Isorhamnetin Inhibits Proliferation and Invasion and Induces Apoptosis through the Modulation of Peroxisome Proliferator-activated Receptor γ Activation Pathway in Gastric Cancer

Gastric cancer (GC) is a lethal malignancy and the second most common cause of cancer-related deaths. Although treatment options such as chemotherapy, radiotherapy, and surgery have led to a decline in the mortality rate due to GC, chemoresistance remains as one of the major causes for poor prognosis and high recurrence rate. In this study, we investigated the potential effects of isorhamnetin (IH), a 3′-O-methylated metabolite of quercetin on the peroxisome proliferator-activated receptor γ (PPAR-γ) signaling cascade using proteomics technology platform, GC cell lines, and xenograft mice model. We observed that IH exerted a strong antiproliferative effect and increased cytotoxicity in combination with chemotherapeutic drugs. IH also inhibited the migratory/invasive properties of GC cells, which could be reversed in the presence of PPAR-γ inhibitor. We found that IH increased PPAR-γ activity and modulated the expression of PPAR-γ regulated genes in GC cells. Also, the increase in PPAR-γ activity was reversed in the presence of PPAR-γ-specific inhibitor and a mutated PPAR-γ dominant negative plasmid, supporting our hypothesis that IH can act as a ligand of PPAR-γ. Using molecular docking analysis, we demonstrate that IH formed interactions with seven polar residues and six nonpolar residues within the ligand-binding pocket of PPAR-γ that are reported to be critical for its activity and could competitively bind to PPAR-γ. IH significantly increased the expression of PPAR-γ in tumor tissues obtained from xenograft model of GC. Overall, our findings clearly indicate that antitumor effects of IH may be mediated through modulation of the PPAR-γ activation pathway in GC.     

Phosphorylation of the gastric tumor suppressor RUNX3 following H. pylori infection results in its localization to the cytoplasm

As H. pylori infection progresses, intestinal metaplasia (IM), a key event in gastric carcinogenesis, develops in the stomach. The mechanism by which H. pylori infection causes the trans-differentiation of gastric cells to intestinal-type cells remains an important question. In the current study, we found that RUNX3 is deregulated in all human IM specimens examined by either down regulation or mislocalization; Aberrant localization of a gastric tumor suppressor RUNX3 is observed in most human cases of IM with concurrent H. pylori infection, and RUNX3 is down-regulated in most cases of IM without H. pylori-infection. The cytoplasmic mislocalization of a RUNX3 was associated with H. pylori-induced c-Src activation and RUNX tyrosine phosphorylation. Moreover, gastric epithelial cells of Runx3(-/-) mice expressed the intestinal markers Muc2 and Li-Cadherin, which suggests that the deregulation of Runx3 is a key event in the intestinalization of the gastric epithelium. Collectively, the results of the current study suggest that RUNX3 deregulation is associated with H. pylori-induced pathogenesis and the development of IM.     

Human nuclear clusterin mediates apoptosis by interacting with Bcl-XL through C-terminal coiled coil domain

Clusterin (CLU), a glycoprotein, is involved in apoptosis, producing two alternatively spliced isoforms in various cell types. The pro-apoptotic CLU appears to be a nuclear isoform (nuclear clusterin; nCLU), and the secretory CLU (sCLU) is thought to be anti-apoptotic. The detailed molecular mechanism of nCLU as a pro-apoptotic molecule has not yet been clear. In the current study, overexpressed nCLU induced apoptosis in human kidney cells. Biochemical studies revealed that nCLU sequestered Bcl-XL via a putative BH3 motif in the C-terminal coiled coil (CC2) domain, releasing Bax, and promoted apoptosis accompanied by activation of caspase-3 and cytochrome c release. These results suggest a novel mechanism of apoptosis mediated by nCLU as a pro-apoptotic molecule.     

Structure of solid-supported lipid membrane probed by noble metal nanoparticle deposition

Direct deposition of a noble metal layer onto a solid-supported membrane was proposed as an indirect microscopy tool to visually observe different lipid phases that may develop in the lipid membrane. The method relied on the different permeability of the lipid membrane towards the incident atoms during deposition. Liquid state or structural defects such as phase boundaries, step ledges in a multi-lamellar stack, and pores permitted the metal atoms to penetrate and nucleate inside the membrane whereas rigid gel state was relatively impermeable to the incident atoms, thus enabling visualization of liquid phase or structural defects inside the gel state. Based on the proposed method, we demonstrated the phase states resulting from thermotropic transitions of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), dioleoylphosphatidylethanolamine (DOPE)/DPPC mixture, and 1,2-dioleoyl-3-trimethylammonium propane (DOTAP). Although the proposed method does not allow in-situ observation of equilibrium states, the method should be an excellent complementary tool for visualizing the lipid phases as the method can resolve fine structural details (up to tens of nanometer scale) as seen in the DPPC membrane while providing macroscopic images (up to several micrometers).     

The Ras-ERK MAPK regulatory network controls dedifferentiation in Caenorhabditis elegans germline

How a committed cell can be reverted to an undifferentiated state is a central question in stem cell biology. This process, called dedifferentiation, is likely to be important for replacing stem cells as they age or get damaged. Tremendous progress has been made in understanding this fundamental process, but its mechanisms are poorly understood. Here we demonstrate that the aberrant activation of Ras-ERK MAPK signaling promotes cellular dedifferentiation in the Caenorhabditis elegans germline. To activate signaling, we removed two negative regulators, the PUF-8 RNA-binding protein and LIP-1 dual specificity phosphatase. The removal of both of these two regulators caused secondary spermatocytes to dedifferentiate and begin mitotic divisions. Interestingly, reduction of Ras-ERK MAPK signaling, either by mutation or chemical inhibition, blocked the initiation of dedifferentiation. By RNAi screening, we identified RSKN-1/P90(RSK) as a downstream effector of MPK-1/ERK that is critical for dedifferentiation: rskn-1 RNAi suppressed spermatocyte dedifferentiation and instead induced meiotic divisions. These regulators are broadly conserved, suggesting that similar molecular circuitry may control cellular dedifferentiation in other organisms, including humans.     

Involvement of GDH3-encoded NADP+-dependent Glutamate Dehydrogenase in Yeast Cell Resistance to Stress-induced Apoptosis in Stationary Phase Cells

Glutamate metabolism is linked to a number of fundamental metabolic pathways such as amino acid metabolism, the TCA cycle, and glutathione (GSH) synthesis. In the yeast Saccharomyces cerevisiae, glutamate is synthesized from α-ketoglutarate by two NADP⁺-dependent glutamate dehydrogenases (NADP-GDH) encoded by GDH1 and GDH3. Here, we report the relationship between the function of the NADP-GDH and stress-induced apoptosis. Gdh3-null cells showed accelerated chronological aging and hypersusceptibility to thermal and oxidative stress during stationary phase. Upon exposure to oxidative stress, Gdh3-null strains displayed a rapid loss in viability associated with typical apoptotic hallmarks, i.e. reactive oxygen species accumulation, nuclear fragmentation, DNA breakage, and phosphatidylserine translocation. In addition, Gdh3-null cells, but not Gdh1-null cells, had a higher tendency toward GSH depletion and subsequent reactive oxygen species accumulation than did WT cells. GSH depletion was rescued by exogenous GSH or glutamate. The hypersusceptibility of stationary phase Gdh3-null cells to stress-induced apoptosis was suppressed by deletion of GDH2. Promoter swapping and site-directed mutagenesis of GDH1 and GDH3 indicated that the necessity of GDH3 for the resistance to stress-induced apoptosis and chronological aging is due to the stationary phase-specific expression of GDH3 and concurrent degradation of Gdh1 in which the Lys-426 residue plays an essential role.     

Competition between sumoylation and ubiquitination of serine hydroxymethyltransferase 1 determines its nuclear localization and its accumulation in the nucleus

Serine hydroxymethyltransferase 1 (SHMT1) expression limits rates of de novo dTMP synthesis in the nucleus. Here we report that SHMT1 is ubiquitinated at the small ubiquitin-like modifier (SUMO) consensus motif and that ubiquitination at that site is required for SHMT1 degradation. SHMT1 protein levels are cell cycle-regulated, and Ub-SHMT1 levels are lowest at S phase when SHMT1 undergoes SUMO modification and nuclear transport. Mutation of the SUMO consensus motif increases SHMT1 stability. SHMT1 interacts with components of the proteasome in both the nucleus and cytoplasm, indicating that degradation occurs in both compartments. Ubc13-mediated ubiquitination is required for SHMT1 nuclear export and increases stability of SHMT1 within the nucleus, whereas Ubc9-mediated modification with Sumo2/3 is involved in nuclear degradation. These data demonstrate that SUMO and ubiquitin modification of SHMT1 occurs on the same lysine residue and determine the localization and accumulation of SHMT1 in the nucleus.     

TNF-α mediates the stimulation of sclerostin expression in an estrogen-deficient condition

Obesity consists in fat accumulation leading to increase in adipose cells number and size. Adipocyte membrane biophysical properties are critical to maintain cellular viability in metabolically healthy obesity. This study investigated the effect of the genetic background and dietary protein restriction on fat tissue lipid composition, adipocyte membrane fluidity and water permeability using the pig as experimental model. Twenty-four male pigs from distinct genotypes, lean and obese, were fed on normal and reduced protein diets within a 2 × 2 factorial arrangement (two genotypes and two diets). Backfat thickness was twofold higher in obese than in lean pigs but unrelated to dietary protein level. In contrast, total fatty acids in the subcutaneous adipose tissue were dependent on both breed and diet, with increased lipid content promoted by the fatty genotype and by the restriction of dietary protein. Adipose membranes isolated from obese pig's subcutaneous fat tissue showed higher permeability to water, in line with an increased fluidity. Moreover, the reduced content of dietary protein influenced positively the fluidity of adipose membranes. Neither genotype nor diet affected total cholesterol concentration in the adipose membranes. Membrane-saturated fatty acids' content was influenced by genotype, while membrane-polyunsaturated fatty acids, particularly from the n-6 family, was influenced by diet. The ratio of oleic (18:1c9)/linoleic (18:2n-6) acids was positively correlated with membrane fluidity. All together, these findings reinforce the genetic background as a determinant player on adipose membrane biophysical properties and point to the dietary protein level as an important factor for subcutaneous lipid deposition as well as for regulation of membrane function, factors that may have impact on human obesity and metabolic syndrome.