Emerging Drugs in the Treatment of Inflammatory Bowel Disease: Beyond Anti-TNF-alpha / 대한소화기학회지

Understanding of the pathophysiology of inflammatory bowel disease (IBD) is constantly evolving and, recently, a number of biologic agents have been developed. They selectively target specific molecule or pathways and correct the imbalance of the gut immune system. Among them, antibody to tumor necrosis factor (anti-TNF-alpha) is the first developed drugs, and it dramatically improved the IBD management. However, more than one-third of the patients do not respond to the drugs due to antibody formation. To increase treatment efficacy, enormous effort to develop novel anti-cytokines which can be an alternative to anti-TNF-alpha has been made. They are anti CD4+ T cell cytokine including interleukin (IL)-12/23 and IL-17 blockers, selective anti-adhesion molecule known as natalizumab, vedolizumab and alicaforsen, T-cell proliferation inhibitor, anti-inflammatory cytokine, immune stimulator, growth factor, and mitogen-activated protein kinase inhibitor. The efficacy and safety of each drugs are under investigation. Some drugs reported very promising data, however, others showed disappointing and different results. In addition, most of the trials were done in a very small number of patients, and there is no trial comparing to anti-TNF-alpha. The present paper reviews the action mechanism, short or long term efficacy and safety of variable drugs other than anti-TNF-alpha in IBD.

Methylation of eukaryotic elongation factor 2 induced by basic fibroblast growth factor via mitogen-activated protein kinase

Protein arginine methylation is important for a variety of cellular processes including transcriptional regulation, mRNA splicing, DNA repair, nuclear/cytoplasmic shuttling and various signal transduction pathways. However, the role of arginine methylation in protein biosynthesis and the extracellular signals that control arginine methylation are not fully understood. Basic fibroblast growth factor (bFGF) has been identified as a potent stimulator of myofibroblast dedifferentiation into fibroblasts. We demonstrated that symmetric arginine dimethylation of eukaryotic elongation factor 2 (eEF2) is induced by bFGF without the change in the expression level of eEF2 in mouse embryo fibroblast NIH3T3 cells. The eEF2 methylation is preceded by ras-raf-mitogen-activated protein kinase kinase (MEK)-extracellular signal-regulated kinase (ERK1/2)-p21(Cip/WAF1) activation, and suppressed by the mitogen-activated protein kinase (MAPK) inhibitor PD98059 and p21(Cip/WAF1) short interfering RNA (siRNA). We determined that protein arginine methyltransferase 7 (PRMT7) is responsible for the methylation, and that PRMT5 acts as a coordinator. Collectively, we demonstrated that eEF2, a key factor involved in protein translational elongation is symmetrically arginine-methylated in a reversible manner, being regulated by bFGF through MAPK signaling pathway.

Methylation of eukaryotic elongation factor 2 induced by basic fibroblast growth factor via mitogen-activated protein kinase

Protein arginine methylation is important for a variety of cellular processes including transcriptional regulation, mRNA splicing, DNA repair, nuclear/cytoplasmic shuttling and various signal transduction pathways. However, the role of arginine methylation in protein biosynthesis and the extracellular signals that control arginine methylation are not fully understood. Basic fibroblast growth factor (bFGF) has been identified as a potent stimulator of myofibroblast dedifferentiation into fibroblasts. We demonstrated that symmetric arginine dimethylation of eukaryotic elongation factor 2 (eEF2) is induced by bFGF without the change in the expression level of eEF2 in mouse embryo fibroblast NIH3T3 cells. The eEF2 methylation is preceded by ras-raf-mitogen-activated protein kinase kinase (MEK)-extracellular signal-regulated kinase (ERK1/2)-p21(Cip/WAF1) activation, and suppressed by the mitogen-activated protein kinase (MAPK) inhibitor PD98059 and p21(Cip/WAF1) short interfering RNA (siRNA). We determined that protein arginine methyltransferase 7 (PRMT7) is responsible for the methylation, and that PRMT5 acts as a coordinator. Collectively, we demonstrated that eEF2, a key factor involved in protein translational elongation is symmetrically arginine-methylated in a reversible manner, being regulated by bFGF through MAPK signaling pathway.

Reactive oxygen species regulate the generation of urokinase plasminogen activator in human hepatoma cells via MAPK pathways after treatment with hepatocyte growth factor

Tumor cells are known to produce larger amounts of reactive oxygen species (ROS) than normal cells. Although numerous reports have indicated the importance of ROS in urokinase plasminogen activator (uPA) production, the precise mechanisms remain controversial. In our study, we investigated the effect of ROS on uPA generation in human hepatoma cells, HepG2 and Hep 3B. We determined the effects of hepatocyte growth factor (HGF) on the regulation of ROS, which resulted in suppression of ROS production, as measured with the fluorescent probe, 2'-7'-dichlorofluorescein diacetate. The role of HGF in modulating ROS production, particularly that regulated by Rac-1, was determined. HGF suppressed the increment in Rac-1-regulated ROS in both cell lines. Treatment with 200 microM of H2O2 showed a 1.6-2.1 fold increment in HGF, but a little increment occurred at 500 microM of H2O2. It looks no dose dependent manner. Combined treatment with H2O2 and HGF, resulted in a slightly increased production of HGF compared to no treatment (control). Also, H2O2 upregulated uPA expression in both hepatoma cell lines. To identify the downstream pathways regulated by ROS, we treated cells with PD 98059, an MEK inhibitor, and SB 203580, a p38 inhibitor, after treatment with H2O2, and showed negative control between ERK and p38 kinase activities for uPA regulation. We found that HGF modulate Rac-1-regulated ROS production through activation of Akt and ROS regulates uPA production via MAP kinase, which provides a novel clue to clarify the mechanism underlying hepatoma progression.

Regulation of hepatocyte growth factor-mediated urokinase plasminogen activator secretion by MEK/ERK activation in human stomach cancer cell lines

The regulatory mechanisms for the proliferation and the particular invasive phenotypes of stomach cancers are not still fully understood. Up-regulations of hepatocytes growth factor (HGF), its receptor (c-Met), and urokinase-type plasminogen activator (uPA) are correlated with the development and metastasis of cancers. In order to investigate roles of HGF/c-Met signaling in tumor progression and metastasis in stomach cancers, we determined effects of a specific MEK1 inhibitor (PD098059) and a p38 kinase inhibitor (SB203580) on HGF-mediated cell proliferation and uPA expression in stomach cancer cell lines (NUGC-3 and MKN-28). HGF treatment induced the phosphorylations of ERK and p38 kinase in time- and dose- dependent manners. Pre-treatment with PD098059 reduced HGF-mediated cell proliferation and uPA secretion. In contrast, SB203580 pre-treatment enhanced cell proliferation and uPA secretion due to induction of ERK phosphorylation. Stable expression of dominant negative-MEK1 in NUGC-3 cells showed a decrease in HGF-mediated uPA secretion. These results suggest that interaction of a MEK/ERK and a p38 kinase might play an important role in proliferation and invasiveness of stomach cancer cells.

Epigallocatechin-3-gallate Suppresses Galactose-alpha1,4-galactose-beta1,4-glucose Ceramide Expression in TNF-alpha Stimulated Human Intestinal Epithelial Cells Through Inhibition of MAPKs and NF-kappa B

Intestinal epithelial cells (IECs) have been known to produce galactose-alpha1,4-galactose-beta1,4-glucose ceramide (Gb3) that play an important role in the mucosal immune response. The regulation of Gb3 is important to prevent tissue damage causing shiga like toxin. Epigallocatechin-3-gallate (EGCG) has been studied as anti-carcinogenic, anti-oxidant, anti-angiogenic, and anti-viral activities, and anti-diabetic. However, little is known between the expressions of Gb3 on IECs. The aim of this study was to examine the inhibitory effect of EGCG, a major ingredient of green tea, on Gb3 production via mitogen-activated protein kinases (MAPKs) and nuclear factor-kappa B (NF-kappa B) in the TNF-alpha stimulated human colon epithelial cells, HT29. To investigate how Gb3 is regulated, ceramide glucosyltransferase (CGT), lactosylceramide synthase (GalT2), and Gb3 synthase (GalT6) were analyzed by RT-PCR in HT 29 cells exposed to TNF-alpha in the presence or absence of EGCG. EGCG dose-dependently manner, inhibits TNF-alpha induced Gb3 expression by blocking in both the MAPKs and NF-kappaB pathways in HT29 cells. TNF-alpha enhanced CGT, GalT2 and GalT6 mRNA levels and EGCG suppressed the level of these enzymes enhanced by TNF-alpha treatment.

Neurotoxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin in cerebellar granule cells

An environmental pollutant, tetrachloro dibenzo dioxin (TCDD) is known to illicit the cognitive disability and motor dysfunction in the developing brain. TCDD induced effects leading to neurodevelopmental and neurobehavioral deficit may have been defined, however underlying molecular mechanism and possible intracellular targets remain to be elucidated. In this study, we attempted to analyze TCDD-induced neurotoxic effects in the granule cells from cerebellum where certain cognitive abilities and motor function command are known to be excuted. [3H]PDBu, (phorbol 12,13-dibutyrate) binding assay indicated that TCDD induced a dose-dependent increase of total PKC activity and its induction was the aryl hydrocarbon receptor (AhR) dependent and N-methyl-D-aspartate receptor (NMDAR) independent. TCDD also caused the translocation of both PKC-alpha and -epsilon in a dose-dependent manner but associated with different receptors; PKC-alpha via AhR but not PKC-epsilon indicating an isozyme-specific pattern of the induction. Increase of the ROS formation was also observed in the cells treated with TCDD in a dose-dependent and an AhR-dependent manner. The treatment of the cells with the diamino dicyano-bis(2-aminophenylthio) butadiene (U0126, MEK-1/2 inhibitor), dizocilpine maleate (MK-801, non-competitive N-methyl-D-aspartate glutamate receptor antagonist) and vitamin E attenuated the TCDD-induced ROS production indicating that TCDD-induced ROS formation may be associated with activation of ERK-1/2 in the MAP kinase pathway or the NMDA receptor. TCDD also increased [Ca2+]i, which is associated with ROS formation and PKC activation in the cerebellar granule cells. It is suggested that TCDD activates the NMDA receptor, which may induce a sustained increase of [Ca2+]i in neurons followed by the ROS formation. Our findings may contribute to understanding the mechanism of TCDD-related neurotoxicity, thereby improving the health risk assessment of neurotoxic compounds in humans.

Dysferlin in a hyperCKaemic patient with caveolin 3 mutation and in C2C12 cells after p38 MAP kinase inhibition

Dysferlin is a plasma membrane protein of skeletal muscle whose deficiency causes Miyoshi myopathy, limb girdle muscular dystrophy 2B and distal anterior compartment myopathy. Recent studies have reported that dysferlin is implicated in membrane repair mechanism and coimmunoprecipitates with caveolin 3 in human skeletal muscle. Caveolin 3 is a principal structural protein of caveolae membrane domains in striated muscle cells and cardiac myocytes. Mutations of caveolin 3 gene (CAV3) cause different diseases and where caveolin 3 expression is defective, dysferlin localization is abnormal. We describe the alteration of dysferlin expression and localization in skeletal muscle from a patient with raised serum creatine kinase (hyperCKaemia), whose reduction of caveolin 3 is caused by a CAV3 P28L mutation. Moreover, we performed a study on dysferlin interaction with caveolin 3 in C2C12 cells. We show the association of dysferlin to cellular membrane of C2C12 myotubes and the low affinity link between dysferlin and caveolin 3 by immunoprecipitation techniques. We also reproduced caveolinopathy conditions in C2C12 cells by a selective p38 MAP kinase inhibition with SB203580, which blocks the expression of caveolin 3. In this model, myoblasts do not fuse into myotubes and we found that dysferlin expression is reduced. These results underline the importance of dysferlin-caveolin 3 relationship for skeletal muscle integrity and propose a cellular model to clarify the dysferlin alteration mechanisms in caveolinopathies.

Expression and Regulation of Endothelial Nitric Oxide Synthase by Vascular Endothelial Growth Factor in ECV 304 Cells

Nitric oxide (NO) seems to play a pivotal role in the vascular endothelial growth factor (VEGF)-induced endothelial cell proliferation. This study was designed to investigate the role and intracellular signal pathway of endothelial nitric oxide synthase (eNOS) activation induced by VEGF. ECV 304 cells were treated with betaVEGF(165) and then cell proliferation, eNOS protein and mRNA expression levels were analyzed to elucidate the functional role of eNOS in cell proliferation induced by VEGF. After exposure of cells to betaVEGF(165) , eNOS activity and cell growth were increased by approximately two-fold in the betaVEGF(165) -treated cells compared to the untreated cells. In addition, VEGF stimulated eNOS expression at both the mRNA and protein levels in a dose-dependent manner. Phosphatidylinositol-3 kinase (PI-3K) inhibitors were used to assess PI-3K involvement in eNOS regulation. LY294002 was found to attenuate VEGF-stimulated eNOS expression. Wortmannin was not as effective as LY294002, but the reduction effect was detectable. Cells activated by VEGF showed increased ERK1/2 levels. Moreover, the VEGF-induced eNOS expression was reduced by the PD98059, MAPK pathway inhibitor. This suggests that eNOS expression might be regulated by PI-3K and the ERK1/2 signaling pathway. In conclusion, betaVEGF(165) induces ECV 304 cell proliferation via the NO produced by eNOS. In addition, eNOS may be regulated by the PI-3K or mitogen-activated protein kinase pathway.