MicroRNA-3614 regulates inflammatory response via targeting TRAF6-mediated MAPKs and NF-κB signaling in the epicardial adipose tissue with coronary artery disease.

OBJECTIVE: The inflammatory status of epicardial adipose tissue (EAT) is one of the factors leading to the development of related diseases such as coronary artery disease (CAD). The thickness of CAD EAT increases and is accompanied with increased macrophage infiltration and heightened inflammatory responses. However, microRNAs (miRNAs) regulating the inflammatory responses of macrophages in CAD EAT remain unclear. METHOD: miRNA expression profiles of CAD EATs and non-CAD EATs were determined by miRNA microarrays. Quantitative real-time reverse transcription-polymerase chain reaction, Western blotting, immunohistochemical assay, and fluorescence in-situ hybridization were adopted to detect miR-3614 expression and function in EATs and macrophages. The interaction between miR-3614 and tumor necrosis factor receptor-associated factor 6 (TRAF6) was identified using an online website combined with a dual-luciferase reporter assay. Enzyme-linked immunosorbent assay was performed to detect the expression of inflammatory cytokines. RESULTS: The decreased expression of miR-3614 was identified in CAD EAT. The level of miR-3614 was down-regulated by lipopolysaccharide (LPS) in macrophages, whereas LPS-induced inflammatory injury can be reduced by miR-3614 overexpression. TRAF6 was predicted and verified to be a target of miR-3614. The phosphorylated levels of kinases in the mitogen-activated protein kinase (MAPK) and nuclear factor (NF)-κB pathways were inhibited by miR-3614 overexpression. Importantly, the knockdown of TRAF6 inhibited the LPS-induced inflammatory cytokine expressions in cells. CONCLUSION: A novel negative feedback loop by miR-3614 possibly contribute to the regulation of inflammatory processes via targeting the TRAF6/MAPK/NF-κB pathway in EATs and prevents an overwhelming inflammatory response.

Next-Generation Sequencing of Synchronous Multiple Primary Lung Cancers in a Patient with Squamous Cell Carcinoma and Small Cell Lung Cancer.

The incidence of synchronous multiple primary malignancies is low. The presence of different lung tumor types in one patient is rare. Here, we report a rare case of synchronous lung squamous cell cancer and small cell lung cancer in a 60-year-old man. Because of the presence of two different tumor types, the proper treatment must be determined. To identify treatment targets, the genetic features of primary tumor tissues from the lungs were analyzed by next-generation sequencing (NGS). The objective was to analyze the origin and evolution of multiple primary lung cancers. NGS can find the genetic mutation sites of patients to guide treatment and promote the advancement of precision medicine. The effects of standard treatments were evaluated by response evaluation criteria in solid tumors. The results suggest that early treatment of synchronous multiple primary malignancies is a favorable outcome.

A chirality-dependent action of vitamin C in suppressing Kirsten rat sarcoma mutant tumor growth by the oxidative combination: Rationale for cancer therapeutics.

Kirsten rat sarcoma (KRAS) mutant cancers, which constitute the vast majority of pancreatic tumors, are characterized by their resistance to established therapies and high mortality rates. Here, we developed a novel and extremely effective combinational therapeutic approach to target KRAS mutant tumors through the generation of a cytotoxic oxidative stress. At high concentrations, vitamin C (VC) is known to provoke oxidative stress and selectively kill KRAS mutant cancer cells, although its effects are limited when it is given as monotherapy. We found that the combination of VC and the oxidizing drug arsenic trioxide (ATO) is an effective therapeutic treatment modality. Remarkably, its efficiency is dependent on chirality of VC as its enantiomer d-optical isomer of VC (d-VC) is significantly more potent than the natural l-optical isomer of VC. Thus, our results demonstrate that the oxidizing combination of ATO and d-VC is a promising approach for the treatment of KRAS mutant human cancers.

One new 19-nor cucurbitane-type triterpenoid from the stems of Momordica charantia.

One new 19-nor cucurbitane-type triterpenoid (3ß,9ß,25-trihydroxy-7ß-methoxy-19-nor-cucurbita-5,23(E)-diene) (1), together with other six known cucurbitane-type triterpenoids (2-7), were isolated from the stems of Momordica charantia L. The chemical structure of 1 was elucidated by extensive 1D NMR and 2D NMR (HSQC, HMBC, COSY and ROESY), MS experiments. Using MTT assay, compound 1 exhibited weak cytotoxicity against HL-60, A-549, and SK-BR-3 cell lines with the IC50 values at 27.3, 32.7 and 26.6 µM, respectively.

Three new iridoids from leaves of Cornus officinalis.

Three new iridoids, cornifins A-C (1-3), together with a known iridoid, were obtained from EtOAc layer of leaves of Cornus officinalis. The structures of new compounds were elucidated on the basis of extensive spectroscopic analyses. Compound 2 showed weak inhibitory activity against lung cancer cell line A-549 with IC50 value of 29.1 µM.

HFE interacts with the BMP type I receptor ALK3 to regulate hepcidin expression.

Mutations in HFE are the most common cause of hereditary hemochromatosis (HH). HFE mutations result in reduced expression of hepcidin, a hepatic hormone, which negatively regulates iron absorption from the duodenum and iron release from macrophages. However, the mechanism by which HFE regulates hepcidin expression in hepatocytes is not well understood. It is known that the bone morphogenetic protein (BMP) pathway plays a central role in controlling hepcidin expression in the liver. Here we show that HFE overexpression increased Smad1/5/8 phosphorylation and hepcidin expression, whereas inhibition of BMP signaling abolished HFE-induced hepcidin expression in Hep3B cells. HFE was found to associate with ALK3, inhibiting ALK3 ubiquitination and proteasomal degradation and increasing ALK3 protein expression and accumulation on the cell surface. The 2 HFE mutants associated with HH, HFE C282Y and HFE H63D, regulated ALK3 protein ubiquitination and trafficking differently, but both failed to increase ALK3 cell-surface expression. Deletion of Hfe in mice resulted in a decrease in hepatic ALK3 protein expression. Our results provide evidence that HFE induces hepcidin expression via the BMP pathway: HFE interacts with ALK3 to stabilize ALK3 protein and increase ALK3 expression at the cell surface.

Hepcidin regulation by BMP signaling in macrophages is lipopolysaccharide dependent.

Hepcidin is an antimicrobial peptide, which also negatively regulates iron in circulation by controlling iron absorption from dietary sources and iron release from macrophages. Hepcidin is synthesized mainly in the liver, where hepcidin is regulated by iron loading, inflammation and hypoxia. Recently, we have demonstrated that bone morphogenetic protein (BMP)-hemojuvelin (HJV)-SMAD signaling is central for hepcidin regulation in hepatocytes. Hepcidin is also expressed by macrophages. Studies have shown that hepcidin expression by macrophages increases following bacterial infection, and that hepcidin decreases iron release from macrophages in an autocrine and/or paracrine manner. Although previous studies have shown that lipopolysaccharide (LPS) can induce hepcidin expression in macrophages, whether hepcidin is also regulated by BMPs in macrophages is still unknown. Therefore, we examined the effects of BMP signaling on hepcidin expression in RAW 264.7 and J774 macrophage cell lines, and in primary peritoneal macrophages. We found that BMP4 or BMP6 alone did not have any effect on hepcidin expression in macrophages although they stimulated Smad1/5/8 phosphorylation and Id1 expression. In the presence of LPS, however, BMP4 and BMP6 were able to stimulate hepcidin expression in macrophages, and this stimulation was abolished by the NF-κB inhibitor Ro1069920. These results suggest that hepcidin expression is regulated differently in macrophages than in hepatocytes, and that BMPs regulate hepcidin expression in macrophages in a LPS-NF-κB dependent manner.

Human ether-à-go-go gene potassium channels are regulated by EGFR tyrosine kinase.

Human ether á-go-go gene potassium channels (hEAG1 or Kv10.1) are expressed in brain and various human cancers and play a role in neuronal excitement and tumor progression. However, the functional regulation of hEAG channels by signal transduction is not fully understood. The present study was therefore designed to investigate whether hEAG1 channels are regulated by protein tyrosine kinases (PTKs) in HEK 293 cells stably expressing hEAG1 gene using whole-cell patch voltage-clamp, immunoprecipitation, Western blot, and mutagenesis approaches. We found that the selective epidermal growth factor receptor (EGFR) kinase inhibitor AG556 (10 µM), but not the platelet growth factor receptor (PDGFR) kinase inhibitor AG1295 (10 µM) or the Src-family inhibitor PP2 (10 µM), can inhibit hEAG1 current, and the inhibitory effect can be reversed by the protein tyrosine phosphatase (PTP) inhibitor orthovanadate. Immunoprecipitation and Western blot analysis revealed that tyrosine phosphorylation level of hEAG1 channels was reduced by AG556, and the reduction was significantly countered by orthovanadate. The hEAG1 mutants Y90A, Y344A and Y485A, but not Y376A and Y479A, exhibited reduced response to AG556. Interestingly, the inhibition effect of AG556 was lost in triple mutant hEAG1 channels at Y90, Y344, and Y485 with alanine. These results demonstrate for the first time that hEAG1 channel activity is regulated by EGFR kinase at the tyrosine residues Tyr90, Try344, and Try485. This effect is likely involved in regulating neuronal activity and/or tumor growth.

The adaptor function of TRAPPC2 in mammalian TRAPPs explains TRAPPC2-associated SEDT and TRAPPC9-associated congenital intellectual disability.

BACKGROUND: The TRAPP (Transport protein particle) complex is a conserved protein complex functioning at various steps in vesicle transport. Although yeast has three functionally and structurally distinct forms, TRAPPI, II and III, emerging evidence suggests that mammalian TRAPP complex may be different. Mutations in the TRAPP complex subunit 2 (TRAPPC2) cause X-linked spondyloepiphyseal dysplasia tarda, while mutations in the TRAPP complex subunit 9 (TRAPPC9) cause postnatal mental retardation with microcephaly. The structural interplay between these subunits found in mammalian equivalent of TRAPPI and those specific to TRAPPII and TRAPPIII remains largely unknown and we undertook the present study to examine the interaction between these subunits. Here, we reveal that the mammalian equivalent of the TRAPPII complex is structurally distinct from the yeast counterpart thus leading to insight into mechanism of disease. PRINCIPAL FINDINGS: We analyzed how TRAPPII- or TRAPPIII- specific subunits interact with the six-subunit core complex of TRAPP by co-immunoprecipitation in mammalian cells. TRAPPC2 binds to TRAPPII-specific subunit TRAPPC9, which in turn binds to TRAPPC10. Unexpectedly, TRAPPC2 can also bind to the putative TRAPPIII-specific subunit, TRAPPC8. Endogenous TRAPPC9-positive TRAPPII complex does not contain TRAPPC8, suggesting that TRAPPC2 binds to either TRAPPC9 or TRAPPC8 during the formation of the mammalian equivalents of TRAPPII or TRAPPIII, respectively. Therefore, TRAPPC2 serves as an adaptor for the formation of these complexes. A disease-causing mutation of TRAPPC2, D47Y, failed to interact with either TRAPPC9 or TRAPPC8, suggesting that aspartate 47 in TRAPPC2 is at or near the site of interaction with TRAPPC9 or TRAPPC8, mediating the formation of TRAPPII and/or TRAPPIII. Furthermore, disease-causing deletional mutants of TRAPPC9 all failed to interact with TRAPPC2 and TRAPPC10. CONCLUSIONS: TRAPPC2 serves as an adaptor for the formation of TRAPPII or TRAPPIII in mammalian cells. The mammalian equivalent of TRAPPII is likely different from the yeast TRAPPII structurally.