Activation Of NF-kappaB Signaling Pathway Research Articles

The microRNA-520a-3p inhibits invasion and metastasis by targeting NF-kappaB signaling pathway in non-small cell lung cancer.

To identify the expression of miR-520a-3p and AKT1 in non-small cell lung cancer cells (NSCLC) and the mechanism in inhibiting cell invasion and metastasis by targeting NF-kappaB signaling pathway. Bioinformatics analysis and dual luciferase reporter gene assay were used to predict and verify the targeting relationship between miR-520a-3p and AKT1. EdU assay was used to detect the proliferation of NSCLC cells. Flow cytometry detected the apoptosis of NSCLC cells. Transwell assay tested the invasion ability of NSCLC cells. qRT-PCR measured the expression of miR-520a-3p and AKT1 mRNA in NSCLC cells; while western blotting was adopted to detect the protein expressions of AKT1, Ki67, CyclinD1, Bax, Bcl-2, MMP-2, MMP-9, NF-kB p65, IkBs kinase (IKK), NF-kB inducing kinase (NIK). Bioinformatics analysis suggested that miR-520a-3p could target AKT1. miR-520a-3p could regulate the expression of AKT1 negatively. Compared to mimic-NC group, miR-520a-3p mimic group had increased expressions of miR-520a-3p and Bax, while decreased expressions of AKT1, Ki67, CyclinD1, Bcl-2, MMP-2, MMP-9, NF-kB p65, IKK and NIK, reduced cell proliferation, invasion, and increased cell apoptosis rate (all P < 0.05). Compared to inhibitor NC group, miR-520a-3p inhibitor group had decreased expressions of miR-520a-3p and Bax, but increased expressions of AKT1, Ki67, CyclinD1, Bcl-2, MMP-2, MMP-9, NF-kB p65, IKK and NIK, promoted cell proliferation, invasion, and suppressed cell apoptosis rate (all P < 0.05). Overexpression of miR-520a-3p can target and downregulate the expression of AKT1 to inhibit the invasion and metastasis of NSCLC via suppressing the activation of NF-kappaB signaling pathway.

Raf Kinase Inhibitor Protein Attenuates Ischemic-Induced Microglia Cell Apoptosis and Activation Through NF-κB Pathway

Background: Acute ischemic stroke is one of the most important factors leading to disability and death with the characterization of accumulated neuron death and injured supportive neurovascular structures. Raf-1 kinase inhibitory protein (RKIP) is a key molecule in cell response to survival or death stimuli. However, the role of RKIP in stroke is worthy to be further studied. Methods: We used lentivirus mediated RKIP knockdown and overexpression to investigate the effect of RKIP on animal models of focal cerebral ischemia. Cell Counting Kit-8 assay, lactate dehydrogenase release analysis, and Annexin V-APC apoptosis assay were used to detect the effect RKIP on microglial cell apoptosis and survival. Transwell migration assay was carried out to evaluate the migration of microglia cells. The releases of inflammatory cytokines were determined by ELISA. The activation of NF-kappaB signaling pathway was determined by western blot. Results: Overexpression of RKIP reduced focal cerebral ischemia injury. RKIP knockdown and overexpression regulated survival, activation, and motility via the NF-κB pathway. NF-κB inhibitor BAY 11-7082 blocked the changes caused by RKIP down-regulation after oxygen-glucose deprivation (OGD). RKIP overexpression inhibited the upregulation of phosphorylation of NF-κB induced by OGD and cerebral ischemia. Conclusions: The present study showed that RKIP protects against ischemic stroke through inhibition of microglial excessive activation, inhibits its motility, and promotes neuronal survival partly though IKKβ-IκBα-NF-κB signaling axis and indicate that RKIP is a new target for the treatment of ischemic stroke.

Geniposide inhibits high glucose-induced cell adhesion through the NF-κB signaling pathway in human umbilical vein endothelial cells

To investigate whether geniposide, an iridoid glucoside extracted from gardenia jasminoides ellis fruits, inhibits cell adhesion to human umbilical vein endothelial cells (HUVECs) induced by high glucose and its underlying mechanisms. HUVECs were isolated from human umbilical cords and cultured. The adhesion of monocytes to HUVECs was determined using fluorescence-labeled monocytes. The mRNA and protein levels of vascular cell adhesion molecule-1 (VCAM-1) and endothelial selectin (E-selectin) were measured using real-time RT-PCR and ELISA. Reactive oxygen species (ROS) production was measured using a fluorescent probe. The amounts of nuclear factor-kappa B (NF-kappaB) and inhibitory factor of NF-kappaB (IkappaB) were determined using Western blot analysis. The translocation of NF-kappaB from the cytoplasm to the nucleus was determined using immunofluorescence. Geniposide (10-20 mumol/L) inhibited high glucose (33 mmol/L)-induced adhesion of monocytes to HUVECs in a dose-dependent manner. This compound (5-40 mumol/L) also inhibited high glucose-induced expression of VCAM-1 and E-selectin at the gene and protein levels. Furthermore, geniposide (5-20 micromol/L) decreased ROS production and prevented IkappaB degradation in the cytoplasm and NF-kappaB translocation from the cytoplasm to the nucleus in HUVECs. Geniposide inhibits the adhesion of monocytes to HUVECs and the expression of CAMs induced by high glucose, suggesting that the compound may represent a new treatment for diabetic vascular injury. The mechanism underlying this inhibitory effect may be related to the inhibition of ROS overproduction and NF-kappaB signaling pathway activation by geniposide.

Growth-stimulatory effect of resveratrol in human cancer cells

Earlier studies have shown that resveratrol could induce death in several human cancer cell lines in culture. Here we report our observation that resveratrol can also promote the growth of certain human cancer cells when they are grown either in culture or in athymic nude mice as xenografts. At relatively low concentrations (</=5 microM), resveratrol exerted a significant growth-stimulatory effect in the MDA-MB-435s human cancer cells, but this effect was not observed in several other human cell lines tested. Analysis of cell signaling molecules showed that resveratrol induced the activation of JNK, p38, Akt, and NF-kappaB signaling pathways in these cells. Further analysis using pharmacological inhibitors showed that only the NF-kappaB inhibitor (BAY11-7082) abrogated the growth-stimulatory effect of resveratrol in cultured cells. In athymic nude mice, resveratrol at 16.5 mg/kg body weight enhanced the growth of MDA-MB-435s xenografts compared to the control group, while resveratrol at the 33 mg/kg body weight dose did not have a similar effect. Additional analyses confirmed that resveratrol stimulated cancer cell growth in vivo through activation of the NF-kappaB signaling pathway. Taken together, these observations suggest that resveratrol at low concentrations could stimulate the growth of certain types of human cancer cells in vivo. This cell type-specific mitogenic effect of resveratrol may also partly contribute to the procarcinogenic effect of alcohol consumption (rich in resveratrol) in the development of certain human cancers.

Tumor Necrosis Factor-like Weak Inducer of Apoptosis Inhibits Skeletal Myogenesis through Sustained Activation of Nuclear Factor-κB and Degradation of MyoD Protein

In this study we have investigated the effect and the mechanisms by which tumor necrosis factor-like weak inducer of apoptosis (TWEAK) modulates myogenic differentiation. Treatment of C2C12 myoblasts with TWEAK inhibited their differentiation evident by a decrease in the expression of creatine kinase, myosin heavy chain-fast twitch, myogenin, and the formation of multinucleated myotubes. TWEAK also inhibited the differentiation of mouse primary myoblasts. Conversely, the proliferation of C2C12 myoblasts and the expression of a cell-cycle regulator cyclin D1 were increased in response to TWEAK treatment. Inhibition of cellular proliferation using hydroxyurea only partially reversed the inhibitory effect of TWEAK on myogenic differentiation. Treatment of C2C12 myoblasts with TWEAK resulted in the activation of nuclear factor-kappaB (NF-kappaB), the (IkappaB) IkappaB kinase (IKK) complex, and the phosphorylation and degradation of IkappaBalpha protein. Inhibition of NF-kappaB activity by overexpression of a dominant negative mutant of IkappaBalpha (IkappaBalphaDeltaN) significantly increased the myogenic differentiation in TWEAK-treated C2C12 cultures. Furthermore, overexpression of a dominant negative mutant of IKKbeta (IKKbetaK44A) but not IKKalpha (IKKalphaK44M) reversed the inhibitory effect of TWEAK on myogenesis. TWEAK inhibited the expression of myogenic regulatory factors MyoD and myogenin and also induced the degradation of MyoD protein. Finally, inhibition of NF-kappaB activation through overexpression of IKKbetaK44A prevented the degradation of MyoD protein. Overall, our data suggest that TWEAK inhibits myogenesis through the activation of NF-kappaB signaling pathway and degradation of MyoD protein.

Central tolerance and autoimmune diseases

Central tolerance is established by the repertoire selection of immature T lymphocytes in the thymus, avoiding autoimmune responses to self-antigens. Differential ligand-TCR interactions that result in positive and negative selection initiate differential intracellular signals that, in turn, lead to the survival-or-death decision of immature thymocytes. TCR signal dysregulation due to the mutation of ZAP-70 or defective apoptosis of autoreactive thymocytes due to the deficiency of pro-apoptotic protein Bim impair tolerance and cause autoimmunity. Thymic repertoire selection also induces the development of CD25(+)CD4(+) regulatory T cells, which play important roles for maintaining peripheral tolerance. Furthermore, the establishment of central tolerance requires the development of thymic medulla that is mediated by the activation of NF-kappaB signaling pathway, promiscuous expression of tissue-specific self-antigens by medullary epithelial cells that is regulated by AIRE, and cortex-to-medulla migration of developing thymocytes that is regulated by CCR7-mediated chemokine signals.

Ethanol‐Induced Sensitization to Endotoxin in Kupffer Cells Is Dependent upon Oxidative Stress

Activation of Kupffer cells by gut-derived endotoxin plays a pivotal role in alcoholic liver injury. On the other hand, it was reported that acute ethanol administration reduced activation of Kupffer cells. We found that Kupffer cells isolated from rat treated only once with ethanol were sensitized to endotoxin 24 hrs later correlatively with CD14 expression. Moreover, it was shown that Kupffer cell activation by endotoxin via Toll-like receptor (TLR)-4 is involved in alcohol-induced liver injury and ethanol-induced oxidative stress is important in the regulation of transcription factor NFkappaB activation and cytokine production by Kupffer cells. Here, we show that IRAK, one of signaling molecules of TLR-4, regulates tolerance and sensitization to LPS and acute ethanol increases in IRAK expression through a mechanism dependent upon oxidant production. Female C57BL/6 mice were given ethanol (5 g/kg) intragastrically, and LPS was injected 1 or 21 hrs later. Serum transaminase levels were measured. Moreover, some mice were treated with NADPH oxidase inhibitor diphenyleneiodonium sulfate (DPI, 1 mg/kg/day) or infected with adenovirus (1 x 10 plaque-forming units, intravenously) containing IkappaB superrepressor gene, which prevent NFkappaB activation of Kupffer cells, for three days. Kupffer cells were isolated from mice 1 hr and 21 hrs after ethanol treatment. After the addition of LPS, TNF-alpha in the media was measured using ELISA, Electrophoretic mobility shift assay (EMSA) was performed to analyze DNA binding activity of NFkappaB. Further, expression of Interleukin-1 receptor-associated kinase (IRAK) was evaluated by Western blotting. LPS-induced increases in transaminases were blunted in mice treated with ethanol before 1 hr. However, ethanol treatment 21 hrs earlier augmented LPS-increased transaminases three-fold over controls. Pretreatment with nonabsorbable antibiotics blocked these effects of ethanol. LPS-induced TNF-alpha production by Kupffer cells isolated from mice 1 hr after ethanol was reduced to about 60% of values from control Kupffer cells, while LPS-induced TNF-alpha production by Kupffer cells isolated from mice treated with ethanol 21 hrs earlier increased 1.5-fold over control Kupffer cells. In Kupffer cells from mice 1 hr after ethanol treatment, expression of IRAK was decreased, and LPS-induced activation of NFkappaB was decreased correlatively. In contrast, ethanol treatment to mice increased expression of IRAK in Kupffer cells 21hrs later and LPS-induced activation of NFkappaB was elevated significantly. On the other hands, DPI treatment for three days prior to ethanol did not prevent decreases in IRAK expression due to ethanol treatment for 1 hr. However, DPI treatment blunted ethanol-induced increases in IRAK expression. Additionally, inhibition of NKkappaB activation with dominant-negative IkappaBalpha blunted ethanol-induced increase in IRAK expression. Contrary, inhibition of NKkappaB did not affect decrease of IRAK expression due to ethanol treatment for 1 hr. Ethanol causes tolerance in the early phase after ethanol consumption, while sensitization was observed later. Both tolerance and sensitization were induced by gut-derived endotoxin. These findings indicate that ethanol-induced both tolerance and sensitization of Kupffer cells to endotoxin involve IRAK expression. Further, NADPH oxidase plays a pivotal role in the increase in IRAK expression due to ethanol via activation of NFkappaB signaling pathway. In conclusion, these data indicate that acute ethanol causes sensitization to endotoxin through mechanisms dependent upon oxidative stress.

Drosophila TAB2 is required for the immune activation of JNK and NF-kappaB

The TAK1 plays a pivotal role in the innate immune response of Drosophila by controlling the activation of JNK and NF-kappaB. Activation of TAK1 in mammals is mediated by two TAK1-binding proteins, TAB1 and TAB2, but the role of the TAB proteins in the immune response of Drosophila has not yet been established. Here, we report the identification of a TAB2-like protein in Drosophila called dTAB2. dTAB2 can interact with dTAK1, and stimulate the activation of the JNK and NF-kB signaling pathway. Furthermore, we have found that silencing of dTAB2 expression by dsRNAi inhibits JNK activation by peptidoglycans (PGN), but not by NaCl or sorbitol. In addition, suppression of dTAB2 blocked PGN-induced expression of antibacterial peptide genes, a function normally mediated by the activation of NF-kappaB signaling pathway. No significant effect on p38 activation by dTAB2 was found. These results suggest that dTAB2 is specifically required for PGN-induced activation of JNK and NF-kappaB signaling pathways.

Lupeol, a fruit and vegetable based triterpene, induces apoptotic death of human pancreatic adenocarcinoma cells via inhibition of Ras signaling pathway

Pancreatic cancer is an exceptionally aggressive disease, the treatment of which has largely been unsuccessful due to higher resistance offered by pancreatic cancer cells to conventional approaches such as surgery, radiation and/or chemotherapy. The aberration of Ras oncoprotein has been linked to the induction of multiple signaling pathways and to the resistance offered by pancreatic cancer cells to apoptosis. Therefore, there is a need for development of new and effective chemotherapeutic agents which can target multiple pathways to induce responsiveness of pancreatic cancer cells to death signals. In this study, human pancreatic adenocarcinoma cells AsPC-1 were used to investigate the effect of Lupeol on cell growth and its effects on the modulation of multiple Ras-induced signaling pathways. Lupeol caused a dose-dependent inhibition of cell growth as assessed by MTT assay and induction of apoptosis as assessed by flow cytometry, fluorescence microscopy and western blotting. Lupeol treatment to cells was found to significantly reduce the expression of Ras oncoprotein and modulate the protein expression of various signaling molecules involved in PKCalpha/ODC, PI3K/Akt and MAPKs pathways along with a significant reduction in the activation of NFkappaB signaling pathway. Our data suggest that Lupeol can adopt a multi-prong strategy to target multiple signaling pathways leading to induction of apoptosis and inhibition of growth of pancreatic cancer cells. Lupeol could be a potential agent against pancreatic cancer, however, further in-depth in vivo studies are warranted.

Troglitazone and 15-deoxy-Δ-prostaglandin J inhibit shear-induced coupling factor 6 release in endothelial cells

We previously showed that mitochondrial coupling factor 6 (CF6), an endogenous inhibitor of prostacyclin synthesis and a vasoconstrictor, is present on the surface of human umbilical vein endothelial cells (HUVEC) and is released outside of the cells by shear stress. We investigated the intracellular signaling mechanism for shear-induced release of CF6 in HUVEC and the effects of troglitazone and 15-deoxy-delta(12,14)-prostaglandin J2 (15d-PGJ2), both peroxisome proliferator-activated receptor (PPAR)-gamma ligands, on it. The release and gene expression of CF6 in HUVEC were enhanced by shear stress at 25 dyn/cm2, measured by radioimmunoassay and real-time RT-PCR, respectively. The intracellular content of CF6 was decreased after exposure to shear stress at 25 dyn/cm2. Transfection experiments with deletional and mutational CF6 promoter constructs, and with dominant negative mutant IkappaB kinase alpha (K44M) demonstrated that shear-induced CF6 transcription was dependent on nuclear factor-kappa B (NF-kappaB) activation. Pretreatment with troglitazone or 15d-PGJ2 inhibited the shear-induced release and gene expression of CF6, whereas fenofibric acid, a PPAR-alpha ligand, had no influence on them. Western blot and immunostaining showed that troglitazone and 15d-PGJ2 inhibited the shear-induced, reactive oxygen species (ROS)-mediated activation of NF-kappaB at the level of IkappaB protein. The shear-induced gene expression and release of CF6 in HUVEC are mediated by the ROS-related activation of NF-kappaB signaling pathway. Troglitazone and 15d-PGJ2 inhibit them at the IkappaB protein level.

Id-1 expression promotes cell survival through activation of NF-kappaB signalling pathway in prostate cancer cells.

The growth-promoting effect of Id-1 (inhibitor of differentiation/DNA binding) has been demonstrated in a number of human cancers. However, the mechanisms responsible for its action are not clear. In this study, we report that in prostate cancer cells, Id-1 promotes cell survival through activation of nuclear factor-kappaB (NF-kappaB) signalling pathway. After stable expression of Id-1 protein in LNCaP cells, we found that the Id-1 transfectants showed increased resistance to apoptosis induced by TNFalpha through inactivation of Bax and caspase 3. In addition, in the LNCaP cells expressing ectopic Id-1 protein, we also observed increased NF-kappaB transactivation activity and nuclear translocation of the p65 and p50 proteins, which was accompanied by upregulation of their downstream effectors Bcl-xL and ICAM-1. These results indicate that the Id-1-induced antiapoptotic effect may be via NF-kappaB signalling transduction pathway in these cells. In addition, inactivation of Id-1 by its antisense oligonucleotide and retroviral construct in DU145 cells resulted in the decrease of nuclear level of p65 and p50 proteins, which was associated with increased sensitivity to TNFalpha-induced apoptosis. Our results strongly suggest that Id-1 may be one of the upstream regulators of NF-kappaB and activation of NF-kappaB signalling pathway may be essential for Id-1 induced cell proliferation through protection against apoptosis. Our findings also suggest a potential therapeutic strategy in which inactivation of Id-1 may lead to sensitization of prostate cancer cells to chemotherapeutic drug-induced apoptosis.