TOPK inhibits TNF-α-induced granulosa cell apoptosis via regulation of SIRT1/p53.

T-LAK cell originated protein kinase (TOPK) has been shown to regulate proliferation, invasion or migration of various cancer cells. However, the role of TOPK in follicle environments remains unknown. Here we reveal that TOPK inhibits TNF-α-induced human granulosa COV434 cell apoptosis. The expression of TOPK were increased in COV434 cells in response to TNF-α. TOPK inhibition also decreased TNF-α-induced SIRT1 expression but promoted TNF-α-induced p53 acetylation and expression of PUMA or NOXA. Accordingly, TOPK inhibition attenuated TNF-α-mediated SIRT1 transcriptional activity. In addition, SIRT1 inhibition augmented acetylation of p53 or expression of PUMA and NOXA in response to TNF-α, leading to COV434 cell apoptosis. We conclude that TOPK suppresses TNF-α-induced COV434 granulosa cell apoptosis via regulation of p53/SIRT1 axis, suggesting a potential role of TOPK in regulation of ovarian follicular development.

TOPK mediates hypoxia-induced epithelial-mesenchymal transition and the invasion of nonsmall-cell lung cancer cells via the HIF-1α/snail axis.

Hypoxia has been suggested to induce epithelial-mesenchymal transition (EMT) in various cancer types via the transcription factor hypoxia-inducible factor-1 alpha (HIF-1α). Here, we demonstrated that TOPK upregulates EMT and the invasion of H460 nonsmall-cell lung cancer cells through the induction of the HIF-1α/Snail axis and hypoxic signaling. The expression of endogenous TOPK, phosphorylated TOPK, HIF-1α and Snail was significantly increased upon hypoxia exposure, but TOPK depletion markedly abrogated the induced mRNA and protein levels of HIF-1α and Snail. Interestingly, TOPK knockdown restored the hypoxia-induced suppression of E-cadherin and diminished hypoxia-induced N-cadherin expression. In addition, Snail depletion suppressed hypoxia-induced N-cadherin expression, which was attenuated by TOPK knockdown. Moreover, knockdown of Snail decreased hypoxia-induced nonsmall-cell lung cancer cell migration and invasion, which were suppressed by TOPK depletion. In summary, we conclude that TOPK positively regulates HIF-1α expression through hypoxia signaling and thereby promotes Snail expression, leading to EMT and the invasion of nonsmall-cell lung cancer cells. These findings suggest that TOPK plays a critical role as a novel mediator of hypoxia signaling that regulates nonsmall-cell lung cancer development.

Role of the SIRT1/p53 regulatory axis in oxidative stress­mediated granulosa cell apoptosis.

Oxidative stress has been suggested to induce granulosa cell apoptosis, which contributes to follicular atresia. However, the mechanism via which oxidative stress mediates granulosa cell apoptosis remains elusive. Therefore, the aim of this study was to elucidate the molecular mechanisms regulating oxidative stress­induced granulosa cell apoptosis. The present study demonstrated that reactive oxygen species induced by H2O2 resulted in human granulosa COV434 cell apoptosis via the regulation of sirtuin 1 (SIRT1)­mediated p53 activity. Endogenous SIRT1 expression was alleviated by H2O2 treatment of COV434 cells in a time­dependent manner. In addition, knockdown or inhibition of SIRT1 promoted H2O2­induced poly(ADP­ribose) polymerase (PARP) cleavage and p53 acetylation, which led to an increase in COV434 cell apoptosis. Treatment with H2O2 enhanced the expression levels of the p53­dependent proteins, p53­upregulated modulator of apoptosis (PUMA) and phorbol­12­myristate­13­acetate­induced protein 1 (PMAIP1), as well as those of p53; however, knockdown of p53 decreased cleaved PARP, PUMA and PMAIP1 expression levels induced by H2O2 treatment. Moreover, knockdown of PUMA or PMAIP1 attenuated the H2O2 induction of PARP cleavage and COV434 cell apoptosis. In conclusion, the present findings suggested that H2O2­induced oxidative stress causes granulosa COV434 cell apoptosis via the upregulation of p53 activity by SIRT1 suppression, indicating a mechanistic role of the SIRT1/p53 axis in H2O2­induced granulosa cell apoptosis.

Verbascoside-Rich Abeliophyllum distichum Nakai Leaf Extracts Prevent LPS-Induced Preterm Birth Through Inhibiting the Expression of Proinflammatory Cytokines from Macrophages and the Cell Death of Trophoblasts Induced by TNF-α.

Background: Preterm birth is a known leading cause of neonatal mortality and morbidity. The underlying causes of pregnancy-associated complications are numerous, but infection and inflammation are the essential high-risk factors. However, there are no safe and effective preventive drugs that can be applied to pregnant women. Objective: The objectives of the study were to investigate a natural product, Abeliophyllum distichum leaf (ADL) extract, to examine the possibility of preventing preterm birth caused by inflammation. Methods: We used a mouse preterm birth model by intraperitoneally injecting lipopolysaccharides (LPS). ELISA, Western blot, real-time PCR and immunofluorescence staining analyses were performed to confirm the anti-inflammatory efficacy and related mechanisms of the ADL extracts. Cytotoxicity and cell death were measured using Cell Counting Kit-8 (CCK-8) analysis and flow cytometer. Results: A daily administration of ADL extract significantly reduced preterm birth, fetal loss, and fetal growth restriction after an intraperitoneal injection of LPS in mice. The ADL extract prevented the LPS-induced expression of TNF-α in maternal serum and amniotic fluid and attenuated the LPS-induced upregulation of placental proinflammatory genes, including IL-1ß, IL-6, IL-12p40, and TNF-α and the chemokine gene CXCL-1, CCL-2, CCL3, and CCL-4. LPS-treated THP-1 cell-conditioned medium accelerated trophoblast cell death, and TNF-α played an essential role in this effect. The ADL extract reduced LPS-treated THP-1 cell-conditioned medium-induced trophoblast cell death by inhibiting MAPKs and the NF-κB pathway in macrophages. ADL extract prevented exogenous TNF-α-induced increased trophoblast cell death and decreased cell viability. Conclusions: We have demonstrated that the inhibition of LPS-induced inflammation by ADL extract can prevent preterm birth, fetal loss, and fetal growth restriction.

mTOR-Dependent Role of Sestrin2 in Regulating Tumor Progression of Human Endometrial Cancer.

Oncogenic activation of the mammalian target of rapamycin complex 1 (mTORC1) leads to endometrial cancer cell growth and proliferation. Sestrin2 (SESN2), a highly conserved stress-inducible protein, is involved in homeostatic regulation via inhibition of reactive oxygen species (ROS) and mTORC1. However, the role of SESN2 in human endometrial cancer remains to be investigated. Here, we investigated expression, clinical significance, and underlying mechanisms of SESN2 in endometrial cancer. SESN2 was upregulated more in endometrial cancer tissues than in normal endometrial tissues. Furthermore, upregulation of SESN2 statistically correlated with shorter overall survival and disease-free survival in patients with endometrial cancer. SESN2 expression strongly correlated with mTORC1 activity, suggesting its impact on prognosis in endometrial cancer. Additionally, knockdown of SESN2 promoted cell proliferation, migration, and ROS production in endometrial cancer cell lines HEC-1A and Ishikawa. Treatment of these cells with mTOR inhibitors reversed endometrial cancer cell proliferation, migration, and epithelial-mesenchymal transition (EMT) marker expression. Moreover, in a xenograft nude mice model, endometrial cancer growth increased by SESN2 knockdown. Thus, our study provides evidence for the prognostic significance of SESN2, and a relationship between SESN2, the mTORC1 pathway, and endometrial cancer growth, suggesting SESN2 as a potential therapeutic target in endometrial cancer.

TOPK inhibition accelerates oxidative stress­induced granulosa cell apoptosis via the p53/SIRT1 axis.

It has been suggested that oxidative stress involving reactive oxygen species (ROS) induces granulosa cell apoptosis, leading to follicular atresia, and that T­lymphokine­activated killer cell­originated protein kinase (TOPK) suppresses cancer cell apoptosis induced by several stimuli. However, it remains to be determined whether TOPK affects oxidative stress­induced granulosa cell apoptosis. The present study demonstrates that TOPK inhibition increases human granulosa COV434 cell apoptosis induced by hydrogen peroxide (H2O2). Co­treatment with the TOPK inhibitor, OTS514, in combination with H2O2 increased p53 acetylation and its expression, whereas it decreased Sirtuin 1 (SIRT1) expression, contributing to the promotion of apoptosis. In addition, the SIRT1 activator, resveratrol, or the SIRT1 inhibitor, Ex527, reduced or elevated H2O2­induced COV434 cell apoptosis, respectively. Furthermore, the p53 inhibitor, Pifithrin­µ, diminished the augmentation in poly(ADP­ribose) polymerase (PARP) cleavage induced by OTS514 plus H2O2, while the Mdm2 antagonist, Nutlin 3, increased PARP cleavage. Moreover, OTS514 further decreased the SIRT1 transcriptional activity decreased by H2O2, but promoted the H2O2­induced p53 or p21 transcriptional activity. Notably, the expression of exogenous p53 reduced SIRT1 transcriptional activity. Taken together, the findings of the present study demonstrate that TOPK inhibition promotes p53­mediated granulosa cell apoptosis through SIRT1 downregulation in response to H2O2. Therefore, it can be concluded that TOPK suppresses H2O2­induced apoptosis through the modulation of the p53/SIRT1 axis, suggesting a potential role of TOPK in the regulation of human granulosa cell apoptosis, leading to the promotion of abnormal follicular development.

Activation of NF-κB by TOPK upregulates Snail/Slug expression in TGF-ß1 signaling to induce epithelial-mesenchymal transition and invasion of breast cancer cells.

TGF-ß1 is known to induce epithelial-mesenchymal transition (EMT), which is a prerequisite for cancer cell invasion. Here we reveal that TOPK upregulates EMT and invasion of human breast cancer MDA-MB-231 or Hs578T cells via NF-κB-dependent Snail/Slug in TGF-ß1 signaling. Endogenous TOPK expression was significantly increased in response to TGF-ß1 and TOPK knockdown mitigated TGF-ß1-induced breast cancer cell invasion. Interestingly, TOPK knockdown restored TGF-ß1 suppression of E-cadherin expression and markedly reduced N-cadherin induced by TGF-ß1. Also, NF-κB activity or expression of EMT markers Snail and Slug induced by TGF-ß1 was decreased by TOPK knockdown. Meanwhile, knockdown of Snail or TOPK attenuated TGF-ß1-induced breast cancer cell invasion. Taken, we conclude that TOPK mediates TGF-ß1-induced EMT and invasion in breast cancer cells via NF-κB/Snail signaling, suggesting novel role of TOPK as therapeutic target in TGF-ß1-mediated breast cancer development.

Red Ginseng Inhibits Tau Aggregation and Promotes Tau Dissociation In Vitro.

Tau, a microtubule-associated protein expressed in mature neurons, interacts with tubulin to promote the assembly and stabilization of microtubules. However, abnormally hyperphosphorylated tau dissociates from microtubules and self-aggregates. Tau aggregates, including paired helical filaments and neurofibrillary tangles, promote neuronal dysfunction and death and are the defining neuropathological feature of tauopathies. Therefore, suppressing tau aggregation or stimulating the dissociation of tau aggregates has been proposed as an effective strategy for treating neurodegenerative diseases associated with tau pathology such as Alzheimer's disease (AD) and frontotemporal dementia. Interestingly, ginsenosides extracted from Panax ginseng reduced the hippocampal and cortical expression of phosphorylated tau in a rat model of AD. However, no studies have been conducted into the effect of red ginseng (RG) and its components on tau pathology. Here, we evaluated the effect of Korean red ginseng extract (KRGE) and its components on the aggregation and disassociation of tau. Using the thioflavin T assay, we monitored the change in fluorescence produced by the aggregation or disassociation of tau K18, an aggregation-prone fragment of tau441 containing the microtubule-binding domain. Our analysis revealed that KRGE not only inhibited tau aggregation but also promoted the dissociation of tau aggregates. In addition, the KRGE fractions, such as saponin, nonsaponin, and nonsaponin fraction with rich polysaccharide, also inhibited tau aggregation and promoted the dissociation of tau aggregates. Our observations suggest that RG could be a potential therapeutic agent for the treatment of neurodegenerative diseases associated with tauopathy.

Neuroprotective and Anti-Inflammatory Effects of Low-Moderate Dose Ionizing Radiation in Models of Alzheimer's Disease.

Alzheimer's disease (AD) is the most common cause of dementia. The neuropathological features of AD include amyloid-ß (Aß) deposition and hyperphosphorylated tau accumulation. Although several clinical trials have been conducted to identify a cure for AD, no effective drug or treatment has been identified thus far. Recently, the potential use of non-pharmacological interventions to prevent or treat AD has gained attention. Low-dose ionizing radiation (LDIR) is a non-pharmacological intervention which is currently being evaluated in clinical trials for AD patients. However, the mechanisms underlying the therapeutic effects of LDIR therapy have not yet been established. In this study, we examined the effect of LDIR on Aß accumulation and Aß-mediated pathology. To investigate the short-term effects of low-moderate dose ionizing radiation (LMDIR), a total of 9 Gy (1.8 Gy per fraction for five times) were radiated to 4-month-old 5XFAD mice, an Aß-overexpressing transgenic mouse model of AD, and then sacrificed at 4 days after last exposure to LMDIR. Comparing sham-exposed and LMDIR-exposed 5XFAD mice indicated that short-term exposure to LMDIR did not affect Aß accumulation in the brain, but significantly ameliorated synaptic degeneration, neuronal loss, and neuroinflammation in the hippocampal formation and cerebral cortex. In addition, a direct neuroprotective effect was confirmed in SH-SY5Y neuronal cells treated with Aß1-42 (2 µM) after single irradiation (1 Gy). In BV-2 microglial cells exposed to Aß and/or LMDIR, LMDIR therapy significantly inhibited the production of pro-inflammatory molecules and activation of the nuclear factor-kappa B (NF-κB) pathway. These results indicate that LMDIR directly ameliorated neurodegeneration and neuroinflammation in vivo and in vitro. Collectively, our findings suggest that the therapeutic benefits of LMDIR in AD may be mediated by its neuroprotective and anti-inflammatory effects.

PBK attenuates paclitaxel-induced autophagic cell death by suppressing p53 in H460 non-small-cell lung cancer cells.

PDZ-binding kinase (PBK) has previously been shown to mediate chemoresistance of cancer cells to anticancer drugs. However, it remains unclear how PBK regulates paclitaxel-induced cancer cell death. Here, we demonstrate that PBK hinders paclitaxel-mediated autophagic cell death in H460 non-small-cell lung cancer cells. PBK knockdown increased apoptosis, autophagy, p53 level, and LC3 puncta upon paclitaxel treatment. Moreover, p53 expression facilitated an increase in the LC3-II/LC3-I ratio in response to paclitaxel, and PBK knockdown augmented paclitaxel-mediated p53 transcriptional activity. Meanwhile, paclitaxel induced PBK-mediated p53 nuclear export and its subsequent ubiquitination in control cells, but not in PBK knockdown cells. We conclude that PBK hampers paclitaxel-induced autophagic cell death by suppressing p53, suggesting a potential role of PBK in p53-mediated H460 cell death.

TOPK promotes epithelial-mesenchymal transition and invasion of breast cancer cells through upregulation of TBX3 in TGF-ß1/Smad signaling.

TOPK has been suggested to contribute to invasion of lung, prostate, gastric, pancreatic or breast cancer cells. However, how TOPK mediates TGF-ß1/Smad signaling leading to epithelial-mesenchymal transition (EMT) and invasion of breast cancer cells remains unknown. Here we report that TOPK upregulates T-box transcription factor TBX3 to enhance TGF-ß1-induced EMT and invasion of MDA-MB-231 breast cancer cells. Expression of endogenous TOPK was promoted by TGF-ß1 treatment of MDA-MB-231 cells time-dependently. In addition, knockdown of TOPK attenuated TGF-ß1-induced phosphorylation or transcriptional activity of Smad3. Meanwhile, levels of both mRNA and protein of TBX3 induced by TGF-ß1 were abolished by TOPK depletion. Also, knockdown of TBX3 inhibited TGF-ß1 induction of EMT-related genes Snail, Slug or Fibronectin. Furthermore, ablation of TOPK or TBX3 suppressed TGF-ß1-induced MDA-MB-231 cell invasion. Collectively, we conclude that TOPK positively regulates TBX3 in TGF-ß1/Smad signaling pathway, thereby enhancing EMT and invasion of breast cancer cells, implying a mechanistic role of TOPK in TGF-ß1/Smad signaling.

Role of TOPK in lipopolysaccharide-induced breast cancer cell migration and invasion.

Inflammation has been known to be linked to invasion or metastasis of breast cancer, which has poor prognosis, although the regulatory mechanism remains to be undiscovered. Here we show that T-LAK cell-originated protein kinase (TOPK) mediates pro-inflammatory endotoxin lipopolysaccharide (LPS)-induced breast cancer cell migration and invasion. The mRNA or protein level of TOPK, toll- like receptor4 (TLR4), interleukin (IL)-6, vascular endothelial growth factor (VEGF) or matrix metalloproteinase9 (MMP9) genes related to TLR4 signaling or tumor progression was induced by LPS treatment in MCF7 breast cancer cells, but the induction was abolished by stable knocking down of TOPK in MCF7 cells. Also, TOPK depletion decreased LPS-induced phosphorylation of p38, but not ERK and JNK among mitogen-activated protein kinases (MAPKs). On the other hand, we revealed that TOPK is essential for transcriptional activity of NF-κB or MMP9 promoter triggered by LPS. The induced promoter activity of NF-κB or MMP9 but not AP-1 was inhibited by knocking down of TOPK. Furthermore, we demonstrated that inhibitor of TOPK or MMP9 as well as MMP9 siRNA efficiently blocked LPS-induced migration or invasion of breast cancer cell lines. Interestingly, both of expression of TOPK and TLR4 were markedly increased in high-grade breast cancer. Collectively, we conclude that TOPK functions as a key mediator of LPS/TLR4-induced breast cancer cell migration and invasion through regulation of MMP9 expression or activity, implying a potential role of TOPK as a therapeutic target linking LPS-induced inflammation to breast cancer development.

Withaferin A inhibits inflammatory responses induced by Fusobacterium nucleatum and Aggregatibacter actinomycetemcomitans in macrophages.

Periodontitis is a progressive chronic inflammatory disease and a major cause of tooth loss in humans. As a withanolides, withaferin A (WA) is known to exhibit strong anti­inflammatory activity. The present study examined whether WA inhibited inflammatory responses in macrophages in response to two representative periodontal pathogens, Fusobacterium nucleatum and Aggregatibacter actinomycetemcomitans. Murine bone marrow­derived macrophages (BMDMs) were used in this study and cytokine production in culture supernatants was measured by enzyme­linked immunosorbent assays. Western blot analysis was performed to determine the activation of nuclear factor­κB and mitogen­activated protein kinases (MAPKs) and the expression of inducible nitric oxide synthase (iNOS), toll­like receptor (TLR) 2 and TLR4. The production of nitric oxide (NO) was determined by the Griess reaction. WA treatment was shown to decrease interleukin (IL)­6 and tumor necrosis factor (TNF)­α production in BMDMs in response to F. nucleatum and A. actinomycetemcomitans in a dose­dependent manner. The phosphorylation of IκB­α and MAPKs (p38, extracellular signal­regulated kinases and c­Jun N­terminal kinases) induced by F. nucleatum and A. actinomycetemcomitans was also inhibited by WA. F. nucleatum and A. actinomycetemcomitans induced iNOS expression and NO production in BMDMs, which was inhibited by WA in a dose­dependent manner. WA also reduced endogenous and induced expression of TLR2 and TLR4 in these cells. These results suggest that WA may be a potential therapeutic agent or preventive additive for periodontitis control.

A role for Toll-like receptor 4 in the host response to the lung infection of Yersinia pseudotuberculosis in mice.

Although a Yersinia pseudotuberculosis (Yptb) lung infection model has been developed to study Y. pestis pathogenesis, it is still necessary to establish a new animal model to mimic the pathophysiological features induced by Y. pestis infection. Here, we provide a new lung infection model using the Yptb strain, IP2777, which displayed rapid spread of bacteria to the liver, spleen, and blood. In addition, we examined whether TLR4 is involved in Yptb-induced pathogenesis in the lung infection model of mice we generated. Following lung infection of WT and TLR4-deficient mice with the Yptb strain IP2777, the survival rate, bacterial colonization, histopathology, and level of cytokines and chemokines in the lung, spleen, liver, and blood were analyzed. TLR4-deficient mice had a lower survival rate than WT mice in response to Yptb lung infection. Although the bacterial colonization and pathology of the lung were comparable between WT and TLR4-deficient mice, those of the spleen and liver were more severe in TLR4-deficient mice. In addition, the levels of TNF-α and CXCL2 in the liver and IL-6 and CXCL2 in the blood were higher in TLR4-deficient mice than in WT mice. Our results demonstrate that TLR4 is necessary for optimal host protection against Yptb lung infection and TLR4-deficient mice may serve as a better genetic model of Yptb infection for mimicking Y. pestis infection.

Toll/IL-1 domain-containing adaptor inducing IFN-ß (TRIF) mediates innate immune responses in murine peritoneal mesothelial cells through TLR3 and TLR4 stimulation.

Mesothelial cells are composed of monolayer of the entire surface of serosal cavities including pleural, pericardial, and peritoneal cavity. Although mesothelial cells are known to express multiple Toll-like receptors (TLRs) which contribute to trigger innate immune responses against infections, the precise molecular mechanism remains still unclear. In the present study, we investigated the role of Toll/IL-1 domain-containing adaptor inducing IFN-ß (TRIF), one of the two major TLRs-adaptor molecules, on innate immune response induced by TLR3 and TLR4 stimulation in murine peritoneal mesothelial cells (PMCs). TRIF was strongly expressed in PMCs and its deficiency led to impaired production of cytokines and chemokines by poly I:C and LPS in the cells. Activation of NF-κB or MAPKs through poly I:C and LPS stimulation was reduced in TRIF-deficient PMCs as compared to the WT cells. TRIF was also necessary for optimal nitric oxide synthesis and gene expression of inducible nitric oxide synthase (iNOS) and IFN-ß in PMCs in response to poly I:C and LPS. Furthermore, both Escherichia coli and Pseudomonas aeruginosa induced high level of IL-6, CXCL1, and CCL2 production in PMCs, which was significantly impaired by TRIF deficiency. These results demonstrated that TRIF is required for optimal activation of innate immune responses in mesothelial cells against microbial infections.

The role of nucleotide-binding oligomerization domain 1 during cytokine production by macrophages in response to Mycobacterium tuberculosis infection.

Tuberculosis due to Mycobacterium tuberculosis infection is a leading cause of death worldwide. Recognition of this pathogen is crucial for the activation of innate and adaptive immune responses. Nucleotide-binding oligomerization domain (Nod)1 and Nod2 are cytoplasmic receptors that can detect unique muropeptides of bacterial peptidoglycan. Nod2 is critical for the initiation of the host immune response against M. tuberculosis infection, however the role of Nod1 remains largely unknown. We investigated the role of Nod1 with respect to cytokine production by bone marrow-derived macrophages (BMDMs) in response to M. tuberculosis infection. Production of proinflammatory cytokines, such as IL-6, TNF-α, and IL-1ß were induced in BMDMs; cytokine levels were not affected by a deficiency in Nod1. Activation of NF-κB and MAPKs was also comparable between wild-type and Nod1-deficient BMDMs. Levels of IL-6 and IL-1ß were reduced in Nod1/Nod2 double-deficient BMDMs to a greater extent than in Nod2-deficient cells. Furthermore, when signaling of Toll-like receptors (TLRs) was inhibited by lipopolysaccharide pre-treatment, cytokine production was diminished in Nod1-deficient BMDMs. Our results indicate that Nod1 cooperates with Nod2 or TLRs to produce cytokines in macrophages in response to M. tuberculosis infection.

Inhibitory effect of withaferin A on Helicobacter pylori­induced IL­8 production and NF­κB activation in gastric epithelial cells.

Withaferin A (WA), a withanolide purified from Withania somnifera, has been known to exert anti-inflammatory effects. The present study sought to determine the effects of WA on Helicobacter (H.) pylori-mediated inflammation in the AGS gastric epithelial cell line. Cellular production of interleukin (IL)-8 and vascular endothelial growth factor (VEGF) was measured by ELISA. Western blot analysis was performed to determine the activation of nuclear factor (NF)-κB and mitogen-activated protein kinases (MAPKs) as well as hypoxia-inducible factor 1α stabilization. Bacterial growth was also examined by measuring the optical density. Pre-treatment or co-treatment with WA efficiently reduced IL-8 production by AGS cells in response to H. pylori infection. H. pylori-induced activation of NF-κB, but not MAPKs, was also inhibited by pre-treatment of WA in the cells. However, WA did not affect VEGF production and HIF-1α stabilization induced by H. pylori in AGS cells. In addition, WA did not influence the growth of H. pylori, suggesting that the anti-inflammatory effect of WA was not due to any bactericidal effect. These findings indicate that WA is a potential preventive or therapeutic agent for H. pylori-mediated gastric inflammation.

Pc2-mediated SUMOylation of WWOX is essential for its suppression of DU145 prostate tumorigenesis.

Tumor suppressor WW domain-containing oxidoreductase (WWOX) is depleted in various cancer types. Here we report that WWOX is modified by small ubiquitin-like modifier (SUMO) proteins and represses DU145 prostate cancer tumorigenesis in a SUMOylation-dependent manner. Ectopic WWOX was shown to associate with SUMO2/3 or E2 Ubc9. Furthermore, we revealed that WWOX SUMOylation was promoted by E3 ligase polycomb2 (Pc2), and that WWOX associated with Pc2. Meanwhile, anisomycin-induced activator protein-1 (AP-1) activity was markedly diminished by co-expression of SUMO and WWOX. Also, WWOX wild type (WT), but not WWOX SUMO mutant (K176A) markedly reduced both DU145 prostate cancer cell proliferation and xenograft tumorigenesis. Collectively, our findings demonstrate that SUMO modification of WWOX is essential for its suppressive activity for DU145 prostate cancer tumorigenesis.

Withaferin A Inhibits Helicobacter pylori-induced Production of IL-1ß in Dendritic Cells by Regulating NF-κB and NLRP3 Inflammasome Activation.

Helicobacter pylori infection is associated with chronic gastritis, peptic ulcer, and gastric cancer. There is evidence that IL-1ß is associated with the development of gastric cancer. Therefore, downregulation of H. pylori-mediated IL-1ß production may be a way to prevent gastric cancer. Withaferin A (WA), a withanolide purified from Withania somnifera, is known to exert anti-inflammatory and anti-tumor effects. In the present study, we explored the inhibitory activity of WA on H. pylori-induced production of IL-1ß in murine bone marrow-derived dendritic cells (BMDCs) and the underlying cellular mechanism. Co-treatment with WA decreased IL-1ß production by H. pylori in BMDCs in a dose-dependent manner. H. pylori-induced gene expression of IL-1ß and NLRP3 (NOD-like receptor family, pyrin domain containing 3) were also suppressed by WA treatment. Moreover, IκB-α phosphorylation by H. pylori infection was suppressed by WA in BMDCs. Western blot analysis revealed that H. pylori induced cleavage of caspase-1 and IL-1ß, as well as increased procaspase-1 and pro IL-1ß protein levels, and that both were suppressed by co-treatment with WA. Finally, we determined whether WA can directly inhibit ac tivation of the NLRP3 inflammasome. NLRP3 activators induced IL-1ß secretion in LPS-primed macrophages, which was inhibited by WA in a dose-dependent manner, whereas IL-6 production was not affected by WA. Moreover, cleavage of IL-1ß and caspase-1 by NLRP3 activators was also dose-dependently inhibited by WA. These findings suggest that WA can inhibit IL-1ß production by H. pylori in dendritic cells and can be used as a new preventive and therapeutic agent for gastric cancer.

Suppression of NF-κB by dieckol extracted from Ecklonia cava negatively regulates LPS induction of inducible nitric oxide synthase gene.

Dieckol, extracted from brown algae, Ecklonia cava, is suggested to elicit anti-inflammatory or anti-tumorigenic activities. However, dieckol-mediated regulatory mechanism for inflammatory response still remains elusive. Here, we show that dieckol suppressed lipopolysaccharide (LPS)-induced inducible nitric oxide synthase (iNOS) expression in mouse leukemic macrophage Raw264.7 cells. Also, dieckol decreased LPS-induced both nitric oxide (NO) production and iNOS promoter-driven transcriptional activity in a dose-dependent manner. On the other hand, LPS-mediated NF-κB activity was inhibited by dieckol treatment. Moreover, results revealed that dieckol diminished LPS-mediated p65 nuclear translocation or IκBα phosphorylation dose-dependently, and reduced LPS-induced phosphorylation of mitogen-activated protein kinases (MAPKs), significantly p38MAPK. Collectively, these findings suggest that dieckol acts as a negative regulator of LPS-mediated iNOS induction through suppression of NF-κB activity, implying a mechanistic role of dieckol in regulation of inflammatory response.