Indacaterol Inhibits Tumor Cell Invasiveness and MMP-9 Expression by Suppressing IKK/NF-κB Activation

The β₂ adrenergic receptor (ADRB2) is a G protein-coupled transmembrane receptor expressed in the human respiratory tract and widely recognized as a pharmacological target for treatments of asthma and chronic obstructive pulmonary disorder (COPD). Although a number of ADRB2 agonists have been developed for use in asthma therapy, indacaterol is the only ultra-long-acting inhaled β₂-agonist (LABA) approved by the FDA for relieving the symptoms in COPD patients.The precise molecular mechanism underlying the pharmacological effect of indacaterol, however, remains unclear. Here, we show that β-arrestin-2 mediates the internalization of ADRB2 following indacaterol treatment. Moreover, we demonstrate that indacaterol significantly inhibits tumor necrosis factor-α (TNF-α)-induced NF-κB activity by reducing levels of both phosphorylated-IKK and -IκBα, thereby decreasing NF-κB nuclear translocation and the expression of MMP-9, an NF-κB target gene. Subsequently, we show that indacaterol significantly inhibits TNF-α/NF-κB-induced cell invasiveness and migration in a human cancer cell line. In conclusion, we propose that indacaterol may inhibit NF-κB activity in a β-arrestin2-dependent manner, preventing further lung damage and improving lung function in COPD patients.     

Neurotropin Suppresses Inflammatory Cytokine Expression and Cell Death through Suppression of NF-κB and JNK in Hepatocytes

Inflammatory response and cell death in hepatocytes are hallmarks of chronic liver disease, and, therefore, can be effective therapeutic targets. Neurotropin® (NTP) is a drug widely used in Japan and China to treat chronic pain. Although NTP has been demonstrated to suppress chronic pain through the descending pain inhibitory system, the action mechanism of NTP remains elusive. We hypothesize that NTP functions to suppress inflammatory pathways, thereby attenuating disease progression. In the present study, we investigated whether NTP suppresses inflammatory signaling and cell death pathways induced by interleukin-1β (IL-1β) and tumor necrosis factor-α (TNFα) in hepatocytes. NTP suppressed nuclear factor-κB (NF-κB) activation induced by IL-1β and TNFα assessed by using hepatocytes isolated from NF-κB-green fluorescent protein (GFP) reporter mice and an NF-κB-luciferase reporter system. The expression of NF-κB target genes, Il6, Nos2, Cxcl1, ccl5 and Cxcl2 induced by IL-1β and TNFα was suppressed after NTP treatment. We also found that NTP suppressed the JNK phosphorylation induced by IL-1β and TNFα. Because JNK activation contributes to hepatocyte death, we determined that NTP treatment suppressed hepatocyte death induced by IL-1β and TNFα in combination with actinomycin D. Taken together, our data demonstrate that NTP attenuates IL-1β and TNFα-mediated inflammatory cytokine expression and cell death in hepatocytes through the suppression of NF-κB and JNK. The results from the present study suggest that NTP may become a preventive or therapeutic strategy for alcoholic and non-alcoholic fatty liver disease in which NF-κB and JNK are thought to take part.     

Fisetin Inhibits Human Melanoma Cell Invasion through Promotion of Mesenchymal to Epithelial Transition and by Targeting MAPK and NFκB Signaling Pathways

Malignant melanoma is responsible for approximately 75% of skin cancer-related deaths. BRAF plays an important role in regulating the mitogen-activated protein kinase (MAPK) signaling cascade in melanoma with activating mutations in the serine/threonine kinase BRAF occurring in 60–70% of malignant melanomas. The BRAF-MEK-ERK (MAPK) pathway is a key regulator of melanoma cell invasion. In addition, activation of NFκB via the MAPK pathway is regulated through MEK-induced activation of IKK. These pathways are potential targets for prevention and treatment of melanoma. In this study, we investigated the effect of fisetin, a phytochemical present in fruits and vegetables, on melanoma cell invasion and epithelial-mesenchymal transition, and delineated the underlying molecular mechanism. Treatment of multiple human malignant melanoma cell lines with fisetin (5–20 µM) resulted in inhibition of cell invasion. BRAF mutated melanoma cells were more sensitive to fisetin treatment, and this was associated with a decrease in the phosphorylation of MEK1/2 and ERK1/2. In addition, fisetin inhibited the activation of IKK leading to a reduction in the activation of the NFκB signaling pathway. Treatment of cells with an inhibitor of MEK1/2 (PD98059) or of NFκB (caffeic acid phenethyl ester) also reduced melanoma cell invasion. Furthermore, treatment of fisetin promoted mesenchymal to epithelial transition in melanoma cells, which was associated with a decrease in mesenchymal markers (N-cadherin, vimentin, snail and fibronectin) and an increase in epithelial markers (E-cadherin and desmoglein). Employing three dimensional skin equivalents consisting of A375 cells admixed with normal human keratinocytes embedded onto a collagen-constricted fibroblast matrix, we found that treatment of fisetin reduced the invasive potential of melanoma cells into the dermis and increased the expression of E-cadherin with a concomitant decrease in vimentin. These results indicate that fisetin inhibits melanoma cell invasion through promotion of mesenchymal to epithelial transition and by targeting MAPK and NFκB signaling pathways.     

Curcumin Inhibits Non-Small Cell Lung Cancer Cells Metastasis through the Adiponectin/NF-κb/MMPs Signaling Pathway

Adipose tissue is now considered as an endocrine organ involved in metabolic and inflammatory reactions. Adiponectin, a 244–amino acid peptide hormone, is associated with insulin resistance and carcinogenesis. Curcumin (diferuloylmethane) is the principal curcuminoid of the popular Indian spice, turmeric. Curcumin possesses antitumor effects, including the inhibition of neovascularization and regulation of cell cycle and apoptosis. However, the effects of adiponectin and curcumin on non-small cell lung cancer (NSCLC) remain unclear. In this study, we evaluated the expression of adiponectin in paired tumors and normal lung tissues from 77 patients with NSCLC using real-time polymerase chain reaction, western blotting, and immunohistochemistry. Kaplan–Meier survival analysis showed that patients with low adiponectin expression ratio (<1) had significantly longer survival time than those with high expression ratio (>1) (p = 0.015). Curcumin inhibited the migratory and invasive ability of A549 cells via the inhibition of adiponectin expression by blocking the adiponectin receptor 1. Curcumin treatment also inhibited the in vivo tumor growth of A549 cells and adiponectin expression. These results suggest that adiponectin can be a prognostic indicator of NSCLC. The effect of curcumin in decreasing the migratory and invasive ability of A549 cells by inhibiting adiponectin expression is probably mediated through NF-κB/MMP pathways. Curcumin could be an important potential adjuvant therapeutic agent for lung cancer in the future.     

Suppression of NF-κB and NF-κB-Regulated Gene Expression by Apigenin through IκBα and IKK Pathway in TRAMP Mice

Aberrant Nuclear Factor-κappaB (NF-κB) activation due to rapid IκBα turnover and high basal IκBα kinase (IKK) activity has been frequently observed in prostate cancer. Apigenin, a naturally occurring plant flavone, exhibits anti-proliferative, anti-inflammatory and anti-carcinogenic activities by inhibiting NF-κB pathway, through a mechanism not fully understood. We found that apigenin feeding in microgram doses (bioavailable in humans) inhibited prostate tumorigenesis in TRAMP mice by interfering with NF-κB signaling. Apigenin feeding to TRAMP mice (20 and 50 μg/mouse/day, 6 days/week for 20 weeks) exhibited significant decrease in tumor volumes of the prostate and completely abolished metastasis, which correlated with inhibition of NF-κB activation and binding to the DNA. Apigenin intake blocked phosphorylation and degradation of IκBα by inhibiting IKK activation, which in turn led to suppression of NF-κB activation. The expression of NF-κB-regulated gene products involved in proliferation (cyclin D1, and COX-2), anti-apoptosis (Bcl-2 and Bcl-xL), and angiogenesis (vascular endothelial growth factor) were also downregulated after apigenin feeding. These events correlated with the induction of apoptosis in tumor cells, as evident by increased cleaved caspase-3 labeling index in the dorsolateral prostate. Our results provide convincing evidence that apigenin inhibits IKK activation and restores the expression of IκBα, preventing it’s phosphorylation in a fashion similar to that elicited by IKK and proteasomal inhibitors through suppression of NF-κB signaling pathway.     

Activation of NF-κB by alloferon through down-regulation of antioxidant proteins and IκBα

Alloferon is a 13-amino acid peptide isolated from the bacteria-challenged larvae of the blow fly Calliphora vicina. The pharmaceutical value of the peptide has been well demonstrated by its capacity to stimulate NK cytotoxic activity and interferon (IFN) synthesis in animal and human models, as well as to enhance antiviral and antitumor activities in mice. Antiviral and the immunomodulatory effectiveness of alloferon have also been supported clinically proved in patients suffering with herpes simplex virus (HSV) and human papilloma virus (HPV) infections. To elucidate molecular response to alloferon treatment, we initially screened a model cell line in which alloferon enhanced IFN synthesis upon viral infection. Among the cell lines tested, Namalva was chosen for further proteomic analysis. Fluorescence difference gel electrophoresis (DIGE) revealed that the levels of a series of antioxidant proteins decreased after alloferon treatment, while at least three glycolytic enzymes and four heat-shock proteins were increased in their expression levels. Based on the result of our proteomic analysis, we speculated that alloferon may activate the NF-kappaB signaling pathway. IkappaB kinase (IKK) assay, Western blot analysis on IkappaBalpha and its phosphorylated form at Ser 32, and an NF-kappaB reporter assay verified our proteomics-driven hypothesis. Thus, our results suggest that alloferon potentiates immune cells by activating the NF-kappaB signaling pathway through regulation of redox potential. Since NF-kappaB activation is involved in IFN synthesis, our results provide further clues as to how the alloferon peptide may stimulate IFN synthesis.     

Roles of TLR7 in Activation of NF-κB Signaling of Keratinocytes by Imiquimod

Imiquimod is known to exert its effects through Toll-like receptor 7 (TLR7) and/or TLR8, resulting in expression of proinflammatory cytokines and chemokines. Keratinocytes have not been reported to constitutively express TLR7 and TLR8, and the action of imiquimod is thought to be mediated by the adenine receptor, not TLR7 or TLR8. In this study, we revealed the expression of TLR7 in keratinocytes after calcium-induced differentiation. After addition of calcium to cultured keratinocytes, the immunological responses induced by imiquimod, such as activation of NF-κB and induction of TNF-α and IL-8, were more rapid and stronger. In addition, imiquimod induced the expression TLR7, and acted synergistically with calcium to induce proinflammatory cytokines. We confirmed that the responses induced by imiquimod were significantly inhibited by microRNAs suppressing TLR7 expression. These results suggest that TLR7 expressed in keratinocytes play key roles in the activation of NF-κB signaling by imiquimod, and that their modulation in keratinocytes could provide therapeutic potential for many inflammatory skin diseases.     

Lupeol Is One of Active Components in the Extract of Chrysanthemum indicum Linne That Inhibits LMP1-Induced NF-κB Activation

We have previously reported that seventy percent ethanol extract of Chrysanthemum indicum Linne (CIE) strongly reduces Epstein-Barr virus (EBV)-transformed lymphoblastoid cell line (LCL) survival by inhibiting virus-encoded latent infection membrane protein 1 (LMP1)-induced NF-κB activation. To identify an active compound(s) in CIE that inhibits LMP1-induced NF-κB activation, activity-guided fractionation was employed. The CH₂Cl₂ fraction of CIE strongly reduced LMP1-induced NF-κB activation and LCL viability with relatively low cytotoxic effects on primary human foreskin fibroblast (HFF), HeLa or Burkitt’s lymphoma (BL41) cells. Furthermore, lupeol, a pentacyclic triterpene, was identified in the CH₂Cl₂ fraction of CIE to attenuate LMP1-induced NF-κB activation and LCL viability. This study demonstrates that lupeol is one of active compounds in the CH₂Cl₂ fraction of CIE that inhibits LMP1-induced NF-κB activation and reduces NF-κB-dependent LCL viability.     

IFN-γ Induces Gastric Cancer Cell Proliferation and Metastasis Through Upregulation of Integrin β3-Mediated NF-κB Signaling

Interferon γ (IFN-γ), a multifunctional cytokine, was upregulated in the resected gastric cancer tissue. However, whether IFN-γ is involved in the regulation of gastric cancer has not been well elucidated. Herein, we aimed to investigate the effects and mechanism of IFN-γ on gastric cancer. In this study, we found a vital role of IFN-γ in enhancing proliferation, inhibiting apoptosis, and promoting cell migration and invasion in gastric cancer cells SGC-7901 and MGC-803. Additionally, IFN-γ activated nuclear factor κB (NF-κB) signaling pathway by upregulating the phosphorylation expression of p65 and IκBα, and induced the expression of integrin β3 in vitro. Therefore, to further investigate the relationship between IFN-γ and integrin β3, SGC-7901 cells were transfected with integrin β3 siRNA. And then cells expressed lower cell viability, migration, and invasion rates, while cell apoptosis was significantly enhanced. Meanwhile, expression of integrin β3, MMP-2, MMP-9, and NF-κB, including p65 and IκBα, and the nuclear translocation of NF-κB/p65 were dramatically repressed, whereas IFN-γ significantly improved the effects. Moreover, in vivo, the experiment of xenograft model and pulmonary metastasis model also retarded in integrin β3 siRNA group. And the expression of integrin β3, MMP-2, MMP-9, and NF-κB was repressed. However, the treatment with IFN-γ improved tumor volume, lung/total weight, tumor nodules, and the protein expression described above compared with integrin β3 siRNA group. Overall, the results indicated that IFN-γ induces gastric cancer cell proliferation and metastasis partially through the upregulation of integrin β3-mediated NF-κB signaling. Hence, the inhibition of IFN-γ or integrin β3 may be the key for the treatment of gastric cancer.     

A Splicing Variant of NME1 Negatively Regulates NF-κB Signaling and Inhibits Cancer Metastasis by Interacting with IKKβ

IKKβ functions as a principal upstream activator of the canonical NF-κB pathway by phosphorylating IκB, leading to its proteasomal degradation. Because IKKβ is considered a therapeutic target, understanding its regulation may facilitate the design of efficient regulators of this molecule. Here, we report a novel IKKβ-interacting molecule, NME1L, a splicing variant of the NME1 protein. NME1 has attracted attention in cancer research because of its antimetastatic activity and reduced expression in multiple aggressive types of cancer. However, the effect was just moderate but not dramatic in anti-cancer activities. We found that only NME1L interacts with IKKβ. Exogenous expression of NME1L resulted in a potent decrease in TNFα-stimulated NF-κB activation, whereas knockdown of NME1/NME1L with shRNA enhanced activity of NF-κB. NME1L down-regulates IKKβ signaling by blocking IKKβ-mediated IκB degradation. When NME1L was introduced into highly metastatic HT1080 cells, the mobility was efficiently inhibited. Furthermore, in a metastasis assay, NME1L-expressing cells did not colonize the lung. Based on these results, NME1L is a potent antimetastatic protein and may be a useful weapon in the fight against cancers.     

Notch-1 Signaling Promotes the Malignant Features of Human Breast Cancer through NF-κB Activation

The aberrant activation of Notch-1 signaling pathway has been proven to be associated with the development and progression of cancers. However, the specific roles and the underlying mechanisms of Notch-1 signaling pathway on the malignant behaviors of breast cancer are poorly understood. In this study, using multiple cellular and molecular approaches, we demonstrated that activation of Notch-1 signaling pathway promoted the malignant behaviors of MDA-MB-231 cells such as increased cell proliferation, colony formation, adhesion, migration, and invasion, and inhibited apoptosis; whereas deactivation of this signaling pathway led to the reversal of the aforementioned malignant cellular behaviors. Furthermore, we found that activation of Notch-1 signaling pathway triggered the activation of NF-κB signaling pathway and up-regulated the expression of NF-κB target genes including MMP-2/-9, VEGF, Survivin, Bcl-xL, and Cyclin D1. These results suggest that Notch-1 signaling pathway play important roles in promoting the malignant phenotype of breast cancer, which may be mediated partly through the activation of NF-κB signaling pathway. Our results further suggest that targeting Notch-1 signaling pathway may become a newer approach to halt the progression of breast cancer.     

Fisetin Inhibits Migration and Invasion of Human Cervical Cancer Cells by Down-Regulating Urokinase Plasminogen Activator Expression through Suppressing the p38 MAPK-Dependent NF-κB Signaling Pathway

Fisetin (3,3’,4’,7-tetrahydroxyflavone), a naturally occurring flavonoid, has been reported to inhibit proliferation and induce apoptosis in several cancer types. However, its effect on the anti-metastatic potential of cervical cancer cells remains unclear. In the present study, we found that fisetin inhibits the invasion and migration of cervical cancer cells. The expression and activity of urokinase plasminogen activator (uPA) was significantly suppressed by fisetin in a dose-dependent manner. We also demonstrated that fisetin reduces the phosphorylation of p38 MAPK, but not that of ERK1/2, JNK1/2, or AKT. Addition of a p38 MAPK inhibitor, SB203580, further enhanced the inhibitory effect of fisetin on the expression and activity of uPA and the invasion and motility in cervical cancer cells. Fisetin suppressed the TPA (tetradecanoylphorbol-13-acetate)-induced activation of p38 MAPK and uPA, and inhibited the TPA-enhanced migratory and invasive abilities. Furthermore, the promoter activity of the uPA gene was dramatically repressed by fisetin, which disrupted the nuclear translocation of NF-κB and its binding amount on the promoter of the uPA gene, and these suppressive effects could be further enhanced by SB203580. This study provides strong evidence for the molecular mechanism of fisetin in inhibiting the aggressive phenotypes by repression of uPA via interruption of p38 MAPK-dependent NF-κB signaling pathway in cervical cancer cells and thus contributes insight to the potential of using fisetin as a therapeutic strategy against cervical cancer by inhibiting migration and invasion.