Contrasting Effects of Cancer-Associated Mutations in EphA3 and EphB2 Kinases.

Erythropoietin-producing hepatoma (Eph) receptors are a family of tyrosine kinases that can act as tumor promoters or tumor suppressors, depending on the receptor and cancer cell type. Cancer-associated somatic mutations have been identified in all Eph receptors, but in most cases, the functional effects of the mutations are unknown. In this study, we expressed and purified the kinase domains of wild-type (WT) EphA3 and EphB2 along with 16 cancer-associated mutants. We identified mutations that decrease EphA3 activity and both activating and inhibitory mutations in EphB2. To shed light on the mechanisms by which the mutations altered kinase activity, we measured the thermal stabilities of the enzymes and performed steady-state kinetic experiments. We also expressed the full-length receptors in HEK293T cells to determine the cellular effects. WT EphB2 promoted downstream ERK signaling, while a kinase-inactive mutant (S706F) was similar to the control cells. In contrast, WT EphA3 (but not loss-of-function mutants) inhibited ERK signaling. The reciprocal effects of EphB2 and EphA3 on ERK phosphorylation in HEK293T cells were also evident in Ras-GTP loading. Thus, consistent with the dual roles of Eph receptors as tumor promoters and tumor suppressors, somatic mutations have the potential to increase or decrease Eph function, resulting in changes in the downstream signaling transduction.

Colorectal cancer-associated mutations impair EphB1 kinase function.

Erythropoietin-producing hepatoma (Eph) receptor tyrosine kinases regulate the migration and adhesion of cells that are required for many developmental processes and adult tissue homeostasis. In the intestinal epithelium, Eph signaling controls the positioning of cell types along the crypt-villus axis. Eph activity can suppress the progression of colorectal cancer (CRC). The most frequently mutated Eph receptor in metastatic CRC is EphB1. However, the functional effects of EphB1 mutations are mostly unknown. We expressed and purified the kinase domains of WT and five cancer-associated mutant EphB1 and developed assays to assess the functional effects of the mutations. Using purified proteins, we determined that CRC-associated mutations reduce the activity and stability of the folded structure of EphB1. By mammalian cell expression, we determined that CRC-associated mutant EphB1 receptors inhibit signal transducer and activator of transcription 3 and extracellular signal-regulated kinases 1 and 2 signaling. In contrast to the WT, the mutant EphB1 receptors are unable to suppress the migration of human CRC cells. The CRC-associated mutations also impair cell compartmentalization in an assay in which EphB1-expressing cells are cocultured with ligand (ephrin B1)-expressing cells. These results suggest that somatic mutations impair the kinase-dependent tumor suppressor function of EphB1 in CRC.

Biochemical Studies of Systemic Lupus Erythematosus-Associated Mutations in Nonreceptor Tyrosine Kinases Ack1 and Brk.

Tyrosine kinases (TKs) play essential roles in signaling processes that regulate cell survival, migration, and proliferation. Dysregulation of tyrosine kinases underlies many disorders, including cancer, cardiovascular and developmental diseases, as well as pathologies of the immune system. Ack1 and Brk are nonreceptor tyrosine kinases (NRTKs) best known for their roles in cancer. Here, we have biochemically characterized novel Ack1 and Brk mutations identified in patients with systemic lupus erythematosus (SLE). These mutations are the first SLE-linked polymorphisms found among NRTKs. We show that two of the mutants are catalytically inactive, while the other three have reduced activity. To understand the structural changes associated with the loss-of-function phenotype, we solved the crystal structure of one of the Ack1 kinase mutants, K161Q. Furthermore, two of the mutated residues (Ack1 A156 and K161) critical for catalytic activity are highly conserved among other TKs, and their substitution in other members of the kinase family could have implications in cancer. In contrast to canonical gain-of-function mutations in TKs observed in many cancers, we report loss-of-function mutations in Ack1 and Brk, highlighting the complexity of TK involvement in human diseases.

Acai berry extract as a regulator of intestinal inflammation pathways in a Caco-2 and RAW 264.7 co-culture model.

The aim of this study was to assess the anti-inflammatory effects of acai berries in a Caco-2 and RAW 264.7 macrophage co-culture model. The acai berry extract (ABE) was prepared using 70% ethanol, and total anthocyanin, polyphenol, and flavonoid contents in ABE were analyzed. To the antioxidant activity of ABE, we measured radical scavenging activity as well as ferric reducing antioxidant power values. Prior to inducing inflammation, Caco-2 cells were co-cultured with RAW 264.7. Inflammation was induced using lipopolysaccharides (LPS) in RAW 264.7 cells. The transepithelial electrical resistance value was significantly recovered and the mRNA level of tight junction proteins, including ZO-1, JAM-1, and claudin-4, tended to increase compared with that in the LPS group. LPS-induced interleukin (IL)-6, IL-8, and prostaglandin E2 levels reduced significantly following treatment with the highest ABE concentration. In the highest ABE concentration, the phosphorylation of p65, p38 mitogen-activated protein kinase, and c-Jun N-terminal kinase was downregulated compared with the LPS group. The phosphorylation of extracellular signal-regulated kinase showed a decreased tendency. These results suggest that acai berry may improve gastrointestinal health. PRACTICAL APPLICATIONS: Acai berry is known to have abundant anthocyanin, which has many biological activities, including anti-inflammatory, antioxidant, antihypertensive, and anticytotoxic/cytoprotective activities. This study demonstrated the anti-inflammatory effects of acai berry extracts via TEER value, expression of tight junction protein, and production of inflammatory mediators and cytokines in the Caco-2 and RAW 264.7 co-culture model. Therefore, acai berry has the potential to prevent intestinal inflammatory diseases.

Mitochondrial DNA alterations underlie an irreversible shift to aerobic glycolysis in fumarate hydratase-deficient renal cancer.

Understanding the mechanisms of the Warburg shift to aerobic glycolysis is critical to defining the metabolic basis of cancer. Hereditary leiomyomatosis and renal cell carcinoma (HLRCC) is an aggressive cancer characterized by biallelic inactivation of the gene encoding the Krebs cycle enzyme fumarate hydratase, an early shift to aerobic glycolysis, and rapid metastasis. We observed impairment of the mitochondrial respiratory chain in tumors from patients with HLRCC. Biochemical and transcriptomic analyses revealed that respiratory chain dysfunction in the tumors was due to loss of expression of mitochondrial DNA (mtDNA)-encoded subunits of respiratory chain complexes, caused by a marked decrease in mtDNA content and increased mtDNA mutations. We demonstrated that accumulation of fumarate in HLRCC tumors inactivated the core factors responsible for replication and proofreading of mtDNA, leading to loss of respiratory chain components, thereby promoting the shift to aerobic glycolysis and disease progression in this prototypic model of glucose-dependent human cancer.

Differential responses of endothelial integrity upon the intake of microencapsulated garlic, tomato extract or a mixture: a single-intake, randomized, double-blind, placebo-controlled crossover trial.

This study investigated the effect of microencapsulated garlic and/or tomato on endothelial dysfunction induced by the PhenFlex test (PFT) in healthy male smokers. In a randomized, double-blind, placebo-controlled crossover trial, 41 healthy male smokers were randomly assigned to one of four groups to receive the test groups (in microencapsulated garlic powder, tomato extract and a mixture thereof) or the placebo group. Proteomic biomarkers related to endothelial integrity were measured in plasma. Microencapsulated garlic, tomato extract and the mixture affected endothelial integrity biomarkers differently. Garlic consumption increased prothrombin time and decreased SAA and IL-12. Tomato extract intake increased activated partial thrombin time and decreased d-dimer, SAA, sVCAM-1, IL-13 and MCP-3 levels. Consumption of the mixture increased sE-selectin and lowered D-dimer, SAA, IL-13 and IL-10 responses after PFT challenge for 6 h. The different responses became clearer under high compliance in the dietary restriction groups. This single-intake clinical trial addressed the different responses of biomarkers related to endothelial integrity.

Ginger Extract Suppresses Inflammatory Response and Maintains Barrier Function in Human Colonic Epithelial Caco-2 Cells Exposed to Inflammatory Mediators.

The beneficial effects of ginger in the management of gastrointestinal disturbances have been reported. In this study, the anti-inflammatory potential of ginger extract was assessed in a cellular model of gut inflammation. In addition, the effects of ginger extract and its major active compounds on intestinal barrier function were evaluated. The response of Caco-2 cells following exposure to a mixture of inflammatory mediators [interleukin [IL]-1ß, 25 ng/mL; lipopolysaccharides [LPS], 10 ng/mL; tumor necrosis factor [TNF]-α, 50 ng/mL; and interferon [INF]-γ, 50 ng/mL] were assessed by measuring the levels of secreted IL-6 and IL-8. In addition, the mRNA levels of cyclooxygenase-2 and inducible nitric oxide synthase were measured. Moreover, the degree of nuclear factor (NF)-κB inhibition was examined, and the intestinal barrier function was determined by measuring the transepithelial electrical resistance (TEER) and fluorescein isothiocyanate (FITC)-dextran transfer. It was observed that ginger extract and its constituents improved inflammatory responses by decreasing the levels of nitrite, PGE2, IL-6, and IL-8 via NF-κB inhibition. The ginger extract also increased the TEER and decreased the transfer of FITC-dextran from the apical side of the epithelium to the basolateral side. Taken together, these results show that ginger extract may be developed as a functional food for the maintenance of gastrointestinal health.

Quercetin-glutamic acid conjugate with a non-hydrolysable linker; a novel scaffold for multidrug resistance reversal agents through inhibition of P-glycoprotein.

Previously, we have reported remarkable effect of a quercetin-glutamic acid conjugate to reverse multidrug resistance (MDR) of cancer cells to a broad spectrum of anticancer agents through inhibition of P-glycoprotein (Pgp)-mediated drug efflux. Due to the hydrolysable nature, MDR-reversal activity of the quercetin conjugate was attributed to its hydrolysis product, quercetin. However, several lines of evidence demonstrated that the intact quercetin-glutamic acid conjugate has stronger MDR-reversal activity than quercetin. In order to evaluate this hypothesis and to identify a novel scaffold for MDR-reversal agents, we prepared quercetin conjugates with a glutamic acid attached at the 7-O position via a non-hydrolysable linker. Pgp inhibition assay, Pgp ATPase assay, and MDR-reversal activity assay were performed, and the non-hydrolysable quercetin conjugates showed significantly higher activities compared with those of quercetin. Unfortunately, the quercetin conjugates were not as effective as verapamil in Pgp-inhibition and thereby reversing MDR, but it is worth to note that the structurally modified quercetin conjugates with a non-cleavable linker showed significantly improved MDR-reversal activity compared with quercetin. Taken together, the quercetin conjugates with appropriate structural modifications were shown to have a potential to serve as a scaffold for the design of novel MDR-reversal agents.

Antioxidant activities of ethanolic and acidic ethanolic extracts of astringent persimmon in H2O2-stimulated Caco-2 human colonic epithelial cells.

The aim of this study was to investigate the protective effects of ethanol (EAP) and acidic ethanol extracts (AEAP) of astringent persimmon fruits (Diospyros kaki) against hydrogen peroxide (H2O2)-induced oxidative stress in Caco-2 epithelial cells. Levels of reactive oxygen species (ROS) and reduced glutathione (GSH), activities of superoxide dismutase (SOD) and catalase (CAT), as well as protein and DNA damage were measured. Treatment with H2O2 induced oxidative stress in Caco-2 cells but had no effect on SOD activity. Both EAP and AEAP protected the cells against oxidative damage and exhibited similar effects on cellular ROS scavenging activity and protein carbonyl levels. However, AEAP was more potent than EAP in normalizing CAT activity and cytosolic GSH level, and in protecting against DNA damage. These results demonstrated that astringent persimmon fruit extracts were biologically active, and that the tannin-rich fraction (AEAP) possesses stronger protective activity against H2O2-induced oxidative stress in Caco-2 cells.

Comparison of digital fundus photographic and echographic measurements for maximal linear dimension from eyes with choroidal melanoma.

PURPOSE: Accurate assessment of tumor size is crucial in the treatment of choroidal melanoma. Digital fundus photographic systems can create a montage that clearly documents the tumor margins. These images can be used to measure tumor maximal linear dimension (MLD). This study evaluates the potential value of a digital imaging system compared with echography in measuring the MLD of choroidal melanoma. METHODS: Echographic measurements were performed in a standardized fashion following Collaborative Ocular Melanoma Study (COMS) methods. For photographic measurements, the tumor margin was outlined with an electronic pen and the MLD were calculated by software (ANKA-ProView, Topcon Medical Systems, Paramus, NJ). RESULTS: The mean MLD before I-125 brachytherapy was 9.32 mm by echography and 10.57 mm by digital photography analysis. This difference was statistically significant (P < 0.001). The 2 measurements of MLD agreed within 1.1 mm for 11 eyes (52%) and 2.2 mm for 20 eyes (95%). Digital photographic analysis estimated a larger MLD in 19 of the 21 eyes compared with echography. CONCLUSION: This discrepancy likely results from the photographic measurement more accurately assessing the flat portion of the tumor. Digital photographic analysis may allow clinicians to determine the MLD of tumors more accurately than echography. This could increase the rate of successful treatment with brachytherapy.

High glucose inhibits gene expression of tyrosyl-tRNA synthetase in osteoblast cells.

It has been suggested that bone metabolism disorders are one of the major complications of diabetes mellitus. However, the exact mechanisms as to how diabetes affects bone metabolism are yet to be determined. In the present study, we have searched for high glucose regulated genes in osteoblast-like UMR-106 cells. UMR-106 cells were treated with normal glucose (5.5 mM), high glucose (16.5 mM or 30.5 mM) and mannitol (16.5 mM) as a hyperosmotic control. Following the isolation of total RNA, GeneFishing differential display-PCR (DDPCR) was carried out and followed by cloning, sequencing and searching in a gene bank data base to identify the high glucose induced gene(s). Through the DD-PCR technique which employs Annealing Control Primer, or ACP, it has been found that expression of a PCR product was significantly decreased by high glucose treatment: it was identified as tyrosyl-tRNA synthetase. Furthermore, reverse transcriptase PCR analysis confirmed that high glucose significantly decreases mRNA expression of tyrosyl-tRNA synthetase, whereas mannitol treatment does not cause any change in such expression. These results suggest that high glucose may play a significant role in the protein synthesis process of osteoblast cells by decreasing expression of tyrosyl-tRNA synthetase. In a Western blot analysis, the protein expression of tyrosyl-tRNA synthetase was also decreased by high glucose treatment. Taken together, these results suggest that high glucose could affect bone metabolism by regulating the expression of tyrosyl-tRNA synthetase genes.