Peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 directly binds and stabilizes Nrf2 in breast cancer.

Peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (Pin1) has been frequently overexpressed in many types of malignancy, suggesting its oncogenic function. It recognizes phosphorylated serine or threonine (pSer/Thr) of a target protein and isomerizes the adjacent proline (Pro) residue, thereby altering folding, subcellular localization, stability, and function of target proteins. The oncogenic transcription factor, Nrf2 harbors the pSer/Thr-Pro motif. This prompted us to investigate whether Pin1 could bind to Nrf2 and influence its stability and function in the context of implications for breast cancer development and progression. The correlation between Pin1 and Nrf2 in the triple-negative breast cancer cells was validated by RNASeq analysis as well as immunofluorescence staining. Interaction between Pin1 and Nrf2 was assessed by co-immunoprecipitation and an in situ proximity ligation assay. We found that mRNA and protein levels of Pin1 were highly increased in the tumor tissues of triple-negative breast cancer patients and the human breast cancer cell line. Genetic or pharmacologic inhibition of Pin1 enhanced the ubiquitination and degradation of Nrf2. In contrast, the overexpression of Pin1 resulted in the accumulation of Nrf2 in the nucleus, without affecting its transcription. Notably, the phosphorylation of Nrf2 at serine 215, 408, and 577 is essential for its interaction with Pin1. We also identified phosphorylated Ser104 and Thr277 residues in Keap1, a negative regulator of Nrf2, for Pin1 binding. Pin1 plays a role in breast cancer progression through stabilization and constitutive activation of Nrf2 by competing with Keap1 for Nrf2 binding.

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.

Alternative regulation of HIF-1α stability through Phosphorylation on Ser451.

The hypoxia-inducible factor (HIF-1α) functions as a master regulator of oxygen homeostasis. Oxygen-dependent hydroxylation of HIF-1α is tightly regulated by prolyl hydroxylase domain containing proteins (PHD1, PHD2, and PHD3). The prolyl hydroxylation facilitates the recruitment of the von Hippel-Lindau (VHL) protein, leading to ubiquitination and degradation of HIF-1α by the proteasomes. Besides prolyl hydroxylation, phosphorylation of HIF-1α is another central post-translational modification, which regulates its stability under hypoxic conditions as well as normoxic conditions. By use of LC/MS/MS-based analysis, we were able to identify a specific serine residue (Ser451) of HIF-1α phosphorylated under hypoxic conditions. Using plasmids expressing wild type (WT), non-phosphorylatable mutant HIF-1α (S451A), and phosphomimetic mutant HIF-1α (S451E), we demonstrated that the phosphorylation at Ser451 is important in maintaining the HIF-1α protein stability. Notably, phosphorylation at S451 interrupts the interaction of HIF-1α with PHD and pVHL. A phosphomimetic construct of HIF-1α at Ser451 (S451E) is significantly more stable than WT HIF-1α under normoxic conditions. Cells transfected with unphosphorylatable HIF-1α exhibited significantly lower HIF-1 transcriptional activity than WT cells and markedly reduced tumor cell migration. Further, tumors derived from the phosphomimetic mutant cells grew faster, whereas the tumors derived from non-phosphorylatable mutant cells grew slower than the control tumors, suggesting that the phosphorylation of HIF-1α at the Ser451 site is critical to promote tumor growth in vivo. Taken together, our data suggest an alternative mechanism responsible for the regulation of HIF-1α stability.

Helicobacter pylori infection induces STAT3 phosphorylation on Ser727 and autophagy in human gastric epithelial cells and mouse stomach.

Helicobacter pylori (H. pylori) infection is considered as one of the principal risk factors of gastric cancer. Constitutive activation of the signal transducer and activator of transcription 3 (STAT3) plays an important role in inflammation-associated gastric carcinogenesis. In the canonical STAT3 pathway, phosphorylation of STAT3 on Tyr705 is a major event of STAT3 activation. However, recent studies have demonstrated that STAT3 phosphorylated on Ser727 has an independent function in mitochondria. In the present study, we found that human gastric epithelial AGS cells infected with H. pylori resulted in localization of STAT3 phosphorylated on Ser727 (P-STAT3Ser727), predominantly in the mitochondria. Notably, H. pylori-infected AGS cells exhibited the loss of mitochondrial integrity and increased expression of the microtubule-associated protein light chain 3 (LC3), the autophagosomal membrane-associated protein. Treatment of AGS cells with a mitophagy inducer, carbonyl cyanide 3-chlorophenylhydrazone (CCCP), resulted in accumulation of P-STAT3Ser727 in mitochondria. In addition, the elevated expression and mitochondrial localization of LC3 induced by H. pylori infection were attenuated in AGS cells harboring STAT3 mutation defective in Ser727 phosphorylation (S727A). We also observed that both P-STAT3Ser727 expression and LC3 accumulation were increased in the mitochondria of H. pylori-inoculated mouse stomach.

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.

Docosahexaenoic acid inhibits Helicobacter pylori-induced STAT3 phosphorylation through activation of PPARγ.

SCOPE: The health beneficial effects of docosahexaenoic acid (DHA) have been attributed to its anti-inflammatory properties. However, the molecular mechanism underlying anti-inflammatory effects of DHA remains largely elusive. METHODS AND RESULTS: In the present study, DHA was found to suppress the phosphorylation and nuclear translocation of signal transducer and activator of transcription 3 (STAT3) induced by Helicobacter pylori infection in human gastric cancer AGS cells. Notably, DHA induced expression of suppressor of cytokine signaling 3 (SOCS3), a negative regulator of STAT3. Knockdown of SOCS3 abolished the suppressive effect of DHA on STAT3(Tyr705) phosphorylation induced by H. pylori infection. DHA also induced nuclear translocation, DNA binding, and transcriptional activities of peroxisome proliferator-activated receptor gamma (PPARγ) in AGS cells. Knockdown of PPARγ inhibited the transcription of SOCS3 and attenuated the suppressive effect of DHA on phosphorylation of STAT3(Tyr705) induced by H. pylori. The PPARγ antagonist bisphenol A diglycidyl ether also mitigated the suppressive effect of DHA on H. pylori-induced phosphorylation of STAT3(Tyr705) . In addition, DHA inhibited the expression of c-Myc, which was attenuated in the AGS cells harboring SOCS3 specific siRNA. DHA also markedly decreased anchorage-independent growth of AGS cells infected by H. pylori. CONCLUSION: DHA inhibits H. pylori-induced STAT3 phosphorylation in a PPARγ/SOCS3-dependent manner.

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.

Peptidyl Prolyl Isomerase PIN1 Directly Binds to and Stabilizes Hypoxia-Inducible Factor-1α.

Peptidyl prolyl isomerase (PIN1) regulates the functional activity of a subset of phosphoproteins through binding to phosphorylated Ser/Thr-Pro motifs and subsequently isomerization of the phosphorylated bonds. Interestingly, PIN1 is overexpressed in many types of malignancies including breast, prostate, lung and colon cancers. However, its oncogenic functions have not been fully elucidated. Here, we report that PIN1 directly interacts with hypoxia-inducible factor (HIF)-1α in human colon cancer (HCT116) cells. PIN1 binding to HIF-1α occurred in a phosphorylation-dependent manner. We also found that PIN1 interacted with HIF-1α at both exogenous and endogenous levels. Notably, PIN1 binding stabilized the HIF-1α protein, given that their levels were significantly increased under hypoxic conditions. The stabilization of HIF-1α resulted in increased transcriptional activity, consequently upregulating expression of vascular endothelial growth factor, a major contributor to angiogenesis. Silencing of PIN1 or pharmacologic inhibition of its activity abrogated the angiogenesis. By utilizing a bioluminescence imaging technique, we were able to demonstrate that PIN1 inhibition dramatically reduced the tumor volume in a subcutaneous mouse xenograft model and angiogenesis as well as hypoxia-induced transcriptional activity of HIF-1α. These results suggest that PIN1 interacting with HIF-1α is a potential cancer chemopreventive and therapeutic target.

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.

Acanthoic acid, a diterpene in Acanthopanax koreanum, protects acetaminophen-induced hepatic toxicity in mice.

The protective effect of a diterpenoid acanthoic acid (AA) isolated from Acanthopanax koreanum Nakai was investigated in acetaminophen (APAP)-induced hepatic toxicity. Drug-induced hepatotoxicity induced by an intraperitoneal (i.p.) injection of 300mg/kg (sub-lethal dose) of APAP. Pretreatment with AA (50 and 100mg/kg) orally 2h before the APAP administration attenuated the APAP-induced acute increase in serum aspartate aminotransferase (AST), and alanine aminotransferase (ALT) activites, replenished the depleted hepatic glutathione (GSH), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px) activities, decreased malondialdehyde (MDA) level and considerably reduced the histopathological alterations in a manner similar to silymarin (Sily). Immunohistochemical analyses also demonstrated that AA could reduce the appearance of necrosis regions as well as caspase-3 and hypoxia inducible factor-1alpha (HIF-1alpha) expression in liver tissue. Our results indicated that AA protected liver tissue from the oxidative stress elicites by APAP-induced liver damage and suggestes that the hepatic protection mechanism of AA would relate to antioxidation and hypoxia factor on APAP-induced hepatotoxicity.