Isothiocyanates (ITCs) derived from cruciferous vegetables, including benzyl isothiocyanate (BITC), phenethyl isothiocyanate (PEITC) and sulforaphane (SFN), exhibit preventative effects against various types of cancers. Yet, the inhibitory effects of ITCs on C6 glioma cell invasion and migration have not been reported. Thus, we aimed to analyze ITC-regulated MMP-9 activation, a crucial enzyme of cancer metastasis that degrades the extracellular matrix, in C6 glioma cells to investigate the inhibitory effects on cancer invasion and migration by ITCs. In the present study, we found that ITCs specifically suppressed PMA-induced MMP-9 secretion and protein expression. The inhibitory effects of ITCs on PMA-induced MMP-9 expression were found to be associated with the inhibition of MMP-9 transcription levels through suppression of nuclear translocation of NF-κB and activator protein-1 (AP-1). It was also confirmed that ITCs decreased MMP-9-mediated signaling such as FAK and JNK, whereas they had no effect on the phosphorylation of ERK and p38. Moreover, wound-healing and Τranswell invasion assays showed that ITCs inhibited the migration and invasion of C6 glioma cells. These results suggest that ITCs could be potential agents for the prevention of C6 glioma cell migration and invasion by decreasing FAK/JNK-mediated MMP-9 expression.

Sirt1, a key regulator of metabolism and longevity, has recently been implicated in the regulation of allergic reactions, although the underlying mechanism remains unclear. Here we show that Sirt1 negatively regulates FcεRI-stimulated mast cell activation and anaphylaxis through two mutually regulated pathways involving AMP-activated protein kinase (AMPK) and protein tyrosine phosphatase 1B (PTP1B). Mast cell-specific knockout of Sirt1 dampened AMPK-dependent suppression of FcεRI signaling, thereby augmenting mast cell activation both in vitro and in vivo. Sirt1 inhibition of FcεRI signaling also involved an alternative component, PTP1B, which attenuated the inhibitory AMPK pathway and conversely enhanced the stimulatory Syk pathway, uncovering a novel role of this phosphatase. Moreover, a Sirt1 activator resveratrol stimulated the inhibitory AMPK axis, with reciprocal suppression of the stimulatory PTP1B/Syk axis, thus potently inhibiting anaphylaxis. Overall, our results provide a molecular explanation for the beneficial role of Sirt1 in allergy and underscore a potential application of Sirt1 activators as a new class of anti-allergic agents.

Background: Nuclear receptor subfamily 4 group A member 1 (NR4A1), an orphan nuclear receptor, has been implicated in several biological events such as metabolism, apoptosis, and inflammation. Recent studies indicate a potential role for NR4A1 in mast cells, yet its role in allergic responses remains largely unknown.Objectives: The aim of this study was to clarify the role of NR4A1 in mast cell activation and anaphylaxis. Methods: To evaluate the function of NR4A1 in mast cells, the impacts of siRNA knockdown, gene knockout, adenoviral overexpression, and pharmacological inhibition of NR4A1 on FcεRI signaling and effector functions in mouse bone marrowderived mast cells (BMMCs) in vitro and on anaphylactic responses in vivo were evaluated. Results: Knockdown or knockout of NR4A1 markedly suppressed degranulation and lipid mediator production by FcεRI-crosslinked BMMCs, while its overexpression augmented these responses. Treatment with a NR4A1 antagonist also blocked mast cell activation to a similar extent as NR4A1 knockdown or knockout. Moreover, mast cell-specific NR4A1-deficient mice displayed dampened anaphylactic responses in vivo. Mechanistically, NR4A1 promoted FcεRI signaling by counteracting the liver kinase B1 (LKB1)/adenosine monophosphate-activated protein kinase (AMPK) axis. Following FcεRI crosslinking, NR4A1 bound to the LKB1/AMPK complex and sequestered it in the nucleus, thereby promoting FcεRI downstream signaling pathways. Silencing or knockout of LKB1/AMPK largely abrogated the effect of NR4A1 on mast cell activation. Additionally, NR4A1 facilitated spleen tyrosine kinase activation independently of LKB1/AMPK.

Abbreviations: A770041, a selective inhibitor of Lck; Ag, antigen; BMMC, bone marrow-derived mast cells; CCL3, C-C motif chemokine ligand 3; DNP-HSA, dinitrophenyl-human serum albumin; eMIP, a 69-amino acid variant of human CCL3; ERK/Erk1/2, extracellular signal-regulated kinase 1/2; Fexo, fexofenadine; GPCR, G protein-coupled receptor; HSP, heat shock proteins; HSP70, heat shock protein 70; IgE, immunoglobulin E; IKK, IκB kinase; IL-6, interleukin-6; IRF, interferon regulatory factor; JNK, c-Jun N-terminal kinase; KIT, c-kit receptor; KO, knockout; LAT, linker for activation of T cells; Lck, lymphocyte-specific protein tyrosine kinase; LTC 4 , leukotriene C 4 ; MAPK, mitogen-activated protein kinase; M-dose, micromolar doses; MyD88, myeloid differentiation factor 99; N-dose, nanomolar doses; NF-κB, nuclear factor-kappa B; NK, natural killer; PCA, passive cutaneous anaphylaxis or anaphylactic; PGD 2 , prostaglandin D 2 ; Phospho-Lck, phosphorylated Lck; Phospho-Syk, phosphorylated Syk; PI3K, phosphatidylinositol 3-kinase; PKC, protein kinase C; PLCγ, phospholipase Cγ; SCF, stem cell factor; SHP-1, Src homology region 2 domain-containing phosphatase-1; Syk, spleen tyrosine kinase; TIR domain, Toll/interleukin-1 receptor homology domain; TIRAP, TIR domain-containing adaptor protein; TLR4, Toll-like receptor 4; TNF-α, tumor necrosis factor-α; TRAF6, TNF receptor-associated factor 6; β-Hex, β-hexosaminidase. AbstractBackground: Signal transduction pathways mediated by various receptors expressed on mast cells are thought to be complex, and inhibitory signals that turn off activating signals are not known. Methods: Upstream signaling cascades mediated by several known receptors in bonemarrow-derived mast cells that lead to degranulation and mediator release were studied by immunoblotting and immunoprecipitation. Small interfering RNAs and knockout mice were used to confirm findings. Results: All ligands tested including IgE/Ag, SCF, HSP70, CCL3, and its valiant eMIP induced phosphorylation of linker for activation of T cells (LAT), which triggered their receptor-mediated downstream signaling cascades that controlled degranulation and mediator release. Phosphorylation of lymphocyte-specific protein kinase (Lck) was induced by each ligand, which commonly played an indispensable role in LAT phosphorylation. In contrast, phosphorylation of spleen tyrosine kinase was additionally induced in cells stimulated only with IgE/Ag and SCF, which is also associated with LAT phosphorylation in part. Degranulation and mediator release induced by IgE/Ag, SCF, or HSP70 were enhanced by nanomolar doses of CCR1 ligands CCL3 and eMIP via enhanced LAT phosphorylation. On the other hand, micromolar doses of CCR1 ligand inhibited degranulation and mediator release from mast cells stimulated with IgE/Ag, SCF, or HSP70 by de-phosphorylation of phosphorylated Lck with Src homology region 2 domain-containing phosphatase-1.

Jellyfish species are widely distributed in the world’s oceans, and their population is rapidly increasing. Jellyfish extracts have several biological functions, such as cytotoxic, anti-microbial, and antioxidant activities in cells and organisms. However, the anti-cancer effect of Jellyfish extract has not yet been examined. We used chronic myelogenous leukemia K562 cells to evaluate the mechanisms of anti-cancer activity of hexane extracts from Nomura’s jellyfish in vitro. In this study, jellyfish are subjected to hexane extraction, and the extract is shown to have an anticancer effect on chronic myelogenous leukemia K562 cells. Interestingly, the present results show that jellyfish hexane extract (Jellyfish-HE) induces apoptosis in a dose- and time-dependent manner. To identify the mechanism(s) underlying Jellyfish-HE-induced apoptosis in K562 cells, we examined the effects of Jellyfish-HE on activation of caspase and mitogen-activated protein kinases (MAPKs), which are responsible for cell cycle progression. Induction of apoptosis by Jellyfish-HE occurred through the activation of caspases-3,-8 and -9 and phosphorylation of p38. Jellyfish-HE-induced apoptosis was blocked by a caspase inhibitor, Z-VAD. Moreover, during apoptosis in K562 cells, p38 MAPK was inhibited by pretreatment with SB203580, an inhibitor of p38. SB203580 blocked jellyfish-HE-induced apoptosis. Additionally, Jellyfish-HE markedly arrests the cell cycle in the G0/G1 phase. Therefore, taken together, the results imply that the anti-cancer activity of Jellyfish-HE may be mediated apoptosis by induction of caspases and activation of MAPK, especially phosphorylation of p38, and cell cycle arrest at the Go/G1 phase in K562 cells.