction, enzyme immunoassay, gelatin zymography, and Western blot analysis. RESULTS: PMA increased MMP-9 and MUC5B expression. MMP-9 increased MUC5B expression. Doxycycline inhibited PMA-induced MUC5B expression, and PMA-induced MMP-9 mRNA expression and protein activity. Doxycycline inhibited phosphorylation of p38 induced by PMA and MMP-9. CONCLUSION: The results of this study suggest that doxycycline inhibited PMA-induced MUC5B mRNA expression and protein production through the MMP-9 and p38 pathways in human NCI-H292 airway epithelial cells.


Subject(s)
Blotting, Western , Doxycycline , Epithelial Cells , Gelatin , Humans , Immunoenzyme Techniques , Inflammation , Matrix Metalloproteinase 9 , Mucins , Phorbols , Phosphorylation , Protein Kinase C , RNA, Messenger , Signal Transduction , Tetradecanoylphorbol Acetate , Thiram
18.
Int. j. morphol ; 28(3): 911-920, Sept. 2010. ilus
Article in English | LILACS | ID: lil-577205

ABSTRACT

Human neutrophils stimulated by phorbol myristate acetate (PMA), an activator of protein kinase C, produce active oxygen by NADPH oxidase in intracellular structures. We added succinimidyl ester of dichlorodihydrofluorescein diacetate (H2DCFDA), which first emits fluorescence when oxidized with active oxygen species, to neutrophils to produce active oxygen, in order to investigate the antioxidant effects of protocatechuic acid, ferulic acid, and caffeic acid which belong to polyphenols and are widely distributed among plants. Particularly, we focused on examining whether these substances capture and eliminate active oxygen inside or outside the neutrophil cytoplasm and whether these substances inhibit NADPH oxidase. Fluorescence microscopy demonstrated that fluorescence-positive intracellular structures were decreased in neutrophils when stimulated by PMA and exposed to an antioxidant. Quantitative measurement by flow cytometry revealed that the fluorescence intensities in neutrophils, exposed to protocatechuic acid, ferulic acid, or caffeic acid, were decreased by 62.9 percent, 71.4 percent, and 86.1 percent, respectively, as compared with those stimulated by PMA but not exposed to an antioxidant. Judging from fluorescence microscopy and dot blots by flow cytometry, these antioxidants had no effects on neutrophil morphology. On the other hand, the fluorescence intensities of the active oxygen released from neutrophils were decreased by 81.4 percent, 46.7 percent, and 27.4 percent, respectively. Diphenylene iodonium, a specific inhibitor of NADPH oxidase, inhibited the enzyme by 92.1 percent in the PMA-stimulated neutrophils. Protocatechuic acid, ferulic acid, and caffeic acid inhibited the enzyme by 36.5 percent, 54.6 percent, and 27.4 percent, respectively. These results demonstrate that protocatechuic acid, ferulic acid, and caffeic acid capture and eliminate active oxygen, produced by PMA-stimulated neutrophils, intracellularly and extracellularly. Furthe...


Los neutrófilos humanos estimulados por forbol-miristato-acetato (PMA), un activador de la proteína quinasa C, producen oxígeno activo por la NADPH oxidasa en las estructuras intracelulares. Hemos añadido diacetato de 2', 7-dihidro dicloro fluoresceína (H2DCFDA), que emite fluorescencia cuando se oxida con las especies de oxígeno activo, a neutrófilos para producir oxígeno activo, a fin de investigar el efecto antioxidante del ácido protocatéquico, el ácido ferúlico y el ácido cafeico que pertenecen a polifenoles y se distribuyen ampliamente entre las plantas. Particularmente, nos enfocamos en examinar si estas sustancias capturan y eliminan el oxígeno activo dentro o fuera del citoplasma de neutrófilos y si estas sustancias inhiben la NADPH oxidasa. La microscopia de fluorescencia demostró que las estructuras intracelulares positivas a fluorescencia disminuyeron en los neutrófilos mediante la estimulación de la PMA y exposición a un antioxidante. La medición cuantitativa por citometría de flujo reveló que la intensidad de fluorescencia en los neutrófilos, expuestos al ácido protocatéquico, el ácido ferúlico, o el ácido cafeico, se redujo un 62,9 por ciento, 71,4 por ciento y 86,1 por ciento, respectivamente, en comparación con las estimuladas por PMA pero no expuestas a un antioxidante. A juzgar desde la microscopía de fluorescencia y la citometría de flujo, estos antioxidantes no tuvieron efectos sobre la morfología de los neutrófilos. Por otra parte, la intensidad de fluorescencia del oxígeno activo liberado por los neutrófilos se redujeron un 81,4 por ciento, 46,7 por ciento y 27,4 por ciento, respectivamente. El DPI (difenileno-iodonio), un inhibidor específico de la NADPH oxidasa, inhibió a la enzima en el 92,1 por ciento en los neutrófilos estimulados por PMA. El ácido protocatéquico, el ácido ferúlico y el ácido caféico inhiben la enzima en un 36,5 por ciento, 54,6 por ciento y 27,4 por ciento, respectivamente. Estos resultados demuestran...


Subject(s)
Antioxidants/pharmacology , Phenols/pharmacology , Neutrophils , Caffeic Acids/pharmacology , Coumaric Acids/pharmacology , Flow Cytometry , Fluorescein , Hydroxybenzoates , Microscopy, Fluorescence , NADPH Oxidases/antagonists & inhibitors , Tetradecanoylphorbol Acetate
19.
Chinese Medical Journal ; (24): 2440-2445, 2010.
Article in English | WPRIM | ID: wpr-237436

ABSTRACT

<p><b>BACKGROUND</b>The CKLF-like MARVEL transmembrane domain-containing family (CMTM) is a novel family of proteins linking chemokines and TM4SF. Different members exhibit diverse biological functions. In this study, the effect of intracellular CMTM2 on regulating human immunodeficiency virus type-1 (HIV-1) transcription was evaluated.</p><p><b>METHODS</b>The effects of CMTM2 on regulating full-length HIV-1 provirus and the HIV-1 long terminal repeat (LTR)-directed transcription were assessed by luciferase assay. Transcription factor assays, using the luciferase reporter plasmids of AP-1, CRE, and NF-κB were conducted to explore the signaling pathway(s) that may be regulated by CMTM2. The potential relationship between CMTM2 and the transcription factor AP-1 was further analyzed by Western blotting analyses to investigate the effect of CMTM2 on PMA-induced ERK1/2 phosphorylation.</p><p><b>RESULTS</b>The results from the current study revealed that CMTM2 acts as a negative regulator of HIV-1 transcription. CMTM2 exerted a suppressive action on both full-length HIV-1 provirus and HIV-1 LTR-directed transcription. Transcription factor assays showed that CMTM2 selectively inhibited basal AP-1 and CREB activity. Co-expression of HIV-1 Tat, a potent AP-1 and CREB activator, can not reverse CMTM2-mediated AP-1 and CREB inhibition, suggesting a potent and specific effect of CMTM2 on negatively regulating these two signaling pathways.</p><p><b>CONCLUSION</b>Intracellular CMTM2 can negatively regulate HIV-1 transcription, at least in part, by targeting the AP-1 and CREB pathways. Exploring the mechanisms further may lead to new ways to control HIV-1 replication.</p>


Subject(s)
Chemokines , Physiology , Cyclic AMP Response Element-Binding Protein , HIV Long Terminal Repeat , HIV-1 , Genetics , Humans , Intracellular Space , Metabolism , Jurkat Cells , MARVEL Domain-Containing Proteins , Tetradecanoylphorbol Acetate , Pharmacology , Transcription Factor AP-1 , Transcription, Genetic , U937 Cells
20.
Pakistan Journal of Medical Sciences. 2010; 26 (2): 352-357
in English | IMEMR | ID: emr-97979

ABSTRACT

To observe the K562 cell line derived from a patient of chronic myelogenous leukemia differentiated into megakaryocytes by growing in the presence of phorbol myristate acetate [PMA]. The differentiation process of K562 cells was monitored by the expression of a platelet cell marker, CD61 through immunocytochemistry using mouse alkaline phosphatase antialkaline phosphatase [APAAP] complex employing fast red TR as substrate, crystal violet and MTT assay used for cell growth analysis. The crystal in the presence of PMA, cells obtained were of large size and less in number as compared to cells incubated without PMA where they were of smaller size and more in number and immunochemical reaction used to detect the presence of CD61, a platelet cell marker that is expressed during differentiation of K562 cells to megakaryocytes. The results showed that the addition of PMA to the growing culture of K562 cell lines induced differentiation, observed through CD61 expression and increase in cell size and cessation of proliferation


Subject(s)
Humans , K562 Cells/drug effects , Tetradecanoylphorbol Acetate , Megakaryocyte Progenitor Cells/drug effects , Leukemia, Myelogenous, Chronic, BCR-ABL Positive
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