ABSTRACT
Hyperglycemic milieu in diabetes mellitus stimulates macrophages for exaggerated pro-inflammatory cytokine response, particularly IL-1ß, IL-6, and TNF-α. Although hyperglycemia causes macrophages to produce pro-inflammatory cytokines, AGEs (advanced glycation end products) active inflammation, produced as a result of chronic hyperglycemia, inducers cause polarization of macrophages into pro-inflammatory M1 phenotype. AGEs in diabetes accelerate atherosclerotic plaque initiation and progression via promoting macrophages polarization towards pro-inflammatory state. Gliclazide (Glz) is a well known antidiabetic drug with excellent safety profile. Its repurposing in the management of diabetes-associated late complications has tremendous merit. The present study demonstrated that Glz retards diabetic atherosclerotic progression, and cytokines storm in a concentration dependent manner over a concentration range of 1-100 µM than those of AGEs (200 µg/ml)-treated cells through a mechanism that alters macrophage M1 polarization state. Glz exerted these beneficial effects, independent of its antidiabetic effect. Glz pretreatment significantly (P < 0.05) inhibited the AGEs-induced pro-inflammatory mediators (NOâ¢, reactive oxygen species, i-NOS), and production of pro-inflammatory cytokines, including IL-1ß, IL-6, and TNF-α. It also significantly (P < 0.05) promoted the production of anti-inflammatory cytokines (IL-10 and TGF-ß) in RAW 264.7 mouse macrophages. Glz pretreatment also effectively abated the AGEs-induced RAGE (~2-fold decrease), and CD86 surface marker expressions (P < 0.001 at 100 µM) on macrophages by inhibiting the NF-kß activation in a concentration dependent manner (1-100 µM) (P < 0.001). In conclusion, our data demonstrates that Glz alleviates the diabetic atherosclerosis progression by ameliorating the AGEs-mediated M1 pro-inflammatory phenotype via blocking AGE-RAGE/TLR4-reactive oxygen species -activated NF-kß nexus in macrophages.
Subject(s)
Atherosclerosis/drug therapy , Diabetes Complications/drug therapy , Gliclazide/pharmacology , Glycation End Products, Advanced/antagonists & inhibitors , Receptor for Advanced Glycation End Products/antagonists & inhibitors , Animals , Antigens, Surface/drug effects , Atherosclerosis/etiology , Cell Survival/drug effects , Cytokines/metabolism , Diabetes Mellitus/metabolism , Macrophage Activation/drug effects , Macrophages/drug effects , Mice , NF-kappa B p50 Subunit/antagonists & inhibitors , NF-kappa B p50 Subunit/metabolism , Nitric Oxide Synthase Type II/drug effects , Nitric Oxide Synthase Type II/metabolism , RAW 264.7 Cells , Reactive Oxygen Species/antagonists & inhibitors , Signal Transduction/drug effects , Toll-Like Receptor 4/antagonists & inhibitorsABSTRACT
Development of chemoresistance in breast cancer patients greatly increases mortality. Thus, understanding mechanisms underlying breast cancer resistance to chemotherapy is of paramount importance to overcome this clinical challenge. Although activated Notch receptors have been associated with chemoresistance in cancer, the specific Notch ligands and their molecular mechanisms leading to chemoresistance in breast cancer remain elusive. Using conditional knockout and reporter mouse models, we demonstrate that tumor cells expressing the Notch ligand Dll1 is important for tumor growth and metastasis and bear similarities to tumor-initiating cancer cells (TICs) in breast cancer. RNA-seq and ATAC-seq using reporter models and patient data demonstrated that NF-κB activation is downstream of Dll1 and is associated with a chemoresistant phenotype. Finally, pharmacological blocking of Dll1 or NF-κB pathway completely sensitizes Dll1+ tumors to chemotherapy, highlighting therapeutic avenues for chemotherapy resistant breast cancer patients in the near future.
Subject(s)
Antineoplastic Combined Chemotherapy Protocols/pharmacology , Breast Neoplasms/drug therapy , Calcium-Binding Proteins/metabolism , Drug Resistance, Neoplasm/genetics , Membrane Proteins/metabolism , NF-kappa B p50 Subunit/metabolism , Animals , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Benzamides/pharmacology , Benzamides/therapeutic use , Breast/pathology , Breast Neoplasms/genetics , Breast Neoplasms/pathology , Calcium-Binding Proteins/antagonists & inhibitors , Calcium-Binding Proteins/genetics , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Proliferation/genetics , Cell Survival/drug effects , Cell Survival/genetics , Datasets as Topic , Disease Models, Animal , Doxorubicin/pharmacology , Doxorubicin/therapeutic use , Drug Resistance, Neoplasm/drug effects , Female , Gene Expression Regulation, Neoplastic , Humans , Mice , Mice, Knockout , NF-kappa B p50 Subunit/antagonists & inhibitors , Neoplastic Stem Cells/drug effects , Neoplastic Stem Cells/metabolism , RNA-Seq , Receptors, Notch/metabolism , Signal Transduction/drug effects , Signal Transduction/geneticsABSTRACT
Malignant gliomas are a heterogeneous group of brain tumors with a poor prognosis, which is largely due to its aggressive invasiveness and angiogenesis. In recent years, it has been found that multiple long noncoding RNAs (lncRNAs) participate in a wide range of biological functions including angiogenesis through the regulation of gene expression in cancers. In this study, we investigate and report the novel role of lncRNA SLC26A4-AS1 in gliomas, with a novel mechanism involving transcription factors NFKB1 and NPTX1. We determined that SLC26A4-AS1 was downregulated in human glioma tissues and cells. Furthermore, overexpression of SLC26A4-AS1 or NPTX1 restrained the aggressiveness of glioma cells and their pro-angiogenic ability. SLC26A4-AS1 was also found to upregulate NPTX1 by recruiting NFKB1 into the NPTX1 promoter. Moreover, silencing of either NPTX1 or NFKB1 restored the aggressive and pro-angiogenic properties of glioma cells in the presence of SLC26A4-AS1. Taken together, we demonstrate that SLC26A4-AS1 promotes NPTX1 transcriptional activity by recruiting NFKB1 and thus exerting antiangiogenic effects on glioma cells. This study provides an experimental basis for the intervention of SLC26A4-AS1 in the treatment of gliomas.
Subject(s)
Brain Neoplasms/genetics , C-Reactive Protein/genetics , Glioblastoma/genetics , NF-kappa B p50 Subunit/genetics , Neovascularization, Pathologic/genetics , Nerve Tissue Proteins/genetics , RNA, Long Noncoding/genetics , Sulfate Transporters/genetics , Animals , Brain Neoplasms/blood supply , Brain Neoplasms/metabolism , Brain Neoplasms/pathology , C-Reactive Protein/antagonists & inhibitors , C-Reactive Protein/metabolism , Case-Control Studies , Cell Line, Tumor , Cell Movement , Cell Proliferation , Gene Expression Regulation, Neoplastic , Glioblastoma/blood supply , Glioblastoma/metabolism , Glioblastoma/pathology , Humans , Mice , Mice, Inbred BALB C , Mice, Nude , NF-kappa B p50 Subunit/antagonists & inhibitors , NF-kappa B p50 Subunit/metabolism , Neoplasm Grading , Neovascularization, Pathologic/metabolism , Neovascularization, Pathologic/pathology , Nerve Tissue Proteins/antagonists & inhibitors , Nerve Tissue Proteins/metabolism , Neuroglia/metabolism , Neuroglia/pathology , Promoter Regions, Genetic , RNA, Long Noncoding/agonists , RNA, Long Noncoding/metabolism , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Signal Transduction , Sulfate Transporters/metabolism , Tumor Burden , Xenograft Model Antitumor AssaysABSTRACT
This study was designed to investigate the effect of naringin in oxygen-glucose deprivation/reoxygenation (OGD/R) model and its mechanism. The target gene of naringin and the enriched pathways of the gene were searched and identified using bioinformatics analysis. Then OGD/R model was built using PC12 cells, after which the cells were treated with different concentrations of naringin. Subsequently, cell proliferation and apoptosis were evaluated by cell counting kit-8 (CCK-8) and flow cytometry assays, respectively. Meanwhile, the expression of NFKB1 in PC12 cells underwent OGD/R-induced injury was detected by qRT-PCR, while apoptosis-related and pathway-related proteins were checked by Western blot. DCF-DA kit was utilized to measure the level of ROS. Our results revealed that NFKB1, which was upregulated in MACO rats and OGD/R-treated PC12 cells, was a target gene of naringin. Naringin could alleviate OGD/R-induced injury via promoting the proliferation, and repressing the apoptosis of PC12 cells through regulating the expression of NFKB1 and apoptosis-associated proteins and ROS level. Besides, the depletion of NFKB1 was positive to cell proliferation but negative to cell apoptosis. Moreover, the depletion of NFKB1 enhanced the influences of naringin on cell proliferation and apoptosis as well as the expression of apoptosis-related proteins and ROS level. Western blotting indicated that both naringin treatment and depletion of NFKB1 could increase the expression of HIF-1α, p-AKT, and p-mTOR compared with OGD/R group. What's more, treatment by naringin and si-NFKB1 together could significantly increase these effects. Nevertheless, the expression of AKT and mTOR among each group was almost not changed. In conclusion, naringin could prevent the OGD/R-induced injury in PC12 cells in vitro by targeting NFKB1 and regulating HIF-1α/AKT/mTOR-signaling pathway, which might provide novel ideas for the therapy of cerebral ischemia-reperfusion (I/R) injury.
Subject(s)
Flavanones/pharmacology , Hypoxia-Inducible Factor 1, alpha Subunit/physiology , NF-kappa B p50 Subunit/antagonists & inhibitors , Proto-Oncogene Proteins c-akt/physiology , Reperfusion Injury/prevention & control , TOR Serine-Threonine Kinases/physiology , Animals , Apoptosis/drug effects , Cell Division/drug effects , Flavanones/therapeutic use , Gene Expression Regulation/drug effects , Gene Ontology , Glucose/pharmacology , Infarction, Middle Cerebral Artery/drug therapy , Infarction, Middle Cerebral Artery/genetics , NF-kappa B p50 Subunit/genetics , NF-kappa B p50 Subunit/physiology , Oxygen/pharmacology , PC12 Cells , Phytotherapy , Proto-Oncogene Proteins c-bcl-2/biosynthesis , Proto-Oncogene Proteins c-bcl-2/genetics , RNA Interference , RNA, Small Interfering/genetics , RNA, Small Interfering/pharmacology , Rats , Reactive Oxygen Species/metabolism , Signal Transduction/physiology , bcl-2-Associated X Protein/biosynthesis , bcl-2-Associated X Protein/geneticsABSTRACT
Currently, combination therapy is considered the most effective solution for a selective chemotherapeutic effect in the treatment of colon cancer. This study investigated the death of both colon cancer HT29 cells and healthy vascular smooth muscle TG-Ha-VSMC cells (VSMCs) induced by naringin combined with endoplasmic reticulum (ER) stress and NF-κB inhibition. Naringin combined with tunicamycin and BAY 11-7082 suppressed the proliferation of HT29 cells in a dose-dependent manner and induced particularly apoptotic death without significantly affecting healthy VSMCs according to Annexin V/PI staining and AO/EB staining analyses. Insufficient antioxidant defense and heat shock response as well as excessive ROS generation were observed in HT29 cells following combination therapy. Quantitative real-time PCR and western blot analysis demonstrated that drug combination-induced mitochondrial apoptosis was activated through the ROS-mediated PERK/eIF2α/ATF4/CHOP pathway. Additionally, naringin combination significantly reduced the sXBP expression induced by tunicamycin+BAY 11-7082 in a dose-dependent manner. In conclusion, this study found that naringin combined with tunicamycin+BAY 11-7082 efficiently induced apoptotic cell death in HT29 colon cancer cells via oxidative stress and the PERK/eIF2α/ATF4/CHOP pathway, suggesting that naringin combined with tunicamycin plus BAY 11-7082 could be a new combination therapy strategy for effective colon cancer treatment with minimal side effects on healthy cells.
Subject(s)
Apoptosis , Endoplasmic Reticulum Stress , Flavanones/pharmacology , NF-kappa B p50 Subunit/antagonists & inhibitors , Oxidative Stress , Signal Transduction , Activating Transcription Factor 4/metabolism , Antioxidants/pharmacology , Caspases/metabolism , Cell Line, Tumor , Cell Survival , Colonic Neoplasms/drug therapy , Endoplasmic Reticulum/metabolism , Endoplasmic Reticulum Stress/drug effects , Eukaryotic Initiation Factor-2/metabolism , Flavanones/administration & dosage , HT29 Cells , Humans , Mitochondria/metabolism , Muscle, Smooth, Vascular/cytology , NF-kappa B p50 Subunit/metabolism , Nitriles/pharmacology , Reactive Oxygen Species , Sulfones/pharmacology , Transcription Factor CHOP/metabolism , Tunicamycin/administration & dosage , eIF-2 Kinase/metabolismABSTRACT
The NF-κB transcription factor is involved in inflammation and cell proliferation, survival, and transformation. It is a heterodimer made of p50 or p52 and a member of the Rel family of proteins. p50 and p52 are derived from limited ubiquitin- and proteasome-mediated proteolytic processing of the larger precursors p105 and p100, respectively. Both precursors can be either processed or completely degraded by the ubiquitin-proteasome system. Previous work in our laboratory identified KPC1 as a ubiquitin ligase that mediates processing of p105 to the p50 subunit. Overexpression of the ligase leads to increased level of p50 with a resultant marked tumor-suppressive effect. In the present study, we identify FBXO7, a known ubiquitin ligase that binds to p105 and ubiquitinates it, but surprisingly, leads to its accumulation and to that of p65 - the Rel partner of p50 - and to increased cell proliferation. Importantly, a ΔF-Box mutant of FBXO7 which is inactive has similar effects on accumulation of p105 and cell proliferation, strongly suggesting that p105 is a pseudo substrate of FBXO7.
Subject(s)
F-Box Proteins/metabolism , NF-kappa B p50 Subunit/metabolism , Amino Acid Sequence , Cell Line , Cell Proliferation/physiology , Enzyme Stability , F-Box Proteins/antagonists & inhibitors , F-Box Proteins/genetics , HEK293 Cells , HeLa Cells , Humans , K562 Cells , Mutant Proteins/genetics , Mutant Proteins/metabolism , NF-kappa B p50 Subunit/antagonists & inhibitors , NF-kappa B p50 Subunit/genetics , Proteasome Endopeptidase Complex/metabolism , Protein Binding , Protein Precursors/metabolism , Protein Processing, Post-Translational , Proteolysis , RNA, Small Interfering/genetics , Substrate Specificity , Transcription Factor RelA/metabolism , Tumor Suppressor Proteins/genetics , Tumor Suppressor Proteins/metabolism , UbiquitinationABSTRACT
ETHNOPHARMACOLOGICAL RELEVANCE: Ferula sinkiangensis K. M. Shen is a traditional Chinese medicine that has a variety of pharmacological properties relevant to neurological disorders and inflammations. Kellerin, a novel compound extracted from Ferula sinkiangensis, exerts a strong anti-neuroinflammatory effect by inhibiting microglial activation. Microglial activation plays a vital role in ischemia-induced brain injury. However, the potential therapeutic effect of kellerin on focal cerebral ischemia is still unknown. AIM OF THE STUDY: To explore the effect of kellerin on cerebral ischemia and clarify its possible mechanisms, we applied the middle cerebral artery occlusion (MCAO) model and the LPS-activated microglia model in our study. MATERIALS AND METHODS: Neurological outcome was examined according to a 4-tiered grading system. Brain infarct size was measured using TTC staining. Brain edema was calculated using the wet weight minus dry weight method. Neuron damage and microglial activation were observed by immunofluorescence in MCAO model in rats. In in vitro studies, microglial activation was examined by flow cytometry and the viability of neuronal cells cultured in microglia-conditioned medium was measured using MTT assay. The levels of pro-inflammatory cytokines were measured by qRT-PCR and ELISA. The proteins involved in NF-κB signaling pathway were determined by western blot. Intracellular ROS was examined using DCFH-DA method and NADPH oxidase activity was measured using the NBT assay. RESULTS: We found that kellerin improved neurological outcome, reduced brain infarct size and decreased brain edema in MCAO model in rats. Under the pathologic conditions of focal cerebral ischemia, kellerin alleviated neuron damage and inhibited microglial activation. Moreover, in in vitro studies of LPS-stimulated BV2 cells kellerin protected neuronal cells from being damaged by inhibiting microglial activation. Kellerin also reduced the levels of pro-inflammatory cytokines, suppressed the NF-κB signaling pathway, and decreased ROS generation and NADPH oxidase activity. CONCLUSIONS: Our discoveries reveal that the neuroprotective effects of kellerin may largely depend on its inhibitory effect on microglial activation. This suggests that kellerin could serve as a novel anti-inflammatory agent which may have therapeutic effects in ischemic stroke.
Subject(s)
Anti-Inflammatory Agents/pharmacology , Brain Ischemia/drug therapy , Ferula/chemistry , Infarction, Middle Cerebral Artery/drug therapy , Neuroprotective Agents/pharmacology , Plant Extracts/pharmacology , Animals , Anti-Inflammatory Agents/therapeutic use , Brain Edema/drug therapy , Brain Ischemia/etiology , Brain Ischemia/pathology , Cell Line, Transformed , Cell Line, Tumor , Cytokines/metabolism , Disease Models, Animal , Humans , Infarction, Middle Cerebral Artery/etiology , Infarction, Middle Cerebral Artery/pathology , Inflammation/drug therapy , Lipopolysaccharides/toxicity , Mice , Microglia/drug effects , Microglia/pathology , NADPH Oxidases/antagonists & inhibitors , NF-kappa B p50 Subunit/antagonists & inhibitors , Neurons/drug effects , Neuroprotective Agents/therapeutic use , Plant Extracts/therapeutic use , Rats, Sprague-Dawley , Reactive Oxygen Species/metabolism , Signal Transduction/drug effectsABSTRACT
ETHNOPHARMACOLOGICAL RELEVANCE: Hard antler extract (HAE) is a traditional Chinese medicine and has potent antitumor, antioxidative, anti-inflammatory, and immunomodulatory activities. Previous studies have demonstrated that HAE can inhibit human prostate cancer metastasis and murine breast cancer proliferation. However, the effect of HAE on human breast cancer cells has not been clarified. AIM OF THE STUDY: To investigate the effects and underlying mechanism of HAE on self-renewal of stem-like cells and spontaneous and transforming growth factor (TGF)-ß1-enhanced wound healing, invasion and epithelial-mesenchymal transition (EMT) in breast cancer cells. METHODS: HAE was prepared from sika deer by sequential enzymatic digestions and the active compounds were determined by HPLC. The effects of HAE on the viability, mammosphere formation, wound healing and invasion of MDA-MB-231 and SK-BR3 cells were determined. The impact of HAE treatment on spontaneous and TGF-ß1-promoted EMT and the nuclear factor (NF)-κB signaling in breast cancer cells was examined by quantitative RT-PCR and western blotting. RESULTS: Treatment with HAE at varying concentrations did not change the viability of breast cancer cells. However, HAE at 0.25 or 0.5 mg/mL significantly reduced the number and size of formed mammospheres, and inhibited spontaneous and TGF-ß1-enhanced wound healing, invasion and EMT in MDA-MB-231 and SK-BR3 cells in a dose-dependent manner. TGF-ß1 treatment significantly decreased IκBα expression and increased NF-kBp65 phosphorylation in breast cancer cells, indicating that TGF-ß1 enhanced NF-κB signaling. In contrast, HAE treatment attenuated the spontaneous and TGF-ß1-enhanced NF-κB signaling in breast cancer cells. CONCLUSION: Our data indicated that HAE inhibited the self-renewal of stem-like cells and spontaneous and TGF-ß1-enhanced wound healing, invasion and EMT in breast cancer cells by attenuating the NF-κB signaling in vitro.
Subject(s)
Antlers/chemistry , Breast Neoplasms/metabolism , Epithelial-Mesenchymal Transition/drug effects , NF-kappa B p50 Subunit/antagonists & inhibitors , Tissue Extracts/chemistry , Tissue Extracts/pharmacology , Triple Negative Breast Neoplasms/metabolism , Animals , Antlers/metabolism , Breast Neoplasms/drug therapy , Breast Neoplasms/pathology , Cell Line, Tumor , Cell Movement/drug effects , Cell Survival/drug effects , Deer , Ethnopsychology , Female , Humans , Neoplastic Stem Cells/drug effects , Receptor, ErbB-2/metabolism , Signal Transduction/drug effects , Spheroids, Cellular/drug effects , Spheroids, Cellular/pathology , Tissue Extracts/isolation & purification , Transforming Growth Factor beta1/toxicity , Triple Negative Breast Neoplasms/drug therapy , Triple Negative Breast Neoplasms/pathology , Wound Healing/drug effectsABSTRACT
Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease characterized by synovial inflammation and progressive joint destruction. Chebulanin is a natural polyphenol acid isolated from the traditional Tibetan medicine Terminalia chebula Retz that has previously been reported to possess anti-inflammatory properties. The present study aimed to investigate the anti-inflammatory and anti-arthritic effects of chebulanin and explore its underlying mechanisms in vivo and in vitro using a collagen-induced arthritis (CIA) mouse model and lipopolysaccharide (LPS) stimulated RAW264.7 cell inflammation model. Arthritis severity scores were assessed twice weekly; the levels of cytokines interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in serum were detected using enzyme-linked immunosorbent assay kits; histopathological assessment was performed using micro computed tomography and hematoxylin and eosin staining. Activation of nuclear factor kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways were assessed using western blotting. The inhibition of translocation of cytosolic p38 and p65 into the nucleus was observed using immunofluorescence staining and western blotting in vitro. Chebulanin significantly suppressed the progression and development of RA in CIA mice by decreasing the arthritis severity scores, attenuating paw swelling and joint destruction, and reducing the levels of IL-6 and TNF-α significantly (p < 0.05). Furthermore, chebulanin reduced the levels of excised phosphorylated (p)-p38, phosphorylated-c-JUN N-terminal kinase (p-JNK), p-p65 and phosphorylated NF-κB inhibitor alpha (p-IκBα) in CIA mice, but did not affect the level of phosphorylated extracellular-signal-regulated kinase (ERK). In addition, chebulanin could inhibit the nuclear translocation of p38 and p65 in LPS-stimulated macrophages in dose-dependent manner. In conclusion, this study demonstrated that chebulanin exerts anti-inflammatory and anti-arthritic effects by inhibiting the activation of NF-κB and MAPK signaling pathways.
Subject(s)
Anti-Inflammatory Agents/pharmacology , Arthritis, Experimental/drug therapy , Hydrolyzable Tannins/pharmacology , MAP Kinase Signaling System/drug effects , NF-kappa B p50 Subunit/antagonists & inhibitors , Animals , Anti-Inflammatory Agents/therapeutic use , Arthritis, Experimental/metabolism , Arthritis, Experimental/pathology , Cartilage Diseases/drug therapy , Cartilage Diseases/pathology , Collagen/toxicity , Enzyme Activation , Hydrolyzable Tannins/therapeutic use , I-kappa B Proteins/pharmacokinetics , Inflammation/drug therapy , Inflammation/pathology , Interleukin-6/blood , JNK Mitogen-Activated Protein Kinases/metabolism , Joints/drug effects , Joints/pathology , Lipopolysaccharides/toxicity , Male , Mice , Mice, Inbred DBA , RAW 264.7 Cells , Synovitis/drug therapy , Transcription Factor RelA/metabolism , Tumor Necrosis Factor-alpha/blood , p38 Mitogen-Activated Protein Kinases/metabolismABSTRACT
We investigate the effects of Clostridium butyricum(CB) on gut microbiota and colitis associated colon cancer(CAC) in mice.6-8 weeks old C57BL/6 mice were randomly divided into control, azoxymethane (AOM) + dextran sodium sulphate (DSS) and AOM + DSS + CB groups. Mice in the latter two groups received an intraperitoneal injection of AOM (12.5 mg/kg), followed by three cycles of DSS diluted in water (2.5% w/v). Mice in treatment group received CB (2 × 108 CFU in 200 ul normal saline) by gavage administration three times one week. Microbiota composition was assessed by 16S rRNA high-throughput sequencing. Colon samples were collected to examine severity of colitis and tumorigenesis. Cytokines including TNF-a, IL-6 and Cyclo-oxygenase-2 (COX-2) were detected by RT-qPCR. Expression of Bcl-2, Bax and the state of components of NF-κB signaling pathway were detected by western blot. The results revealed that CB regulated structure of intestinal flora and changed the microbial composition; decreased Firmicutes/ Bacteroidetes ratio in phylum level and increased the relative abundance of probiotics; decreased colitis, decreased incidence and size of colorectal cancer(CRC) and increased apoptosis of tumor cells; decreased cytokines including TNF-a and IL-6; decreased level of COX-2; decreased phosphorylation of NF-κB; decreased level of Bcl-2 and increased expression of Bax. In conclusion, CB could regulate structure and composition of gut microbiota and reduces colitis associated colon cancer in mice, the mechanism may be inhibiting NF-κB pathway and promoting apoptosis.
Subject(s)
Clostridium butyricum/physiology , Colitis-Associated Neoplasms/microbiology , Colitis-Associated Neoplasms/therapy , Colitis/microbiology , Colitis/therapy , Gastrointestinal Microbiome , Animals , Apoptosis , Azoxymethane/toxicity , Bacteroidetes/metabolism , Body Weight , Cell Proliferation , Colitis/chemically induced , Colitis/pathology , Colitis-Associated Neoplasms/chemically induced , Colitis-Associated Neoplasms/pathology , Cytokines/metabolism , Dextran Sulfate/toxicity , Disease Models, Animal , Feces/microbiology , Firmicutes/metabolism , Inflammation/microbiology , Male , Mice, Inbred C57BL , NF-kappa B p50 Subunit/antagonists & inhibitors , RNA, Ribosomal, 16SABSTRACT
Allergic rhinitis (AR) is a common chronic condition characterized by inflammation of the nasal mucosa. The correlation of microRNAs (miRNAs) in AR has been highlighted particularly due to their roles in regulating inflammatory responses. The aim of this study was to explore the anti-inflammatory mechanism by which miR-345-5p regulates the toll-like receptor 4/nuclear factor-κB (TLR4/NF-κB) pathway in mice with AR. Initially, the putative miR-345-5p binding sites on the 3'untranslated region of TLR4 was predicted and verified. AR models were established using ovalbumin, after which the functional role of miR-345-5p in AR was determined using gain- and loss-of-function approaches. We found that miR-345-5p was poorly expressed in nasal mucosal tissues of mice with AR. Meanwhile, TLR4 expression and the TLR4/NF-κB pathway were identified to be promoted, which were then suppressed in the presence of overexpressed miR-345-5p. In addition, nasal epithelial cell apoptosis and fibrosis were inhibited in response to miR-345-5p overexpression and TLR4 silencing. Furthermore, miR-345-5p overexpression and TLR4 silencing were observed to decrease Th2 cells, expression of pro-inflammatory factors, but to increase Th1 cells and expression of anti-inflammatory factors. This study demonstrates an important role of miR-345-5p in alleviating the inflammatory response in mice with AR by inhibiting the TLR4/NF-κB pathway. Therefore, a better understanding of this process may aid in the development of novel therapeutic agents of AR.
Subject(s)
Anti-Inflammatory Agents/metabolism , MicroRNAs/metabolism , NF-kappa B p50 Subunit/metabolism , Rhinitis, Allergic/metabolism , Toll-Like Receptor 4/metabolism , 3' Untranslated Regions , Animals , Apoptosis/genetics , Disease Models, Animal , Epithelial Cells/metabolism , Epithelial Cells/pathology , Female , Fibrosis/genetics , Inflammation/genetics , Mice, Inbred BALB C , Myeloid Differentiation Factor 88/metabolism , NF-kappa B p50 Subunit/antagonists & inhibitors , Nasal Mucosa/metabolism , Nasal Mucosa/pathology , Ovalbumin , Receptors, Interleukin/metabolism , Rhinitis, Allergic/chemically induced , Signal Transduction , Toll-Like Receptor 4/antagonists & inhibitorsABSTRACT
Polysaccharide is the main active component of okra (Abelmoschus esculentus L.) and it can effectively stimulate the activation of macrophages. However, the immune regulatory mechanism is still not clear. Therefore, the present study aimed to reveal the possible mechanism by investigating the effect of okra polysaccharide-2 (RPS-2) on Toll-like receptor (TLR) 2/4-mediated signal transduction pathways in RAW264.7 murine macrophage cells. In order to confirm whether RPS-2 stimulated macrophages activation via TLR2 or TLR4, RAW264.7 murine macrophage cells were pretreated with TLR2/4 inhibitors for 1 h before RPS-2 treatment, and then the NO, IL-10, TNF-α levels were tested. The results indicated that both TLR2 and TLR4 were the keys of immune regulatory effect of RPS-2. Afterwards, the effect of RPS-2 on NF-κB and MAPKs signaling pathways were studied by western blot analysis. It showed RPS-2 induced the phosphorylation of p65, IκBα, p38, ERK1/2 and JNK. At the same time, the specific inhibitors reduced these phosphorylation levels as well as NO, IL-10 and TNF-α amounts. In a word, RPS-2 activated macrophages by NF-κB and MAPKs signal transduction pathways.
Subject(s)
Abelmoschus/chemistry , Polysaccharides/immunology , Polysaccharides/pharmacology , Signal Transduction/drug effects , Toll-Like Receptor 2/metabolism , Toll-Like Receptor 4/metabolism , Animals , I-kappa B Proteins/metabolism , Interleukin-10/metabolism , Janus Kinases/metabolism , Macrophage Activation/drug effects , Macrophages/drug effects , Mice , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/metabolism , NF-kappa B p50 Subunit/antagonists & inhibitors , NF-kappa B p50 Subunit/drug effects , Nitric Oxide/metabolism , Phagocytosis/drug effects , Polysaccharides/isolation & purification , Polysaccharides/metabolism , RAW 264.7 Cells , Toll-Like Receptor 2/antagonists & inhibitors , Toll-Like Receptor 4/antagonists & inhibitors , Transcription Factor RelA/metabolism , Tumor Necrosis Factor-alpha/metabolismABSTRACT
Angelica sinensis (AS; Dang Gui), a traditional Chinese herb, has for centuries been used for the treatment of bone diseases, including osteoporosis and osteonecrosis. However, the effective ingredient and underlying mechanisms remain elusive. Here, we identified guaiacol as the active component of AS by two-dimensional cell membrane chromatography/C18 column/time-of-flight mass spectrometry (2D CMC/C18 column/TOFMS). Guaiacol suppressed osteoclastogenesis and osteoclast function in bone marrow monocytes (BMMCs) and RAW264.7 cells in vitro in a dose-dependent manner. Co-immunoprecipitation indicated that guaiacol blocked RANK-TRAF6 association and RANK-C-Src association. Moreover, guaiacol prevented phosphorylation of p65, p50, IκB (NF-κB pathway), ERK, JNK, c-fos, p38 (MAPK pathway) and Akt (AKT pathway), and reduced the expression levels of Cathepsin K, CTR, MMP-9 and TRAP. Guaiacol also suppressed the expression of nuclear factor of activated T-cells cytoplasmic 1(NFATc1) and the RANKL-induced Ca2+ oscillation. In vivo, it ameliorated ovariectomy-induced bone loss by suppressing excessive osteoclastogenesis. Taken together, our findings suggest that guaiacol inhibits RANKL-induced osteoclastogenesis by blocking the interactions of RANK with TRAF6 and C-Src, and by suppressing the NF-κB, MAPK and AKT signalling pathways. Therefore, this compound shows therapeutic potential for osteoclastogenesis-related bone diseases, including postmenopausal osteoporosis.
Subject(s)
CSK Tyrosine-Protein Kinase/metabolism , Guaiacol/pharmacology , Osteoclasts/drug effects , Osteogenesis/drug effects , Receptor Activator of Nuclear Factor-kappa B/metabolism , TNF Receptor-Associated Factor 6/metabolism , Adipogenesis , Animals , Bone Marrow Cells/cytology , Bone Resorption , Cell Proliferation , Female , MAP Kinase Signaling System , Mice , Mice, Inbred C57BL , NF-kappa B/metabolism , NF-kappa B p50 Subunit/antagonists & inhibitors , Osteoclasts/metabolism , Proto-Oncogene Proteins c-akt/metabolism , RAW 264.7 Cells , X-Ray MicrotomographyABSTRACT
Recently, microRNAs (miRNAs) have been demonstrated to play important roles in the cardiovascular system, including heart, blood vessels, plasma, and vascular diseases. Deep vein thrombosis (DVT) refers to the formation of blood clot in the deep veins of the human body and is a common peripheral vascular disease. Herein, we explored the mechanism of miR-9-5p in DVT through nuclear factor-κB (NF-κB). The expression of miR-9-5p in DVT rats was measured through the establishment of DVT rat models, followed by the alteration of miR-9-5p and NF-κB p50 in rats through the injection of constructed lentiviral vectors so as to explore the role of miR-9-5p and NF-κB p50 expression in rats. Next, the expression of NF-κB p50 and levels of inflammation-related factors plasminogen activator inhibitor-1 (PAI-1), interleukin-6 (IL-6), tumor necrosis factor α (TNF-α), and interleukin-8 (IL-8) were measured after the injection with lentiviral vectors, followed by the assessment of platelet aggregation and TXB2 content. MiR-9-5p was found to be downregulated in DVT rats. Through dual luciferase reporter gene assay, NF-κB p50 was verified as the target gene of miR-9-5p and miR-9-5p could negatively regulate NF-κB p50. MiR-9-5p over-expression decreased the levels of PAI-1, TNF-α, IL-6, and IL-8 and platelet aggregation as well as TXB2 content, thus inhibiting thrombosis. Meanwhile, over-expressed NF-κB p50 could reverse the anti-inflammatory or anti-thrombotic effect of miR-9-5p. In summary, miR-9-5p over-expression can suppress the NF-κB signaling pathway through p50 downregulation, thus alleviating inflammation and thrombosis in DVT rats. MiR-9-5p could serve as a potential therapeutic target for DVT.
Subject(s)
Inflammation/prevention & control , MicroRNAs/physiology , NF-kappa B p50 Subunit/antagonists & inhibitors , Venous Thrombosis/pathology , Animals , Gene Expression Regulation , Inflammation/etiology , Interleukin-6/blood , Interleukin-8/blood , MicroRNAs/genetics , MicroRNAs/metabolism , Plasminogen Activator Inhibitor 1/blood , Platelet Aggregation , Protective Agents/pharmacology , Rats , Thromboxane B2/blood , Tumor Necrosis Factor-alpha/bloodABSTRACT
Hypericum japonicum Thunb. ex Murray is traditionally used in Nepal to treat several diseases, among whom inflammation and acute pain. Although several secondary metabolites from the same Hypericum species have been already characterized and considered for their pharmacological use, an exhaustive phytochemical characterization of H. japonicum from Nepal is lacking, as well as the assessment of its potential pharmacological properties. Hence, the aims of this study were the characterization of a methanolic extract of H. japonicum (HJME) collected from the Northern region of Nepal by LC-MSn and UPLC-QTOF. The assessment of in vitro inhibition of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and activator protein 1 (AP-1) transcription factors and HJME's cytotoxic effect on human cell lines was performed to evaluate the potential use of this herb as a source of anti-inflammatory and cytotoxic lead compounds. Fifty-seven phytoconstituents were identified, being mainly flavonoids, phloroglucinols, phenolic acids and xanthones. Although compounds characteristic of H. japonicum were detected (quercetin, quercetin-7-O-α-l-rhamnoside, quercitrin and hyperoside), several others are here reported for the first time in this species. The results from bioassays indicated that HJME could significantly reduce the viability of human THP-1 cells (IC50â¯=â¯5.4⯱â¯1.1⯵g mL-1), showing the promising potential of HJME as anti-tumor agent. Furthermore, HJME significantly decreased the activation of both NF-κB and AP-1 at the concentration of 2⯵g mL-1. Overall, these data suggest that H. japonicum from Nepal could be used as a source of potential natural anti-inflammatory and anti-tumor lead compounds.
Subject(s)
Hypericum/chemistry , NF-kappa B p50 Subunit/antagonists & inhibitors , Plant Extracts/pharmacology , Transcription Factor AP-1/antagonists & inhibitors , Anti-Inflammatory Agents/pharmacology , Antineoplastic Agents/pharmacology , Biological Assay , Cell Line , Cell Survival , Chromatography, Liquid , Flavonoids/pharmacology , Humans , Hydroxybenzoates/pharmacology , Inflammation , Inhibitory Concentration 50 , Mass Spectrometry , Nepal , Phloroglucinol/pharmacology , THP-1 Cells , Xanthones/pharmacologyABSTRACT
Various mechanisms for regulated cell death include the formation of oxidative mediators such as lipid peroxides and nitric oxide (NO). In this respect, 15-lipoxygenase-1 (15-LOX-1) is a key enzyme that catalyzes the formation of lipid peroxides. The actions of these peroxides are interconnected with nuclear factor-κB signaling and NO production. Inhibition of 15-LOX-1 holds promise to interfere with regulated cell death in inflammatory conditions. In this study, a novel potent 15-LOX-1 inhibitor, 9c (i472), was developed and structure-activity relationships were explored. In vitro, this inhibitor protected cells from lipopolysaccharide-induced cell death, inhibiting NO formation and lipid peroxidation. Thus, we provide a novel 15-LOX-1 inhibitor that inhibits cellular NO production and lipid peroxidation, which set the stage for further exploration of these mechanisms.
Subject(s)
Indoles/pharmacology , Lipoxygenase Inhibitors/pharmacology , Protective Agents/pharmacology , Animals , Arachidonate 15-Lipoxygenase/chemistry , Arachidonate 15-Lipoxygenase/metabolism , Catalytic Domain , Gene Expression/drug effects , Indoles/chemical synthesis , Indoles/metabolism , Lipid Peroxidation/drug effects , Lipopolysaccharides/pharmacology , Lipoxygenase Inhibitors/chemical synthesis , Lipoxygenase Inhibitors/metabolism , Mice , Molecular Docking Simulation , Molecular Structure , NF-kappa B p50 Subunit/antagonists & inhibitors , Nitric Oxide/antagonists & inhibitors , Nitric Oxide Synthase Type II/genetics , Oxidative Stress/drug effects , Protective Agents/chemical synthesis , Protective Agents/metabolism , Protein Binding , RAW 264.7 Cells , Rabbits , Signal Transduction/drug effects , Structure-Activity RelationshipABSTRACT
NFκB is implicated in cancer and bone remodelling, and we have recently reported that the verified NFκB inhibitor Parthenolide (PTN) reduced osteolysis and skeletal tumour growth in models of metastatic breast cancer. Here, we took advantage of in vitro and ex vivo bone cell and organ cultures to study the effects of PTN on the ability of prostate cancer cells and their derived factors to regulate bone cell activity and osteolysis. PTN inhibited the in vitro growth of a panel of human, mouse and rat prostate cancer cells in a concentration-dependent manner with a varying degree of potency. In prostate cancer cell-osteoclast co-cultures, the rat Mat-Ly-Lu, but not human PC3 or mouse RM1-BT, enhanced RANKL stimulated osteoclast formation and PTN reduced these effects without affecting prostate cancer cell viability. In the absence of cancer cells, PTN reduced the support of Mat-Ly-Lu conditioned medium for the adhesion and spreading of osteoclast precursors, and survival of mature osteoclasts. Pre-exposure of osteoblasts to PTN prior to the addition of conditioned medium from Mat-Ly-Lu cells suppressed their ability to support the formation of osteoclasts by inhibition of RANKL/OPG ratio. PTN enhanced the ability of Mat-Ly-Lu derived factors to increase calvarial osteoblast differentiation and growth. Ex vivo, PTN enhanced bone volume in calvaria organ-Mat-Ly-Lu cell co-culture, without affecting Mat-Ly-Lu viability or apoptosis. Mechanistic studies in osteoclasts and osteoblasts confirmed that PTN inhibit NFκB activation related to derived factors from Mat-Ly-Lu cells. Collectively, these findings suggest that pharmacological inhibition of the skeletal NFκB signalling pathway reduces prostate cancer related osteolysis, but further studies in the therapeutic implications of NFκB inhibition in cells of the osteoblastic lineage are needed.
Subject(s)
NF-kappa B p50 Subunit/antagonists & inhibitors , Osteogenesis/drug effects , Prostatic Neoplasms/drug therapy , Animals , Apoptosis , Cell Adhesion , Cell Line, Tumor , Cell Survival , Coculture Techniques , DNA Fragmentation , Humans , Male , Mice , Mice, Inbred C57BL , Neoplasm Metastasis , Organ Culture Techniques , Osteoblasts/cytology , Osteoblasts/drug effects , Osteoclasts/cytology , Osteoclasts/drug effects , Prostatic Neoplasms/pathology , Rats , Sesquiterpenes/pharmacology , Signal Transduction , X-Ray MicrotomographySubject(s)
Dust , Glucocorticoids/pharmacology , Interleukins/genetics , Interleukins/metabolism , Nasal Mucosa/drug effects , A549 Cells , Cell Survival/drug effects , Cells, Cultured , Dust/analysis , Humans , NF-kappa B p50 Subunit/antagonists & inhibitors , NF-kappa B p50 Subunit/metabolism , Nasal Mucosa/metabolism , Phosphorylation/drug effects , Proto-Oncogene Proteins c-akt/antagonists & inhibitors , Proto-Oncogene Proteins c-akt/metabolism , Sand , Signal Transduction/drug effects , Silicon Dioxide/analysis , Silicon Dioxide/pharmacology , p38 Mitogen-Activated Protein Kinases/antagonists & inhibitors , p38 Mitogen-Activated Protein Kinases/metabolismABSTRACT
Cancer stem cells (CSCs) are rare types of cells responsible for tumor development, relapse, and metastasis. However, current research in CSC biology is largely limited by the difficulty of obtaining sufficient CSCs. Single-cell analysis techniques are promising tools for CSC-related studies. Here, we used the Single-probe mass spectrometry (MS) technique to investigate the metabolic features of live colorectal CSCs at the single-cell level. Experimental data were analyzed using statistical analysis methods, including the t-test and partial least squares discriminant analysis. Our results indicate that the overall metabolic profiles of CSCs are distinct from non-stem cancer cells (NSCCs). Specifically, we demonstrated that tricarboxylic acid (TCA) cycle metabolites are more abundant in CSCs compared to NSCCs, indicating their major energy production pathways are different. Moreover, CSCs have relatively higher levels of unsaturated lipids. Inhibiting the activities of stearoyl-CoA desaturase-1 (SCD1), nuclear factor κB (NF-κB), and aldehyde dehydrogenases (ALDH1A1) in CSCs significantly reduced the abundances of unsaturated lipids and hindered the formation of spheroids, resulting in reduced stemness of CSCs. Our techniques and experimental protocols can be potentially used for metabolomic studies of other CSCs and rare types of cells and provide a new approach to discovering functional biomarkers as therapeutic targets.
Subject(s)
Metabolome , Neoplastic Stem Cells/metabolism , Aldehyde Dehydrogenase 1 Family/antagonists & inhibitors , Citric Acid Cycle/physiology , Discriminant Analysis , Enzyme Inhibitors/pharmacology , Fatty Acids, Unsaturated/analysis , HCT116 Cells , Humans , Least-Squares Analysis , Mass Spectrometry/methods , Metabolomics/methods , NF-kappa B p50 Subunit/antagonists & inhibitors , Neoplastic Stem Cells/drug effects , Retinal Dehydrogenase/antagonists & inhibitors , Sesquiterpenes/pharmacology , Single-Cell Analysis/methods , Spheroids, Cellular/drug effects , Stearoyl-CoA Desaturase/antagonists & inhibitorsABSTRACT
BACKGROUND: Nuclear factor kappa B (NFkB-light-chain-enhancer of activated B-cells) expression and its regulation is a key role in the development of number of malignancies, as NFkB mediates the balance between cell death and its survival. Therefore, NFkB regulation constitutes an attractive target to overcome the resistance to chemotherapeutic agents in anticancer therapy. Curcumin, as a chemopreventive agent, has a potential role in inhibiting cell growth in a variety of malignancies. Thus, this study was aimed to investigate the efficacy of curcumin along with tumor necrosis factor-α-related apoptosis-inducing ligand (TRAIL) in KCL-22 myeloid cells along with an investigation of the mechanism by which both the agents exert their effects. MATERIALS AND METHODS: KCL-22 cells were exposed to different doses of curcumin and TRAIL alone and in combination. Cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, caspase activity by fluorescent method, protein expression by western Blot, and NFkB activity by electrophoretic mobility shift assays, respectively. RESULTS: Cell viability assay revealed that when both the agents, curcumin and TRAIL, were used together, there was reduced cell viability in dose- and time-dependent manner as compared to each agent alone. Curcumin and TRAIL enhanced the caspase-3 activity as compared to caspase-8 and caspase-9. Both the agents induced apoptosis in KCL-22 cells by suppressing the IκB kinase and NFkB activity. CONCLUSION: Our results conclude that curcumin and TRAIL effectively induce the apoptosis through the inhibition of NFkB activity and by enhancing the caspase-3 activity. Thus, curcumin may prove as a potent inhibitor of NFkB by representing its role in cancer pathogenesis, especially in chronic myeloid leukemia cells.