Src Is a Prime Target Inhibited by Celtis choseniana

Celtis choseniana is the traditional plant used at Korea as a herbal medicine to ameliorate inflammatory responses. Although Celtis choseniana has been traditionally used as a herbal medicine at Korea, no systemic research has been conducted on its anti-inflammatory activity. Therefore, the present study explored an anti-inflammatory effect and its underlying molecular mechanism using Celtis choseniana methanol extract (Cc-ME) in macrophage-mediated inflammatory responses. In vitro anti-inflammatory activity of Cc-ME was evaluated using RAW264.7 cells and peritoneal macrophages stimulated by lipopolysaccharide (LPS), pam3CSK4 (Pam3), or poly(I:C). In vivo anti-inflammatory activity of Cc-ME was investigated using acute inflammatory disease mouse models, such as LPS-induced peritonitis and HCl/EtOH-induced gastritis. The molecular mechanism of Cc-ME-mediated anti-inflammatory activity was examined by Western blot analysis and immunoprecipitation using whole cell and nuclear fraction prepared from the LPS-stimulated RAW264.7 cells and HEK293 cells. Cc-ME inhibited NO production and mRNA expression of inducible nitric oxide synthase (iNOS), cyclooxygenase (COX-2), and tumor necrosis factor-alpha (TNF-α) in the RAW264.7 cells and peritoneal macrophages induced by LPS, pam3, or poly(I:C) without cytotoxicity. High-performance liquid chromatography (HPLC) analysis showed that Cc-ME contained anti-inflammatory flavonoids quercetin, luteolin, and kaempferol. Among those, the content of luteolin, which showed an inhibitory effect on NO production, was highest. Cc-ME suppressed the NF-κB signaling pathway by targeting Src and interrupting molecular interactions between Src and p85, its downstream kinase. Moreover, Cc-ME ameliorated the morphological finding of peritonitis and gastritis in the mouse disease models. Therefore, these results suggest that Cc-ME exerted in vitro and in vivo anti-inflammatory activity in LPS-stimulated macrophages and mouse models of acute inflammatory diseases. This anti-inflammatory activity of Cc-ME was dominantly mediated by targeting Src in NF-κB signaling pathway during macrophage-mediated inflammatory responses

Anti-Inflammatory Effect of Piper attenuatum

Piper attenuatum is used as a traditional medicinal plant in India. One of the substances in P. attenuatum has been suggested to have anti-inflammatory effects. However, there is insufficient research about the anti-inflammatory mechanisms of action of P. attenuatum. The effects of P. attenuatum methanol extract (Pa-ME) on the production of inflammatory mediators nitric oxide (NO) and prostaglandin E2 (PGE2), the expression of proinflammatory genes, the translocation level of transcription factors, and intracellular signaling activities were investigated using macrophages. Pa-ME suppressed the production of NO and PGE2 in lipopolysaccharide- (LPS-), pam3CSK4-, and poly(I:C)-stimulated RAW264.7 cells without displaying cytotoxicity. The mRNA expression levels of inducible NO synthase (iNOS) and cyclooxygenase 2 (COX-2) were decreased by Pa-ME. P-ME reduced the translocation of p50/NF-κB and AP-1 (c-Jun and c-Fos), as well as the activity of their upstream enzymes Src, Syk, and TAK1. Immunoprecipitation analysis showed failure of binding between their substrates, phospho- (p-) p85 and p-MKK3/6. p-p85 and p-MKK3/6, which were induced by overexpression of Src, Syk, and TAK1, were also reduced by Pa-ME. Therefore, these results suggest that Pa-ME exerts its anti-inflammatory effects by targeting Src and Syk in the NF-κB signaling pathway and TAK1 in the AP-1 signaling pathway

TRAF6 Mediates IL-1β/LPS-Induced Suppression of TGF-β Signaling through Its Interaction with the Type III TGF-β Receptor

Transforming growth factor-β1 (TGF-β1) is an important anti-inflammatory cytokine that modulates and resolves inflammatory responses. Recent studies have demonstrated that inflammation enhances neoplastic risk and potentiates tumor progression. In the evolution of cancer, pro-inflammatory cytokines such as IL-1β must overcome the anti-inflammatory effects of TGF-β to boost pro-inflammatory responses in epithelial cells. Here we show that IL-1β or Lipopolysaccharide (LPS) suppresses TGF-β-induced anti-inflammatory signaling in a NF-κB-independent manner. TRAF6, a key molecule in IL-1β signaling, mediates this suppressive effect through interaction with the type III TGF-β receptor (TβRIII), which is TGF-β-dependent and requires type I TGF-β receptor (TβRI) kinase activity. TβRI phosphorylates TβRIII at residue S829, which promotes the TRAF6/TβRIII interaction and consequent sequestration of TβRIII from the TβRII/TβRI complex. Our data indicate that IL-1β enhances the pro-inflammatory response by suppressing TGF-βsignaling through TRAF6-mediated sequestration of TβRIII, which may be an important contributor to the early stages of tumor progression

Multi-Faceted Roles of DNAJB Protein in Cancer Metastasis and Clinical Implications

Heat shock proteins (HSPs) are highly conserved molecular chaperones with diverse cellular activities, including protein folding, assembly or disassembly of protein complexes, and maturation process under diverse stress conditions. HSPs also play essential roles in tumorigenesis, metastasis, and therapeutic resistance across cancers. Among them, HSP40s are widely accepted as regulators of HSP70/HSP90 chaperones and an accumulating number of biological functions as molecular chaperones dependent or independent of either of these chaperones. Despite large numbers of HSP40s, little is known about their physiologic roles, specifically in cancer progression. This article summarizes the multi-faceted role of DNAJB proteins as one subclass of the HSP40 family in cancer development and metastasis. Regulation and deregulation of DNAJB proteins at transcriptional, post-transcriptional, and post-translational levels contribute to tumor progression, particularly cancer metastasis. Furthermore, understanding differences in function and regulating mechanism between DNAJB proteins offers a new perspective on tumorigenesis and metastasis to improve therapeutic opportunities for malignant diseases

Connection between inflammation and carcinogenesis in gastrointestinal tract: Focus on TGF-β signaling

Inflammation is a primary defense process against various extracellular stimuli, such as viruses, pathogens, foods, and environmental pollutants. When cells respond to stimuli for short periods of time, it results in acute or physiological inflammation. However, if the stimulation is sustained for longer time or a pathological state occurs, it is known as chronic or pathological inflammation. Several studies have shown that tumorigenesis in the gastrointestinal (GI) tract is closely associated with chronic inflammation, for which abnormal cellular alterations that accompany chronic inflammation such as oxidative stresses, gene mutations, epigenetic changes, and inflammatory cytokines, are shared with carcinogenic processes, which forms a critical cross-link between chronic inflammation and carcinogenesis. Transforming growth factor (TGF)-β is a multi-potent cytokine that plays an important role in regulation of cell growth, apoptosis and differentiation. Most importantly, TGF-β is a strong anti-inflammatory cytokine that regulates the development of effector cells. TGF-β has a suppressive effect on carcinogenesis under normal conditions by inhibiting abnormal cell growth, but on the other hand, many GI cancers originate from uncontrolled cell growth and differentiation by genetic loss of TGF-β signaling molecules or perturbation of TGF-β adaptors. Once a tumor has developed, TGF-β exerts a promoting effect on the tumor itself and stromal cells to enhance cell growth, alter the responsiveness of tumor cells to stimulate invasion and metastasis, and inhibited immune surveillance. Therefore, novel development of therapeutic agents to inhibit TGF-β-induced progression of tumor and to retain its growth inhibitory activities, in addition to anti-inflammatory actions, could be useful in oncology. In this review, we discuss the role of TGF-β in inflammation and carcinogenesis of the GI tract related to abnormal TGF-β signaling

Lower Salinomycin Concentration Increases Apoptotic Detachment in High-Density Cancer Cells

Abstract: The present study identified a novel salinomycin (Sal) sensitization mechanism in cancer. We tested whether Sal reduced proliferation in a high-density population by counting attached cell numbers after Sal treatment. Sal reduced proliferation in high-density cell populations. Longer exposure to Sal further reduced proliferation. Sal concentrations of 0.1 and 5 μM had similar sensitization effects, suggesting that Sal toxicity was minimal with longer exposure to a high-density cell population. The results suggest that Sal can be applied at a relatively low concentration for a longer time to overcome drug-resistant solid tumors. The 0.5 μM Sal treatment resulted in fewer attached cells than that of the 5 μM Sal treatment with a longer exposure. The lower Sal concentration mainly increased the number of easily detachable cells on the surface. In particular, 0.5 μM Sal increased cellular detachment of newly produced daughter cells. The easily-detachable cells were undergoing apoptosis. It seems that the 0.5 μM Sal treatment also increased cellular toxicity. These novel findings may contribute to the development of Sal-based therapy for patients with drug-resistant cancer or a high-density solid tumor. Int. J. Mol. Sci. 2012, 13 1317

SMAD4 deficiency leads to development of arteriovenous malformations in neonatal and adult mice

© 2018 The Authors. Background—Hereditary hemorrhagic telangiectasia (HHT) is a rare genetic vascular disorder caused by mutations in endoglin (ENG), activin receptor-like kinase 1 (ACVRL1; ALK1), or SMAD4. Major clinical symptoms of HHT are arteriovenous malformations (AVMs) found in the brain, lungs, visceral organs, and mucosal surface. Animal models harboring mutations in Eng or Alk1 recapitulate all of these HHT clinical symptoms and have been useful resources for studying mechanisms and testing potential drugs. However, animal models representing SMAD4 mutations have been lacking. The goal of this study is to evaluate Smad4-inducible knockout (iKO) mice as an animal model of HHT and compare the phenotypes with other established HHT animal models. Methods and Results—Global Smad4 deletion was induced at neonatal and adult stages, and hemoglobin levels, gastrointestinal hemorrhage, and presence of aberrant arteriovenous connections were examined. Neonatal Smad4-iKO mice exhibited signs of gastrointestinal bleeding and AVMs in the brain, intestine, nose, and retina. The radial expansion was decreased, and AVMs were detected on both distal and proximal retinal vasculature of Smad4-iKOs. Aberrant smooth muscle actin staining was observed in the initial stage AVMs and their connecting veins, indicating abnormal arterial flow to veins. In adult mice, Smad4 deficiency caused gastrointestinal bleeding and AVMs along the gastrointestinal tract and wounded skin. HHT-related phenotypes of Smad4-iKOs appeared to be comparable with those found in Alk1-iKO and Eng-iKO mice. Conclusions—These data further confirm that SMAD signaling is crucial for normal arteriovenous network formation, and Smad4-iKO will be an alternative animal model of AVM research associated with HHT

Identification and characterization of D410E, a novel mutation in the loop 3 domain of CaSR, in autosomal dominant hypocalcemia and therapeutic approaches with novel calcilytics, AXT914

Background: Autosomal dominant hypocalcemia (ADH) is a rare disorder characterized by benign hypocalcemia, inappropriately low parathyroid hormone (PTH) levels and mostly hypercalciuria. ADH can be caused by activating mutations of the calcium-sensing receptor (CaSR) gene located on chromosome 3q13.3-21. Calcium-sensing receptor plays a pivotal role in the regulation of calcium homeostasis and is abundantly expressed in parathyroid gland, thyroid C cell, and renal tubular system. Activation of CaSR by increased Ca2+ inhibits PTH secretion, stimulates calcitonin secretion, and promotes urinary Ca2+ excretion, and thereby maintains the Ca2+ at the normal level. Herein, we report a novel activating mutation in the CaSR gene in a Korean family with ADH. Meanwhile, antagonist of the CaSR, calcilytics could be a therapeutic option in the treatment of ADH. Method: We identified a 55-yr-old woman with mild hypocalcemia (7.7 mg/dL) and hypercalciuria (24-hr urine Ca: 868 mg/d) caused by missense mutations of the CaSR gene. She showed low normal serum PTH level (10.14 pg/mL). We performed mutational analysis of the genes encoding GCMB, pre-pro-PTH and CaSR using PCR-amplified genomic DNA in her family members. The ability of wild-type and mutant CaSR to activate the MAPK signaling cascade was assessed by examining phosphorylation of ERK1/2. Intacellular Ca2+ concentration was measured by Fura-2 dye. Blocking of CaSR with calcilytics, AXT914 was also tested by Fura-2 with a variety of concentrations. Result: Direct sequencing analysis of the CaSR gene showed that the proband and her daughter possess a T to A transition at nucleotide 1230 resulting in a D410E missense mutation in exon 4 of the CaSR. No mutation was detected in GCMB and Prepro-PTH genes. HEK293 cells were stably transfected with plasmids encoding wild-type or mutant CaSR genes. Escalation of the extracellular Ca2+ concentration from 0.5 to 5.0 mM resulted in increased phosphorylation of ERK1/2 and escalation of the extracellular Ca2+ concentration from 1.0 to 10 mM resulted in increased intracellular Ca2+ detected by Fura-2 in mutant CaSR-expressing cell than wild-type-expressing one. These results indicate that D410E mutation of CaSR is associated with ADH in this family. Finally, AXT914 successively blunted the increased intracellular signaling via CaSR. Conclusion: Over 60 activating mutations in the CaSR gene have been identified to cause ADH so far. Here we add one more activating mutation that causes ADH. This could be of interest because this novel mutation occurred in the loop 3 region of the VFT domain in CaSR where little was known to be important in its function. Further clinical study is needed to validate the effectiveness of calcilytics in the treatment of ADH in vivo