Protective Effects of Silibinin on Helicobacter pylori-induced Gastritis: NF-κB and STAT3 as Potential Targets.

More than half of the world's populations are considered to be infected by Helicobacter pylori. It causes a chronic inflammation of the stomach, which is implicated in the pathogenesis of gastric ulcer and cancer. Silibinin, a polyphenolic flavonoid derived from milk thistle, has been known for its hepatoprotective effects, and recent studies have revealed its chemopreventive potential. In the present study, we examined the anti-inflammatory effects of silibinin in human gastric cancer MKN-1 cells and in the stomach of C57BL/6 mice infected by H. pylori. Pretreatment with silibinin attenuated the up-regulation of COX-2 and inducible nitric oxide synthase (iNOS) in H. pylori-infected MKN-1 cells and mouse stomach. In addition, the elevated translocation and DNA binding of NF-κB and STAT3 induced by H. pylori infection were inhibited by silibinin treatment. Moreover, H. pylori infection in combination with high salt diet resulted in dysplasia and hyperplasia in mouse stomach, and these pathological manifestations were substantially mitigated by silibinin administration. Taken together, these findings suggest that silibinin exerts anti-inflammatory effects against H. pylori infection through suppression of NF-κB and STAT3 and subsequently, expression of COX-2 and iNOS.

Helicobacter pylori infection promotes autophagy through Nrf2-mediated heme oxygenase upregulation in human gastric cancer cells.

It has been reported that Helicobacter pylori (H. pylori) infection is one of the primary causes of gastritis and peptic ulcer diseases. More than 50% of the world's population is supposed to be infected by this bacterium. However, 90% of infected patients do not develop gastric cancer, suggesting the existence of host defence mechanisms. Nrf2 is a transcription factor that plays a key role in cellular defence against oxidative stress and inflammation. Autophagy, an autodigestive process that degrades cellular organelles and proteins, plays an important role in maintaining cellular homeostasis. To investigate the molecular mechanisms responsible for cellular adaptive response to H. pylori induced gastric inflammation, human gastric epithelial cells and mice were infected with H. pylori. H. pylori infection induced expression of microtubule-associated light chain3 (LC3), an autophagic marker, through accumulation of reactive oxygen species and subsequently nuclear translocation of the redox-sensitive transcription factor, Nrf2 in human gastric epithelial AGS cells. Furthermore, Nrf2-induced LC3 up-regulation was mediated by heme oxygenase-1 (HO-1) and its by-product, carbon monoxide. Taken together, the Nrf2-HO-1 axis is considered to play a role in cellular adaptive survival response to H. pylori-induced gactric carcinogenesis by inducing autophagy.

Helicobacter pylori Activates IL-6-STAT3 Signaling in Human Gastric Cancer Cells: Potential Roles for Reactive Oxygen Species.

BACKGROUND: Recent studies have shown that Helicobacter pylori (H. pylori) activates signal transducer and activator of transcription 3 (STAT3) that plays an important role in gastric carcinogenesis. However, the molecular mechanism underlying H. pylori-mediated STAT3 activation is still not fully understood. In this study, we investigated H. pylori-induced activation of STAT3 signaling in AGS human gastric cancer cells and the underlying mechanism. MATERIALS AND METHODS: AGS cells were cocultured with H. pylori, and STAT3 activation was assessed by Western blot analysis, electrophoretic mobility shift assay and immunocytochemistry. To demonstrate the involvement of reactive oxygen species (ROS) in H. pylori-activated STAT3 signaling, the antioxidant N-acetylcysteine was utilized. The expression and production of interleukin-6 (IL-6) were measured by reverse-transcription polymerase chain reaction and enzyme-linked immunosorbent assay (ELISA), respectively. The interaction between IL-6 and IL-6 receptor (IL-6R) was determined by the immunoprecipitation assay. RESULTS: H. pylori activates STAT3 as evidenced by increases in phosphorylation on Tyr(705) , nuclear localization, DNA binding and transcriptional activity of this transcription factor. The nuclear translocation of STAT3 was also observed in H. pylori-inoculated mouse stomach. In the subsequent study, we found that H. pylori-induced STAT3 phosphorylation was dependent on IL-6. Notably, the increased IL-6 expression and the IL-6 and IL-6R binding were mediated by ROS produced as a consequence of H. pylori infection. CONCLUSIONS: H. pylori-induced STAT3 activation is mediated, at least in part, through ROS-induced upregulation of IL-6 expression. These findings provide a novel molecular mechanism responsible for H. pylori-induced gastritis and gastric carcinogenesis.

Helicobacter pylori induces Snail expression through ROS-mediated activation of Erk and inactivation of GSK-3ß in human gastric cancer cells.

Helicobacter pylori (H. pylori) infection has been known to be implicated in human gastric carcinogenesis. Snail, the zinc-finger transcription factor known as a key inducer of changes in the cell shape and morphogenetic movement, is aberrantly overexpressed and correlates with lymph node metastasis in gastric cancer. In the present study, we investigated whether H. pylori could induce Snail activation to provoke these changes. Using a cell scatter assay, we noticed that human gastric cancer AGS cells infected with H. pylori underwent morphological changes as well as disruption of cell-cell interaction, which was then reversed by silencing of Snail by use of small interfering RNA (siRNA). In addition, infection with H. pylori resulted in an increased intracellular level of Snail in gastric cancer cells, which was abrogated in the presence of U0126 and LY294002, inhibitors of MEK/Erk and PI3K/Akt pathways, respectively. Cycloheximide pulse-chase experiments coupled with immunocytochemical analysis revealed that the induction of Snail by H. pylori was regulated at multiple levels, including increased transcription of Snail mRNA, inhibition of protein degradation, and enhancement of nuclear translocation of Snail. Pre-treatment of AGS cells with N-acetylcysteine, a well-known reactive oxygen species (ROS) scavenger, attenuated the H. pylori-induced activation of Erk, its binding to Snail promoter, inactivation of GSK-3ß, and accumulation of Snail. Collectively, these findings suggest that the upregulation of Snail expression induced by H. pylori and transformation to a spindle-like shape as a consequence in gastric cancer cells are attributable to ROS-mediated activation of Erk and the inhibition of GSK-3ß signaling. © 2016 Wiley Periodicals, Inc.

Keap1 cysteine 288 as a potential target for diallyl trisulfide-induced Nrf2 activation.

Diallyl sulfide, diallyl disulfide, and daillyl trisulfide (DATS) are major volatile components of garlic oil. In this study, we assessed their relative potency in inducing antioxidant enzyme expression. Among the three organosulfur compounds, DATS was found to be most potent in inducing heme oxygenase-1 (HO-1) and NAD(P)H: quinone oxidoreductase-1 (NQO1) in human gastric epithelial (AGS) cells. Furthermore, DATS administration by gavage increased the expression of HO-1 and NQO1 in C57BL/6 mouse stomach. Treatment with DATS increased the accumulation of nuclear factor-erythroid-2-related factor-2 (Nrf2) in the nucleus of cultured AGS cells and in mouse stomach in vivo. The DATS-induced expression of HO-1 and NQO1 was abrogated in the cells transiently transfected with Nrf2-siRNA or in the embryonic fibroblasts from Nrf2-null mice, indicating that Nrf2 is a key mediator of the cytoprotective effects of DATS. Pretreatment of AGS cells with N-acetylcysteine or dithiothreitol attenuated DATS-induced nuclear localization of Nrf2 and the expression of HO-1 and NQO1. Cysteine-151, -273 and -288 of Kelch-like ECH-associated protein-1 (Keap1), a cytosolic repressor of Nrf2, have been considered to act as a redox sensor and play a role in Nrf2 activation. To determine whether DATS could inactivate Keap1 through thiol modification, we established cell lines constitutively expressing wild type-Keap1 or three different mutant constructs in which cysteine-151, -273, or -288 of Keap1 was replaced with serine by retroviral gene transfer. DATS failed to activate Nrf2, and to induce expression of HO-1 and NQO1 only in Keap1-C288S mutant cells. LC-ESI-MS/MS analysis of recombinant Keap1 treated with DATS revealed that the peptide fragment containing Cys288 gained a molecular mass of 72.1 Da equivalent to the molecular weight of mono-allyl mono-sulfide. Taken together, these findings suggest that DATS may directly interact with the Cys288 residue of Keap1, which partly accounts for its ability to induce Nrf2 activation and upregulate defensive gene expression.

Diallyl trisulfide suppresses dextran sodium sulfate-induced mouse colitis: NF-κB and STAT3 as potential targets.

Diallyl trisulfide (DATS), one of the volatile constituents of garlic oil, has been reported to possess antioxidant, anti-inflammatory, and anti-carcinogenic properties. In this study, DATS (10µmol) given orally for 7days before and for another 7days after starting administration of 2.5% dextran sulfate sodium (DSS) in drinking water protected against colitis induced by DSS in male ICR mice. DATS significantly inhibited the DSS-induced DNA binding of NF-κB, phosphorylation of IκBα and the expression of pro-inflammatory proteins, such as cyclooxygenase-2 and inducible nitric oxide synthase, which are major target proteins of NF-κB. The DSS-induced DNA binding and phosphorylation at the Tyr 705 residue of signal transducer and activator of transcription 3 (STAT3), and expression of its major target protein cyclin D1 in mouse colonic mucosa were also attenuated by DATS administration. Likewise, DSS-induced phosphorylation of extracellular signal-regulated kinase 1/2 was suppressed by DATS treatment. In conclusion, DATS ameliorates the DSS-induced mouse colitis presumably by blocking inflammatory signaling mediated by NF-κB and STAT3.

15-Hydroxyprostaglandin dehydrogenase as a novel molecular target for cancer chemoprevention and therapy.

Cyclooxygenase-2 (COX-2), a rate-limiting enzyme in arachidonic acid cascade, plays a key role in the biosynthesis of prostaglandin E(2) (PGE(2)) upon inflammatory insults. Overproduction of PGE(2) stimulates proliferation of various cancer cells, confers resistance to apoptosis of cancerous or transformed cells, and accelerates metastasis and angiogenesis. Excess PGE(2) undergoes metabolic inactivation which is catalyzed by NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH). In this context, 15-PGDH has been speculated as a physiological antagonist of COX-2 and a tumor suppressor. Thus, overexpression of 15-PGDH has been known to protect against experimentally induced carcinogenesis and renders the cancerous or transformed cells susceptible to apoptosis by counteracting oncogenic action of PGE(2). In contrast, silence of 15-PGDH is observed in some cancer cells, which is associated with epigenetic modification, such as DNA methylation and histone deacetylation, in the promoter region of 15-PGDH. A variety of compounds capable of inducing the expression of 15-PGDH have been reported, which include the histone deacetylase inhibitors, nonsteroidal anti-inflammatory drugs, and peroxisome proliferator-activated receptor-gamma agonists. Therefore, 15-PGDH may be considered as a novel molecular target for cancer chemoprevention and therapy. This review highlights the role of 15-PGDH in carcinogenesis and its regulation.

Nrf2-Mediated heme oxygenase-1 upregulation as adaptive survival response to glucose deprivation-induced apoptosis in HepG2 cells.

Induction of heme oxygenase-1 (HO-1) represents an important cellular adaptive survival response to oxidative stress and other toxic insults. In the present study, HepG2 cells grown in glucose-free media underwent apoptotic cell death, but they exhibited elevated expression of HO-1 before apoptosis manifested. Treatment of HepG2 cells with SnCl2, a HO-1 inducer, rescued these cells from glucose deprivation-induced apoptosis, while inhibition of the HO activity with zinc protoporphyrin IX exacerbated apoptosis under the same condition. HepG2 cells transfected with a dominant negative Nrf2 were more vulnerable to glucose deprivation-induced apoptosis compared to cells transfected with empty vector alone. To confirm the involvement of Nrf2 in the induction of HO-1 caused by glucose deprivation, we used embryonic fibroblasts prepared from nrf2⁻(/)⁻, nrf2(+/)⁻, and nrf2(+/+) embryos. Compared to the wild-type and the nrf2(+/)⁻ embryonic fibroblasts, nrf2⁻(/)⁻ cells were less prone to induce HO-1 expression upon glucose deprivation. Exposure of HepG2 cells to glucose-deprived media resulted in an elevated accumulation of reactive oxygen species (ROS). Pretreatment with N-acetylcysteine prevented the glucose deprivation-induced ROS accumulation and also the HO-1 expression. In conclusion, the Nrf2-mediated HO-1 upregulation upon glucose deprivation is mediated by ROS in HepG2 cells, and responsible for the adaptive survival response.

Capsaicin induces heme oxygenase-1 expression in HepG2 cells via activation of PI3K-Nrf2 signaling: NAD(P)H:quinone oxidoreductase as a potential target.

Capsaicin (trans-8-methyl-N-vanillyl-6-nonenamide), a major pungent ingredient of red pepper, is reported to have antimutagenic and anticarcinogenic properties. However, the mechanisms underlying its chemoprotective effects remain largely unresolved. In the present study, we found that capsaicin induced expression of heme oxygenase-1 (HO-1) in HepG2 cells. Capsaicin treatment resulted in a transient increase in the phosphorylation of Akt and subsequently nuclear translocation of NF-E2-related factor 2 (Nrf2), enhancing its binding to antioxidant response element (ARE). HepG2 cells treated with capsaicin exhibited increased production of reactive oxygen species (ROS). Prior exposure of cells to N-acetyl-L -cysteine blocked not only the ROS production but also the nuclear translocation of Nrf2 and its ARE binding, as well as HO-1 induction by capsaicin. Immunoblot analysis showed that whereas the level of HO-1 protein was elevated, that of NAD(P)H:quinone oxidoreductase (NQO1) was decreased after the treatment with capsaicin or the inhibitor of NQO1, dicumarol. We hypothesize that quinone metabolites or other reactive forms of capsaicin may bind covalently to NQO1 and thereby inhibit its activity, leading to production of ROS. This, in turn, would trigger the activation of Akt via phosphorylation, increase the nuclear translocation and ARE binding of Nrf2, and upregulate the expression of HO-1.