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1.
Cells ; 12(9)2023 04 26.
Article in English | MEDLINE | ID: mdl-37174663

ABSTRACT

Fibroblast-like synoviocytes (FLS) in rheumatoid arthritis (RA) patients have increased reactive oxygen species (ROS) levels and an impaired redox balance compared with FLS from control patients. Liver kinase B1 (LKB1) plays a key role in ROS scavenging and cellular metabolism in various cancers. Here, we aimed to determine the specific mechanism of LKB1 in RA pathogenesis. FLS were obtained from RA patients (n = 10). siRNA-induced LKB1 deficiency in RA FLS increased ROS levels via NADPH oxidase 4 (NOX4) upregulation. RA FLS migration and expression of inflammatory factors, including interleukin (IL)-1ß, IL-6, IL-8, tumor necrosis factor-alpha (TNF-α), and vascular endothelial growth factor (VEGF), were enhanced by LKB1 deficiency. LKB1-deficient RA FLS showed increased sensitivity to oxidative stress damage caused by hydrogen peroxidase exposure. siRNA-induced solute carrier family 7 member 11 (SLC7A11) deficiency in RA FLS enhanced NOX4 and ROS expression and increased cell migration. When LKB1-deficient RA FLS were stimulated with an AMP-activated protein kinase (AMPK) activator, the LKB1-inhibition-induced cell migration significantly decreased through the restoration of SLC7A11/NOX4 expression. LKB1 regulates the AMPK-mediated SLC7A11-NOX4-ROS pathway to control cell migration and inflammation. Our data indicate that LKB1 is a key regulator of redox homeostasis in RA FLS.


Subject(s)
AMP-Activated Protein Kinases , Arthritis, Rheumatoid , Synoviocytes , Humans , Amino Acid Transport System y+/metabolism , AMP-Activated Protein Kinases/metabolism , Arthritis, Rheumatoid/pathology , Fibroblasts/metabolism , Inflammation/pathology , NADPH Oxidase 4/metabolism , Reactive Oxygen Species/metabolism , Synoviocytes/metabolism , Vascular Endothelial Growth Factor A/metabolism
2.
Biomater Sci ; 10(12): 3223-3235, 2022 Jun 14.
Article in English | MEDLINE | ID: mdl-35579255

ABSTRACT

In osteoarthritis (OA), chondrocytes in cartilage undergo phenotypic changes and senescence, restricting cartilage regeneration and favoring disease progression. Although senescence biomarker p16INK4a expression is known to induce aging by halting the cell cycle, therapeutic applications for p16INK4a targeting are limited. Here, we aimed to reduce cartilage damage and alleviate pain using p16INK4a nanoparticles in OA. The p16INK4a expression of human OA chondrocytes and synoviocytes from patients with knee OA was measured and the levels of p16INK4a, tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, IL-6, and matrix metalloproteinase (MMP) 13 were examined. p16INK4a siRNA was encapsulated into poly (lactic-co-glycolic acid) (PLGA) nanoparticles and characterized. The partial medial meniscectomy (pMMx) model was performed for the OA model which was investigated by molecular analysis and behavioral tests. The expression of p16INK4a was increased in the synovium and articular cartilage from OA patients. p16INK4a siRNA-loaded PLGA nanoparticles (p16 si_NP) reduced the levels of TNF-α, IL-1ß, and IL-6 especially in fibroblast-like synoviocytes (FLSs), and MMP13 in chondrocytes. Rhodamine-tagged NPs injected into the mouse knee joints were found mainly in the synovium. p16 si_NP injection in the pMMx model alleviated pain-associated behavior, and reduced cartilage damage and p16INK4a in the synovium, and MMP13, collagen X, and NITEGE in cartilage. The preferential reduction of p16INK4a in FLSs by the application of RNAi nanomedicine could contribute to the recovery of osteoarthritic cartilage and relieve pain, suggesting that p16INK4a may be a viable future therapeutic candidate.


Subject(s)
Cartilage, Articular , Nanoparticles , Osteoarthritis, Knee , Synoviocytes , Animals , Cartilage, Articular/pathology , Cells, Cultured , Chondrocytes/metabolism , Fibroblasts/metabolism , Humans , Inflammation/pathology , Interleukin-6/metabolism , Matrix Metalloproteinase 13/genetics , Matrix Metalloproteinase 13/metabolism , Mice , Osteoarthritis, Knee/pathology , Pain , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Synoviocytes/metabolism , Tumor Necrosis Factor-alpha/metabolism
3.
Int J Mol Sci ; 23(4)2022 Feb 20.
Article in English | MEDLINE | ID: mdl-35216458

ABSTRACT

This study aimed to examine the role of CD70, which is highly expressed on fibroblast-like synoviocytes (FLS), in rheumatoid arthritis (RA) patients. FLS isolated from RA (n = 14) and osteoarthritis (OA, n = 4) patients were stimulated with recombinant interleukin-17 (IL-17; 5 ng/mL) and tumor necrosis factor alpha (TNF-α; 5 ng/mL) for 24 h. Expression of CD70, CD27/soluble CD27 (sCD27), and hypoxia-inducible factor-2 alpha (HIF-2α) was analyzed by RT-qPCR, flow cytometry, and ELISA assays, respectively. Reactive oxygen species (ROS) expression and cell migration were also examined. The HIF-2α inhibitor PT-2385 and CD70 inhibitor BU69 were used to specifically suppress these pathways. Stimulation with IL-17 and TNF-α significantly induced CD70 expression in RA FLS. Although the synovial fluids from patients with RA contained high levels of sCD27, surface expression of CD27, a ligand of CD70, was rarely detected in RA FLS. Cytokine-induced CD70 expression was significantly decreased following antioxidant treatment. Following HIF-2α inhibition, RA FLS had decreased expression of CD70 and ROS levels. Migration of RA FLS was also inhibited by inhibition of CD70 or HIF-2α. The surface expression of CD70 is regulated by HIF-2α and ROS levels and is a key contributor to cytokine-enhanced migration in RA FLS.


Subject(s)
Arthritis, Rheumatoid , Basic Helix-Loop-Helix Transcription Factors , CD27 Ligand , Osteoarthritis , Oxidative Stress , Synoviocytes , Arthritis, Rheumatoid/metabolism , Basic Helix-Loop-Helix Transcription Factors/metabolism , CD27 Ligand/metabolism , Cells, Cultured , Fibroblasts/metabolism , Humans , Hypoxia/metabolism , Interleukin-17/metabolism , Interleukin-17/pharmacology , Osteoarthritis/metabolism , Reactive Oxygen Species/metabolism , Synovial Membrane/pathology , Synoviocytes/metabolism , Tumor Necrosis Factor-alpha/metabolism , Tumor Necrosis Factor-alpha/pharmacology
4.
Arch Rheumatol ; 37(3): 335-343, 2022 Sep.
Article in English | MEDLINE | ID: mdl-36589618

ABSTRACT

Objectives: This study aims to investigate the role of cluster of differentiation 14 (CD14) expressed monocytes and soluble CD14-mediated pathway in the synovial inflammation of knee osteoarthritis (OA). Patients and methods: Between May 2012 and July 2013, a total of 35 patients with knee OA (9 males, 26 females; mean age: 66.3±8.8 years; range, 52 to 79 years) were included in this cross-sectional study. Synovial fluid was obtained from knee joints of 35 OA patients. The CD14+ monocytes from synovial fluid mononuclear cells (SFMCs) were isolated using the MACS. The fibroblast-like synoviocytes (FLSs) isolated from knee joint tissue were incubated with recombinant CD14 and lipopolysaccharide (LPS) for 24 h. Cytokine profiling was performed with the Luminex® Performance Assay or magnetic bead panel kit. The expression of CD14 and CD16 was analyzed by immunohistochemistry and flow cytometry. Results: The concentration of sCD14 in synovial fluid was correlated with the interleukin-6 (IL-6) level (n=35) (ρ=0.654, p<0.001). The culture supernatants of CD14+ monocytes isolated from SFMC (n=15) showed a correlation between sCD14 and IL-6 (ρ=0.784, p=0.001), along with complement component 3 (ρ=0.756, p=0.010), IL-1b (ρ=0.652, p=0.012), and tumor necrosis factor-alpha (ρ=0.806, p=0.001). Following recombinant CD14 and LPS treatment, OA FLS synergistically enhanced the secretion of IL-6, IL-8, and matrix metalloproteinase 3 (n=3, p<0.05). In five paired-samples from identical patients, the proportions of CD14+ monocytes were significantly elevated in recurred synovial fluid compared to those in initial synovial fluid (p=0.043). When monocyte subsets were analyzed in SFMC (n=26), CD14+CD16+monocytes were abundant (p=0.019) and had higher toll-like receptor 4 expression than CD14+CD16- (p<0.001). Conclusion: Our study results suggest that CD14+ monocytes and the sCD14-mediated pathway play an important role in OA aggravation through inflammatory cytokine secretion.

5.
J Clin Med ; 10(22)2021 Nov 16.
Article in English | MEDLINE | ID: mdl-34830606

ABSTRACT

There is growing evidence that apurinic/apyrimidinic endonuclease 1/redox factor-1 (APE1/Ref-1) regulates inflammatory responses. Rheumatoid arthritis (RA) is an autoimmune disease, which is characterized with synovitis and joint destruction. Therefore, this study was planned to investigate the relationship between APE1/Ref-1 and RA. Serum and synovial fluid (SF) were collected from 46 patients with RA, 45 patients with osteoarthritis (OA), and 30 healthy control (HC) patients. The concentration of APE1/Ref-1 in serum or SF was measured using the sandwich enzyme-linked immunosorbent assay (ELISA). The disease activity in RA patients was measured using the 28-joint disease activity score (DAS28). The serum APE1/Ref-1 levels in RA patients were significantly increased compared to HC and OA patients (0.44 ± 0.39 ng/mL for RA group vs. 0.19 ± 0.14 ng/mL for HC group, p < 0.05 and vs. 0.19 ± 0.11 ng/mL for OA group, p < 0.05). Likewise, the APE1/Ref-1 levels of SF in RA patients were also significantly increased compared to OA patients (0.68 ± 0.30 ng/mL for RA group vs. 0.31 ± 0.12 ng/mL for OA group, p < 0.001). The APE1/Ref-1 concentration in SF of RA patients was positively correlated with DAS28. Thus, APE1/Ref-1 may reflect the joint inflammation and be associated with disease activity in RA.

6.
Int J Mol Sci ; 22(22)2021 Nov 17.
Article in English | MEDLINE | ID: mdl-34830290

ABSTRACT

The production and oxidation mechanism of reactive oxygen species (ROS) are out of balance in rheumatoid arthritis (RA). However, the correlation between ROS and T cell subsets in RA remains unclear. Peripheral blood mononuclear cells (PBMCs) from patients with RA (n = 40) and healthy controls (n = 10) were isolated from whole blood samples. Synovial tissues (n = 3) and synovial fluid (n = 10) were obtained from patients with RA. The repartition of T cell subsets and expression of ROS and cytokines were examined according to RA severity. Fibroblast-like synoviocytes (FLSs) from patients with RA were stimulated with PBMCs and the expression of inflammation-related molecules were measured by RT-PCR and cytokine array. Regulatory T cells from patients with moderate (5.1 > DAS28 ≥ 3.2) RA showed the highest expression of mitochondrial ROS among the groups based on disease severity. Although ROS levels steadily increased with RA severity, there was a slight decline in severe RA (DAS28 ≥ 5.1) compared with moderate RA. The expression of inflammatory cytokines in RA FLSs were significantly inhibited when FLSs were co-cultured with PBMCs treated with ROS inhibitor. These findings provide a novel approach to suppress inflammatory response of FLSs through ROS regulation in PBMCs.


Subject(s)
Arthritis, Rheumatoid/blood , Arthritis, Rheumatoid/immunology , Fibroblasts/immunology , Oxidative Stress , Reactive Oxygen Species/metabolism , Synoviocytes/immunology , T-Lymphocytes, Helper-Inducer/immunology , T-Lymphocytes, Regulatory/immunology , Adult , Aged , Aged, 80 and over , Arthritis, Rheumatoid/pathology , Case-Control Studies , Cells, Cultured , Coculture Techniques , Cytokines/metabolism , Female , Humans , Inflammation/metabolism , Male , Middle Aged , Mitochondria/metabolism , Severity of Illness Index , Synovial Fluid/metabolism
7.
Arthritis Res Ther ; 22(1): 116, 2020 05 15.
Article in English | MEDLINE | ID: mdl-32414400

ABSTRACT

BACKGROUND: Reactive oxygen species (ROS) regulate the migration and invasion of fibroblast-like synoviocytes (FLS), which are key effector cells in rheumatoid arthritis (RA) pathogenesis. Nicotinamide adenine dinucleotide phosphate oxidase 4 (NOX4) induces ROS generation and, consequently, enhances cell migration. Despite the important interrelationship between RA, FLS, and ROS, the effect of NOX4 on RA pathogenesis remains unclear. METHODS: FLS isolated from RA (n = 5) and osteoarthritis (OA, n = 5) patients were stimulated with recombinant interleukin 17 (IL-17; 10 ng/ml) and tumor necrosis factor alpha (TNF-α; 10 ng/ml) for 1 h. Cell migration, invasion, adhesion molecule expression, vascular endothelial growth factor (VEGF) secretion, and ROS expression were examined. The mRNA and protein levels of NOX4 were analyzed by RT-qPCR and western blotting, respectively. The NOX4 inhibitor GLX351322 and NOX4 siRNA were used to inhibit NOX4 to probe the effect of NOX4 on these cellular processes. RESULTS: Migration of RA FLS was increased 2.48-fold after stimulation with IL-17 and TNF-α, while no difference was observed for OA FLS. ROS expression increased in parallel with invasiveness of FLS following cytokine stimulation. When the expression of NOX was examined, NOX4 was significantly increased by 9.73-fold in RA FLS compared to unstimulated FLS. Following NOX4 inhibition, cytokine-induced vascular cell adhesion molecule 1 (VCAM1), VEGF, and migration and invasion capacity of RA FLS were markedly decreased to unstimulated levels. CONCLUSION: NOX4 is a key contributor to cytokine-enhanced migration and invasion via modulation of ROS, VCAM1, and VEGF in RA FLS.


Subject(s)
Arthritis, Rheumatoid/pathology , NADPH Oxidase 4/metabolism , Synoviocytes/cytology , Arthritis, Rheumatoid/metabolism , Cell Movement , Cell Proliferation , Cells, Cultured , Fibroblasts , Humans , Oxidoreductases , Reactive Oxygen Species/metabolism , Synovial Membrane , Vascular Endothelial Growth Factor A
8.
Oral Dis ; 26(1): 111-121, 2020 Jan.
Article in English | MEDLINE | ID: mdl-31677207

ABSTRACT

OBJECTIVE: This study was designed to investigate whether necroptosis is involved in the pathogenesis of chemoradiation-induced oral mucositis in a murine model and whether 1-palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol (PLAG) ameliorates this disorder. MATERIALS AND METHODS: A chemoradiation-induced oral mucositis model was established by treating mice with concurrent 5-fluorouracil (100 mg/kg, i.p.) and head and neck X-irradiation (20 Gy). Phosphate-buffered saline or PLAG (100 mg/kg or 250 mg/kg, p.o.) was administered daily. Body weights were recorded daily, and mice were sacrificed on Day 9 for tongue tissue analysis. RESULTS: On Day 9, chemoradiotherapy-treated (ChemoRT) mice had tongue ulcerations and experienced significant weight loss (Day 0:26.18 ± 1.41 g; Day 9:19.44 ± 3.26 g). They also had elevated serum macrophage inhibitory protein 2 (MIP-2) (control: 5.57 ± 3.49 pg/ml; ChemoRT: 130.14 ± 114.54 pg/ml) and interleukin (IL)-6 (control: 198.25 ± 16.91 pg/ml; ChemoRT: 467.25 ± 108.12 pg/ml) levels. ChemoRT-treated mice who received PLAG exhibited no weight loss (Day 0:25.78 ± 1.04 g; Day 9:26.46 ± 1.68 g) and had lower serum MIP-2 (4.42 ± 4.04 pg/ml) and IL-6 (205.75 ± 30.41 pg/ml) levels than ChemoRT-treated mice who did not receive PLAG. Tongue tissues of mice who received PLAG also displayed lower phosphorylation levels of necroptotic signalling proteins. CONCLUSION: 1-Palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol mitigated chemoradiation-induced oral mucositis by modulating necroptosis.


Subject(s)
Chemoradiotherapy/adverse effects , Diglycerides/pharmacology , Stomatitis/drug therapy , Animals , Chemokine CXCL2/blood , Fluorouracil/adverse effects , Interleukin-6/blood , Male , Mice , Mice, Inbred BALB C , Stomatitis/etiology
9.
Front Immunol ; 10: 2177, 2019.
Article in English | MEDLINE | ID: mdl-31620122

ABSTRACT

Acute lung injury (ALI) is an acute respiratory failure that is associated with excessive neutrophil recruitment and high mortality. To assess the efficacy of 1-palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol (PLAG) as a therapeutic agent for ALI, this compound was administered orally to mice challenged with an intranasal dose of lipopolysaccharide (LPS). Using this model, we found that PLAG promotes resolution of ALI through effective control of LPS-induced neutrophil infiltration, endothelial permeability, and inflammatory chemokine production. In addition, the Toll like Receptor 4 (TLR4) endocytosis/exocytosis cycle was significantly accelerated in Raw 264.7 cells co-treated with PLAG/LPS, as compared to cells treated only with LPS. During this cycle, a PLAG-induced exotoxin clearance pathway was observed to occur through the prompt assembly of nicotinamide adenine dinucleotide phosphate (NADPH) units and production of reactive oxygen species (ROS), which ultimately lead to earlier LPS clearance. We further detected reduced expression, as well as faster return to homeostatic levels, of macrophage inflammatory protein (MIP)-2, in PLAG/LPS- vs. LPS-treated cells. MIP-2 is a main inducer of neutrophil migration that is mainly controlled by interferon regulatory factor 3 (IRF3) activation and is involved in the TLR4 endosomal-signaling pathway. PLAG induced TLR4-mediated TRIF-related adaptor molecules/Toll-interleukin receptor (TIR) domain-containing adaptor protein including interferon (IFN)-ß/IRF3 endosomal signaling, leading to rapid association of TRAM/TRIF and TLR4 and earlier IRF3 phosphorylation in PLAG/LPS-treated vs. LPS-treated cells. PLAG specificity was further verified with PLAG analogs and metabolites known to control excessive neutrophil infiltration, suggesting that this acetylated diacylglycerol has a unique biological role in neutrophil motility. Thus, our data indicate that PLAG may represent a potential therapeutic agent for resolution of LPS-induced lung inflammation through effective MIP-2 modulation.


Subject(s)
Acute Lung Injury/immunology , Diglycerides/pharmacology , Neutrophil Infiltration/drug effects , Acute Lung Injury/chemically induced , Animals , Lipopolysaccharides/toxicity , Male , Mice , Mice, Inbred BALB C , RAW 264.7 Cells
10.
Thromb Res ; 161: 84-90, 2018 01.
Article in English | MEDLINE | ID: mdl-29220692

ABSTRACT

Previously, PLAG (1-palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol, acetylated diglyceride) was reported to have an effect on the proliferation of hematopoietic stem cells (HSCs) or to contribute to the prevention of chemotherapy-induced neutropenia. In this study, we examined the role of PLAG in the differentiation of bone marrow cells from HSCs into progenitor cells in mice. After 15days, the lineage-negative cells, especially megakaryocyte/erythrocyte progenitors (MEP), were significantly increased in mice that received daily PLAG administration compared to those in the untreated mice. Furthermore, we explored the possibility that the PLAG-induced increase in MEP will contribute to reduction of chemotherapy-induced thrombocytopenia (CIT) in a thrombocytopenia mouse model. Mice were administrated 5-fluorouracil (5-FU) and PLAG. After 7days, bone marrow cells were analyzed. Treatment with 5-FU powerfully decreased myeloid precursor populations and treatment with 5-FU/PLAG resulted in reduction of decreased myeloid progenitor cell numbers. In addition, numbers of circulating platelets were also increased by PLAG treatment. Taken together, PLAG plays a role in differentiating HSCs toward MEP and alleviating chemotherapy-induced bone marrow cell reduction. Thus PLAG shows its potential to augment the therapeutic effect of anti-cancer drugs-induced thrombocytopenia.


Subject(s)
Diglycerides/pharmacology , Erythrocytes/drug effects , Megakaryocytes/drug effects , Thrombocytopenia/chemically induced , Thrombocytopenia/drug therapy , Animals , Blood Platelets/drug effects , Blood Platelets/pathology , Cell Differentiation/drug effects , Erythrocytes/pathology , Fluorouracil/adverse effects , Male , Megakaryocytes/pathology , Mice , Thrombocytopenia/blood , Thrombocytopenia/pathology
11.
Front Oncol ; 6: 209, 2016.
Article in English | MEDLINE | ID: mdl-27800302

ABSTRACT

Chemotherapy-induced mucositis can limit the effectiveness of cancer therapy and increase the risk of infections. However, no specific therapy for protection against mucositis is currently available. In this study, we investigated the therapeutic effect of PLAG (1-palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol, acetylated diglyceride) in 5-fluorouracil (5-FU)-induced oral mucositis animal models. Hamsters were administered 5-FU (80 mg/kg) intraperitoneally on days 0, 6, and 9. The animals' cheek pouches were then scratched equally with the tip of an 18-gage needle on days 1, 2, and 7. PLAG was administered daily at 250 mg/kg/day. PLAG administration significantly reduced 5-FU/scratching-induced mucositis. Dramatic reversal of weight loss in PLAG-treated hamsters with mucositis was observed. Histochemical staining data also revealed newly differentiated epidermis and blood vessels in the cheek pouches of PLAG-treated hamsters, indicative of recovery. Whole blood analyses indicated that PLAG prevents 5-FU-induced excessive neutrophil transmigration to the infection site and eventually stabilizes the number of circulating neutrophils. In a mouse mucositis model, mice with 5-FU-induced disease treated with PLAG exhibited resistance to body-weight loss compared with mice that received 5-FU or 5-FU/scratching alone. PLAG also dramatically reversed mucositis-associated weight loss and inhibited mucositis-induced inflammatory responses in the tongue and serum. These data suggest that PLAG enhances recovery from 5-FU-induced oral mucositis and may therefore be a useful therapeutic agent for treating side effects of chemotherapy, such as mucositis and cachexia.

12.
Cancer Lett ; 377(1): 25-31, 2016 07 10.
Article in English | MEDLINE | ID: mdl-27105612

ABSTRACT

Granulocyte colony-stimulating factor (G-CSF) is widely used for preventing neutropenia during chemotherapy. Polyethylene glycol-conjugated granulocyte colony-stimulating factor (PEG-G-CSF, pegfilgrastim) serves the same purpose but has a longer half-life and greater stability than G-CSF. In this study, we investigated whether 1-palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol, acetylated diglyceride (PLAG), augments the therapeutic effect of pegfilgrastim on chemotherapy-induced neutropenia. We compared neutrophil counts in four groups of mice: control mice, gemcitabine-treated mice, gemcitabine/pegfilgrastim-treated mice, and gemcitabine/pegfilgrastim/PLAG-treated mice. PLAG (50 mg/kg) was orally administered every day during the treatment course. CBC analysis showed that the group treated with PLAG experienced a dramatically increased neutrophil counts on the third day following pegfilgrastim treatment. PLAG had no effect on blood cell apoptosis and neutrophil release from bone marrow. Additionally, pegfilgrastim-induced CXCR2 expression in neutrophils was markedly decreased in PLAG-treated animals. These results suggest that PLAG plays a role in inhibiting neutrophil extravasation, giving rise to an increased number of circulating neutrophils when used with pegfilgrastim during gemcitabine treatment. These data support the potential for PLAG to be used with pegfilgrastim to treat or prevent chemotherapy-induced neutropenia by modulating neutrophil transmigration.


Subject(s)
Deoxycytidine/analogs & derivatives , Diglycerides/pharmacology , Granulocyte Colony-Stimulating Factor/pharmacology , Neutropenia/drug therapy , Neutrophils/drug effects , Animals , Biomarkers/blood , Cell Movement/drug effects , Disease Models, Animal , Drug Synergism , Drug Therapy, Combination , Filgrastim , Leukocyte Count , Mice, Inbred BALB C , Neutropenia/blood , Neutropenia/chemically induced , Neutrophils/metabolism , Polyethylene Glycols , Receptors, Interleukin-8B/blood , Recombinant Proteins/pharmacology , Time Factors , Gemcitabine
13.
Immunol Lett ; 173: 69-76, 2016 05.
Article in English | MEDLINE | ID: mdl-26987843

ABSTRACT

Genkwadaphnin (GD), an extract from the flower buds of Daphne genkwa Siebold & Zucc. (Thymelaeaceae) has been reported a significant anti-leukemic activity. However, its functional mechanism has not been defined well. To study the biological mechanism of GD function, we have investigated whether GD affects CD44 expression, which has a role in the regulation of immune cell motilities, and identified the related signaling pathways. GD treatment induced the increase of CD44 expression in a time- and concentration-dependent manner, which was specific for immune cells. GD activated PKD1/NF-κB signaling to induce CD44 expression, and resulted in the increased migration of K562 cells. In invasion assay, cell migratory ability was induced by GD and the transfection with CD44-specific short hairpin RNA resulted in reduction of its cell migration. GD treated human peripheral blood mononuclear cell (PBMC) were also shown the increased CD44 expression and migration. These data suggest that the induction of CD44 expression by GD treatment promotes immune cell transmigration resulting in the enhanced innate immunity.


Subject(s)
Antineoplastic Agents/pharmacology , Diterpenes/pharmacology , Hyaluronan Receptors/metabolism , Leukemia/drug therapy , Leukocytes, Mononuclear/physiology , Daphne/immunology , HEK293 Cells , Humans , Hyaluronan Receptors/genetics , Immunity, Innate , K562 Cells , Leukocytes, Mononuclear/drug effects , Medicine, Chinese Traditional , NF-kappa B/metabolism , Plant Extracts , RNA, Small Interfering/genetics , Signal Transduction/drug effects , TRPP Cation Channels/metabolism , U937 Cells , Up-Regulation
14.
J Sci Food Agric ; 96(8): 2635-40, 2016 Jun.
Article in English | MEDLINE | ID: mdl-26282882

ABSTRACT

BACKGROUND: Euphorbia kansui, a traditional medical herb, has been shown to have anti-tumour and anti-viral activities. Previously, we have reported that E. kansui increases interferon-gamma (IFN-γ) production in natural killer (NK) cells. However, it is not clear how E. kansui regulates IFN-γ secretion by NK cells. RESULTS: In this study, E. kansui was separated into six individual compounds from the same chloroform fraction so that the activity of each compound could be compared. E. kansui compounds induced IFN-γ secretion through the phosphorylation of protein kinase D and IκB kinase pathways. Furthermore, E. kansui compounds activated the translocation of p65, a sub-unit of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), to the nucleus and induced NF-κB at the transcriptional level. CONCLUSION: These findings suggest that E. kansui enhances IFN-γ secretion through the NF-κB pathway in NK cells. © 2015 Society of Chemical Industry.


Subject(s)
Diterpenes/chemistry , Euphorbia/chemistry , Gene Expression Regulation/drug effects , Interferon-gamma/metabolism , NF-kappa B/metabolism , Active Transport, Cell Nucleus , Cell Line , Humans , Signal Transduction , Transcription, Genetic/drug effects
15.
J Cell Biochem ; 117(1): 172-9, 2016 Jan.
Article in English | MEDLINE | ID: mdl-26096175

ABSTRACT

Genkwadaphnin (GD-1) is isolated from the flower buds of Daphne genkwa Siebold et Zuccarini (Thymelaeaceae), and it has been used as a traditional Korean and Chinese medicine. In this study, the authors observe that GD-1 inhibits the growth of the colon cancer cell line, SW620, through the up-regulation of p21 expression in a PRDM1-dependent manner. After treatment with GD-1, the transcriptional repressor PRDM1 is prominently induced in SW620 cells. Furthermore, GD-1 induce the phosphorylation of PKD1 and MEK and subsequently provide PRDM1 enhancement, resulting in the suppression of c-Myc expression and the up-regulation of p21. PKD1 knockdown using siRNA abrogates PRDM1 expression by GD-1 and subsequently disrupts the regulation of c-Myc and p21 expression. Treating SW620 cells with GD-1 inhibits cell-cycle progression and is characterized by the down-regulation of c-Myc followed by the up-regulation of p21 expression. The up-regulation of p21 by GD-1 induces the growth arrest of the SW620 colon cancer cell line. Based on these data, the authors propose that GD-1 has tumor-suppressor activity that may contribute to the anti-tumor effects of PRDM1 in colon cancer.


Subject(s)
Colonic Neoplasms/metabolism , Diterpenes/pharmacology , Repressor Proteins/metabolism , Apoptosis/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , Cyclin-Dependent Kinase Inhibitor p21/metabolism , Gene Expression Regulation, Neoplastic/drug effects , Humans , Phosphorylation/drug effects , Positive Regulatory Domain I-Binding Factor 1
16.
Mol Cancer Ther ; 14(11): 2613-22, 2015 Nov.
Article in English | MEDLINE | ID: mdl-26351320

ABSTRACT

The MET receptor tyrosine kinase, the receptor for hepatocyte growth factor (HGF), has been implicated in cancer growth, invasion, migration, angiogenesis, and metastasis in a broad variety of human cancers, including human hepatocellular carcinoma (HCC). Recently, MET was suggested to be a potential target for the personalized treatment of HCC with an active HGF-MET signaling pathway. However, the mechanisms of resistance to MET inhibitors need to be elucidated to provide effective treatment. Here, we show that HCC cells exhibit different sensitivities to the MET inhibitor PHA665752, depending on the phosphorylation status of FGFR. Treatment of cells expressing both phospho-FGFR and phospho-MET with the inhibitor PHA665752 did not cause growth inhibition and cell death, whereas treatment with AZD4547, a pan-FGFR inhibitor, resulted in decreased colony formation and cleavage of caspase-3. Moreover, silencing of endogenous FGFR1 and FGFR2 by RNAi of HCC cells expressing phospho-FGFR, phospho-FGFR2, and phospho-MET overcame the resistance to PHA665752 treatment. Treatment of primary cancer cells from patients with HCC expressing both phospho-FGFR and phospho-MET with PHA665752 did not induce cell death, whereas AZD4547 treatment induced cell death through the cleavage of caspase-3. In addition, treatment of cells resistant to PHA665752 with AZD4547 abrogated the activation of downstream effectors of cell growth, proliferation, and survival. On the basis of these results, we conclude that the FGFR pathway is critical for HCC survival, and that targeting this pathway with AZD4547 may be beneficial for the treatment of patients with HCC-expressing phospho-FGFR and phospho-MET.


Subject(s)
Proto-Oncogene Proteins c-met/metabolism , Receptor, Fibroblast Growth Factor, Type 1/metabolism , Receptor, Fibroblast Growth Factor, Type 2/metabolism , Signal Transduction/physiology , Antineoplastic Agents/pharmacology , Benzamides/pharmacology , Blotting, Western , Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/metabolism , Carcinoma, Hepatocellular/pathology , Cell Line, Tumor , Cell Survival/drug effects , Cell Survival/genetics , Drug Resistance, Neoplasm/drug effects , Drug Resistance, Neoplasm/genetics , Humans , Indoles/pharmacology , Liver Neoplasms/genetics , Liver Neoplasms/metabolism , Liver Neoplasms/pathology , Microscopy, Confocal , Phosphorylation/drug effects , Piperazines/pharmacology , Proto-Oncogene Proteins c-met/antagonists & inhibitors , Proto-Oncogene Proteins c-met/genetics , Pyrazoles/pharmacology , RNA Interference , Receptor, Fibroblast Growth Factor, Type 1/antagonists & inhibitors , Receptor, Fibroblast Growth Factor, Type 1/genetics , Receptor, Fibroblast Growth Factor, Type 2/antagonists & inhibitors , Receptor, Fibroblast Growth Factor, Type 2/genetics , Signal Transduction/drug effects , Signal Transduction/genetics , Sulfones/pharmacology
17.
J Biol Chem ; 290(16): 9974-85, 2015 Apr 17.
Article in English | MEDLINE | ID: mdl-25635055

ABSTRACT

YM155, which blocks the expression of survivin, a member of the inhibitor of apoptosis (IAP) family, induces cell death in a variety of cancer types, including prostate, bladder, breast, leukemia, and non-small lung cancer. However, the mechanism underlying gastric cancer susceptibility and resistance to YM155 is yet to be specified. Here, we demonstrate that cIAP1 stability dictates resistance to YM155 in human gastric cancer cells. Treatment of human gastric cancer cells with YM155 differentially induced cell death dependent on the stability of cIAP1 as well as survivin. Transfection with cIAP1 expression plasmids decreased cell sensitivity to YM155, whereas knockdown of endogenous cIAP1 using RNA interference enhanced sensitivity to YM155. In addition, double knockdown of survivin and cIAP1 significantly induced cell death in the YM155-resistant cell line, MKN45. We also showed that YM155 induced autoubiquitination and proteasome-dependent degradation of cIAP1. Surprisingly, survivin affected the stability of cIAP1 through binding, contributing to cell sensitivity to YM155. Thus, our findings reveal that YM155 sensitizes human gastric cancer cells to apoptotic cell death by degrading cIAP1, and furthermore, cIAP1 in gastric cancer cells may act as a PD marker for YM155 treatment.


Subject(s)
Antineoplastic Agents/pharmacology , Gastric Mucosa/drug effects , Gene Expression Regulation, Neoplastic , Imidazoles/pharmacology , Inhibitor of Apoptosis Proteins/genetics , Naphthoquinones/pharmacology , Cell Death/drug effects , Cell Line, Tumor , Drug Resistance, Neoplasm/genetics , Gastric Mucosa/metabolism , Gastric Mucosa/pathology , Humans , Inhibitor of Apoptosis Proteins/antagonists & inhibitors , Inhibitor of Apoptosis Proteins/metabolism , Protein Binding , Protein Stability , Proteolysis , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Signal Transduction , Survivin , Ubiquitination
18.
Cell Immunol ; 292(1-2): 78-84, 2014.
Article in English | MEDLINE | ID: mdl-25460082

ABSTRACT

Erythroid differentiation regulator 1 (ERDR1), which is a stress-related survival factor, exhibits anti-cancer effects against melanoma. However, the function of ERDR1 on immune cells has not been examined. We investigated whether ERDR1 regulates the cytotoxic ability of human natural killer (NK) cells, which are known as innate effector lymphocytes. In this study, treatment with recombinant ERDR1 resulted in enhanced NK cell cytotoxicity through the secretion of lytic granules. Furthermore, actin modulation was involved in the ERDR1-enhanced NK cell cytotoxicity. ERDR1 stimulated actin accumulation at the immunological synapse, which was induced by the activation of Vav-1 in NK cells. These findings suggest new insight into the function of ERDR1 function in the human immune system.


Subject(s)
Actins/immunology , Cytotoxicity, Immunologic , Killer Cells, Natural/immunology , Membrane Proteins/immunology , Tumor Suppressor Proteins/immunology , Actins/metabolism , Cells, Cultured , Humans
19.
Biotechnol Appl Biochem ; 60(3): 356-64, 2013.
Article in English | MEDLINE | ID: mdl-23782216

ABSTRACT

Adipose stem cells (ASCs) are pluripotent cells that can generate pure fat tissue for regeneration. Differentiated adipose cells have been generated by a common inducer cocktail composed of dexamethasone, insulin, and isobutylmethylxanthine (DIM). The major drawbacks of adipose cells are their tendency to float on the culture media and their cost. To overcome some of these disadvantages, a new inducer cocktail that includes insulin, dehydroepiandrosterone, and histamine (DH IH) was tested. As a result, lipid accumulation was elevated more than twofold with DH IH than with DIM. Cell adhesion and viability, which are important factors for stable differentiation, were increased with DH IH and were proven through measurement of mRNA expression levels of adhesion marker genes, N-cadherin and vascular cell adhesion molecule, as well as through an alamar blue assay. The expression of adipogenesis-related genes, adiponectin, and glucose transporter type 4 lasted for a long time. To improve the efficiency of grafting, cell adhesion and neovascularization need to be increased. Neovascularization was observed around the transplanted adipose cells, which showed a higher number of vessel formation in DH IH than in DIM. The above results suggest that DH IH can produce pure differentiated adipose cells effectively and enhance their adhesion onto the target location when these differentiated adipose cells were applied as a clinical resource.


Subject(s)
Adipocytes/physiology , Adipogenesis/physiology , Adipose Tissue/transplantation , Cell Differentiation/physiology , Dehydroepiandrosterone/metabolism , Histamine/metabolism , Insulin/metabolism , 3T3-L1 Cells , Adipocytes/metabolism , Adipocytes/transplantation , Adipose Tissue/metabolism , Adipose Tissue/physiology , Animals , Cell Adhesion/physiology , Cell Culture Techniques , Cell Line , Cell Survival/physiology , Mice , Neovascularization, Physiologic/physiology , Regeneration/physiology , Stem Cells/metabolism , Stem Cells/physiology , Tissue Transplantation/methods
20.
Immunobiology ; 216(10): 1155-62, 2011 Oct.
Article in English | MEDLINE | ID: mdl-21742406

ABSTRACT

Thymosin beta 4 (Tß4) is the major G-actin sequestering molecule and is abundant in lymphoid tissues. However, it is not clear what regulates Tß4 expression and what its function is on natural killer (NK) cells. We investigated whether interleukin-18 (IL-18) has a role in Tß4 expression and if enhanced Tß4 influences IL-18-mediated interferon-gamma (IFN-γ) secretion. In this study, recombinant human IL-18 (rhIL-18) enhanced the endogenous level of Tß4 through p38MAPK and JNK signaling pathway in the human NK cell line, NK-92MI. Overexpression of endogeneous Tß4 stimulated IFN-γ expression and secretion. Additionally, pretreatment with an inhibitor for Tß4 decreased IL-18-enhanced IFN-γ secretion, and transfection with Tß4-specific short hairpin RNA resulted in reduction of IFN-γ production in primary NK cells as well as in the human NK cell line. Taken together, these data indicated that Tß4 is regulated by IL-18 and is involved in IL-18-enhanced IFN-γ secretion in NK cells.


Subject(s)
Interferon-gamma/metabolism , Interleukin-18/metabolism , Killer Cells, Natural/immunology , Killer Cells, Natural/metabolism , Thymosin/metabolism , Gene Expression/immunology , Gene Expression Regulation/drug effects , Humans , Interleukin-18/pharmacology , Killer Cells, Natural/drug effects , Thymosin/genetics
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