ANGPTL3 negatively regulates IL-1ß-induced NF-κB activation by inhibiting the IL1R1-associated signaling complex assembly.

Interleukin-1ß (IL-1ß)-induced signaling is one of the most important pathways in regulating inflammation and immunity. The assembly of the receptor complex, consisting of the ligand IL-1ß, the IL-1 receptor (IL-1R) type 1 (IL1R1), and the IL-1R accessory protein (IL1RAP), initiates this signaling. However, how the IL1R1-associated complex is regulated remains elusive. Angiopoietin like 3 (ANGPTL3), a key inhibitor of plasma triglyceride clearance, is mainly expressed in the liver and exists in both intracellular and extracellular secreted forms. Presently, ANGPTL3 has emerged as a highly promising drug target for hypertriglyceridemia and associated cardiovascular diseases. However, most studies have focused on the secreted form of ANGPTL3, while its intracellular role is still largely unknown. Here, we report that intracellular ANGPTL3 acts as a negative regulator of IL-1ß-triggered signaling. Overexpression of ANGPTL3 inhibited IL-1ß-induced NF-κB activation and the transcription of inflammatory genes in HepG2, THP1, and HEK293T cells, while knockdown or knockout of ANGPTL3 resulted in opposite effects. Mechanistically, ANGPTL3 interacted with IL1R1 and IL1RAP through its intracellular C-terminal fibrinogen-like domain (FLD) and disrupted the assembly of the IL1R1-associated complex. Taken together, our study reveals a novel role for ANGPTL3 in inflammation, whereby it inhibits the physiological interaction between IL1R1 and IL1RAP to maintain immune tolerance and homeostasis in the liver.

Emerging roles of angiopoietin-like proteins in inflammation: Mechanisms and potential as pharmacological targets.

Angiopoietin-like proteins (ANGPTLs), a family of eight secreted glycoproteins termed ANGTPL1-8, are involved in angiogenesis, lipid metabolism, cancer progression, and inflammation. Their roles in regulating lipid metabolism have been intensively studied, as some ANGPTLs are promising pharmacological targets for hypertriglyceridemia and associated cardiovascular disease. Recently, the emerging roles of ANGPTLs in inflammation have attracted great attention. First, elevated levels of multiple circulating ANGPTLs in inflammatory diseases make them potential disease biomarkers. Second, multiple ANGPTLs regulate acute or chronic inflammation via various mechanisms, including triggering inflammatory signaling through their action as ligands for integrin or forming homo- /hetero-oligomers to regulate signal transduction via extra- or intracellular mechanisms. As dysregulation of the inflammatory response is a critical trigger in many diseases, understanding the roles of ANGPTLs in inflammation will aid in drug/therapy development. Here, we summarize the roles, mechanisms, and potential therapeutic values for ANGPTLs in inflammation and inflammatory diseases.

Id4 Suppresses the Growth and Invasion of Colorectal Cancer HCT116 Cells through CK18-Related Inhibition of AKT and EMT Signaling.

Id4 is one of the inhibitors of DNA-binding proteins (Id) and involved in the pathogenesis of numerous cancers. The specific mechanism underlying the Id4-mediated regulation of proliferation, invasion, and metastasis of colorectal cancer (CRC) cells is still largely unclear. In the present study, results showed CRC cells had a lower baseline Id4 expression than normal intestinal epithelial NCM460 cells. In order to explore the role of Id4 in the tumorigenicity, CRC HCT116 cells with stable Id4 expression were used, and results showed Id4 overexpression arrested the cell cycle at the G0/G1 phase, inhibited the cell proliferation and the colony formation, as well as suppressed the migration and invasion. In the in vivo model, Id4 overexpression inhibited the tumor growth and metastasis in the nude mice. Furthermore, Id4 overexpression upregulated the expression of proteins associated with cell proliferation, inhibited the PI3K/AKT pathway, and suppressed epithelial-mesenchymal transition (EMT) of HCT116 cells. Moreover, Id4 significantly decreased cytokeratin 18 (CK18) expression, but CK18 overexpression in Id4 expressing HCT116-Id4 cells rescued the activation of AKT, p-AKT, MMP2, MMP7, and E-cadherin. Collectively, our study indicated Id4 may inhibit CRC growth and metastasis through inhibiting the PI3K/AKT pathway in a CK18-dependent manner and suppressing EMT. Id4 may become a target for the treatment of CRC.

[Application of serum proteomic mass spectrum analysis in breast cancer].

OBJECTIVE: To analyze the characteristics of serum proteins mass spectra in healthy controls, benign breast tumors, and CA15-3 negative or CA15-3 positive breast cancer patients by surface enhanced laser desorption ionization time of flight mass spectrometry (SELDI-TOF-MS). METHODS: Tissue samples of 113 cases of breast cancer (93 case of CA15-3 negative, 20 case of CA15-3 positive), 103 cases of benign breast tumor and 92 cases of healthy controls were examined and analyzed by SELDI and protein chip (CM10) techniques. Biomarker Pattern Software (BPS) was used to detect the protein peaks significantly different between them and establish a diagnostic pattern which was further evaluated by a blind test. RESULTS: Twelve significantly different protein peaks were found in serum samples between breast cancer patients and healthy controls. Eleven significantly different peaks were found between benign breast tumor patients and healthy controls. By combined analysis of those three different protein mass spectra, the peak 15 952 was found to be significantly different between breast cancer group and healthy controls, and the peak 7985 was significantly different among breast cancer group, benign breast tumor group and health controls. The blind test with the differential proteins for the serum samples of 93 cases of CA15-3 negative breast cancer and 36 cases of benign breast tumors showed that the sensitivity was 80.6% and specificity was 91.7%. The blind test in 20 cases of CA15-3 positive breast cancer and 36 cases of benign breast tumors showed that the sensitivity was 75.0% and specificity was 91.7%. Four significantly different protein peaks were found between the benign breast tumor patients and CA15-3 negative breast cancer patients. No significantly different protein were found between CA15-3 negative and CA15-3 positive patients. CONCLUSION: Significantly different protein peaks can be screened out in breast cancer, benign breast tumor patients and healthy controls by SELDI-TOF-MS analysis.

Simultaneous determination of melamine, ammelide, ammeline, and cyanuric acid in milk and milk products by gas chromatography-tandem mass spectrometry.

OBJECTIVE: To develop an analytical method for simultaneously qualitative and quantitative determination of melamine and triazine-related by-products including ammelide, ammeline, and cyanuric acid in milk and milk products by gas chromatography-tandem mass spectrometry (GC-MS/MS). METHODS: Melamine and triazine-related by-products namely ammelide, ammeline and cyanuric acid in the samples were extracted in a solvent mixture of diethylamine, water, and acetonitrile (10:40:50, V/V/V). After centrifugation, an aliquot of the supernatant was evaporated to dryness under a gentle stream of nitrogen gas, and then melamine and triazine-related by-products were derivatized using BSTFA with 1% TMCS. The derivatives of melamine and its analogues were determined by gas chromatography/tandem mass spectrometry using multiple reactional monitoring (MRM) with 2, 6-Diamino-4-chloropyrimidine (DACP) being used as an internal standard. RESULTS: The linear detectable ranges were from 0.004 mg/kg to 1.6 mg/kg for melamine, ammelide, ammeline, and cyanuric acid with a correlation coefficient no less than 0.999. The recovery rates of the four compounds in spiked blank milk powder at concentrations 0.5, 1, 2 mg/kg were between 61.4%-117.2%, and the relative standard deviation was no more than 11.5% (n=6). The detection limits of melamine, ammelide, ammeline and cyanuric acid in milk powder were 0.002 mg/kg with a ratio of signal to noise of 3. CONCLUSION: This GC-MS/MS method for simultaneous determination of melamine, ammelide, ammeline, and cyanuric acid in milk and milk products is sensitive and specific.

The cytotoxicity of a Grb2-SH3 inhibitor in Bcr-Abl positive K562 cells.

Chronic myelogenous leukemia (CML) is characterized by the presence of Bcr-Abl oncoprotein. Gleevec has been designed to treat many CML patients by specifically targeting Bcr-Abl, but resistance to it is already apparent in many cases. In CML cells, Bcr-Abl activates several signaling pathways, including the Ras-dependent pathway, in which growth factor receptor binding 2 (Grb2) acts as an adaptor protein. A specific Grb2-SH3 inhibitor (denoted as peptidimer-c) that disrupts Grb2-Sos complex was designed and synthesized in our laboratory. In this study, we investigated the effect and the molecular mechanism of this inhibitor. Peptidimer-c was shown to bind to Grb2 in K562 cells, a cell line over-expressing Bcr-Abl oncoprotein. It caused cytotoxicity in the cells, and inhibited their ability of colony formation in the semi-solid medium. It was shown to induce apoptosis of K562 cells in a dose-dependent mode, the apoptotic effect of peptidimer-c being associated with caspase-3 activation. The effect of peptidimer-c on growth inhibition was also shown to be accompanied by S-phase arrest of cell cycle mediated by down-regulation of cyclin A and Cdk2, as well as phospho-Cdk2. The above results indicated that peptidimer-c may be another potential therapeutic agent for CML, which can induce S-phase arrest in the Bcr-Abl positive K562.