Gold Nanoparticles Downregulate IL-6 Expression/Production by Upregulating microRNA-26a-5p and Deactivating the RelA and NF-κBp50 Transcription Pathways in Activated Breast Cancer Cells.

MicroRNAs (miRNAs) are involved in the modulation of pathogenic genes by binding to their mRNA sequences' 3' untranslated regions (3'UTR). Interleukin-6 (IL-6) is known to promote cancer progression and treatment resistance. In this study, we aimed to explore the therapeutic effects of gold nanoparticles (GNP) against IL-6 overexpression and the modulation of miRNA-26a-5p in breast cancer (BC) cells. GNP were synthesized using the trisodium citrate method and characterized through UV-Vis spectroscopy, dynamic light scattering (DLS), and transmission electron microscopy (TEM). To predict the binding of miR-26a-5p in the IL-6 mRNA's 3'UTR, we utilized bioinformatics algorithms. Luciferase reporter clone assays and anti-miRNA-26a-5p transfection were employed to validate the binding of miR26a-5p in the IL-6 mRNA's 3'UTR. The activity of RelA and NF-κBp50 was assessed and confirmed using Bay 11-7082. The synthesized GNP were spherical with a mean size of 28.3 nm, exhibiting high stability, and were suitable for BC cell treatment. We found that miR-26a-5p directly regulated IL-6 overexpression in MCF-7 cells activated with PMA. Treatment of MCF-7 cells with GNP resulted in the inhibition of IL-6 overexpression and secretion through the increase of miR26a-5p. Furthermore, GNP deactivated NF-κBp65/NF-κBp50 transcription activity. The newly engineered GNP demonstrated safety and showed promise as a therapeutic approach for reducing IL-6 overexpression. The GNP suppressed IL-6 overexpression and secretion by deactivating NF-κBp65/NF-κBp50 transcription activity and upregulating miR-26a-5p expression in activated BC cells. These findings suggest that GNP have potential as a therapeutic intervention for BC by targeting IL-6 expression and associated pathways.

Simultaneously Targeting Two Coupled Signalling Molecules in the Mesenchymal Stem Cell Support Efficiently Sensitises the Multiple Myeloma Cell Line H929 to Bortezomib.

Several studies have shown that diverse components of the bone marrow (BM) microenvironment play a central role in the progression, pathophysiology, and drug resistance in multiple myeloma (MM). In particular, the dynamic interaction between BM mesenchymal stem cells (BM-MSC) and MM cells has shown great relevance. Here we showed that inhibiting both PKC and NF-κB signalling pathways in BM-MSC reduced cell survival in the MM cell line H929 and increased its susceptibility to the proteasome inhibitor bortezomib. PKC-mediated cell survival inhibition and bortezomib susceptibility induction were better performed by the chimeric peptide HKPS than by the classical enzastaurin inhibitor, probably due to its greatest ability to inhibit cell adhesion and its increased capability to counteract the NF-κB-related signalling molecules increased by the co-cultivation of BM-MSC with H929 cells. Thus, inhibiting two coupled signalling molecules in BM-MSC was more effective in blocking the supportive cues emerging from the mesenchymal stroma. Considering that H929 cells were also directly susceptible to PKC and NF-κB inhibition, we showed that treatment of co-cultures with the HKPS peptide and BAY11-7082, followed by bortezomib, increased H929 cell death. Therefore, targeting simultaneously connected signalling elements of BM-MSC responsible for MM cells support with compounds that also have anti-MM activity can be an improved treatment strategy.

Potential role of BAY11-7082, a NF-κB blocker inhibiting experimental autoimmune encephalomyelitis in C57BL/6J mice via declining NLRP3 inflammasomes.

Multiple sclerosis (MS) is an inflammatory autoimmune demyelinating disease of the central nervous system. NOD-like receptor pyrin domain-containing 3 (NLRP3) inflammasome is implicated in the pathogenesis of MS and its animal model, experimental autoimmune encephalomyelitis (EAE). However, the exact mechanism by which NLRP3 inflammasome is involved in the development of MS and EAE is not clear. NF-kappaB (NF-κB) is associated with the activity of NLRP3 inflammasomes, but the role of NF-κB is controversial. We sought to demonstrate that both NF-κB and NLRP3 contribute to development of MS and EAE, and NF-κB pathway is positively correlated with NLRP3 activation in EAE. The inhibitor of NF-κB and NLRP3, BAY11-7082, can prevent and treat EAE. BAY11-7082 (5 and 20 mg/kg/i.p.) was intraperitoneally administered to EAE mice at the time of second injection of pertussis toxin (BAY11-7082 prevention group) or at the onset of symptoms (BAY11-7082 treatment group). mRNA expressions of NLRP3 were determined by qPCR. Protein expressions of NLRP3, NF-κB p65, and phosphorylated p65 were determined by western blotting. Serum levels of inflammatory cytokines were measured by cytometric bead array. Mice treated with BAY11-7082 (both prevention and treatment groups) showed lower clinical scores and attenuated pathological changes. NLRP3 inflammasome and activity of NF-κB in spinal cord of EAE mice was higher than that in control group. However, the level of NLRP3 inflammasome decreased in BAY11-7082 prevention and treatment groups. BAY11-7082 is a promising therapeutic agent for MS. NLRP3 activation in EAE maybe related with NF-κB pathway.

Blockade of Nuclear Factor-Κb (NF-Κb) Pathway Using Bay 11-7082 Enhances Arsenic Trioxide-Induced Antiproliferative Activity in U87 Glioblastoma Cells.

Background: Glioblastoma (GBM), the most aggressive and common form of glioma, accounts for over 13,000 death per year in the United States which indicates the importance of developing novel strategies for the treatment of this fatal malignancy. Although Arsenic trioxide (ATO) hinders the growth and survival of GBM cells, the requirement of concentrations higher than 4 µM for triggering apoptotic cell death has questioned its safety profile. Since the NF-κB signaling pathway plays a crucial role in tumorigenesis and chemo-resistance, targeting this oncogenic pathway may sensitize GBM cells to lower concentrations of ATO. Methods: Anti-tumor effects of ATO as monotherapy and in combination with Bay 11-7082 were determined using MTT, crystal violet staining, Annexin V/PI staining and scratch assays. Quantitative reverse transcription-PCR (qRT-PCR) analysis was applied to elucidate the molecular mechanisms underlying the anti-tumor activity of this combination therapy. Results: Our results revealed that ATO and Bay 11-7082 synergistically inhibited the proliferation and survival of GBM cells. Also, it was revealed that NF-κB inhibition using Bay 11-7082 enhanced the inhibitory effects of ATO on migration of GBM cells via suppressing the expression of NF-κB target genes such as TWIST, MMP2, ICAM-1, and cathepsin B. Furthermore, combination treatment of GBM cells with ATO and Bay 11-7082 significantly induce apoptotic cell death coupled with downregulation of NF-κB anti-apoptotic target genes including Bcl-2 and IAP family members. Conclusion: Altogether, these findings suggest that combination therapy with ATO and Bay 11-7082 may be a promising strategy for the treatment of GBM.

Effects of the NF-κB Signaling Pathway Inhibitor BAY11-7082 in the Replication of ASFV.

African swine fever virus (ASFV) mainly infects the monocyte/macrophage lineage of pigs and regulates the production of cytokines that influence host immune responses. Several studies have reported changes in cytokine production after infection with ASFV, but the regulatory mechanisms have not yet been elucidated. Therefore, the aim of this study was to examine the immune response mechanism of ASFV using transcriptomic and proteomic analyses. Through multi-omics joint analysis, it was found that ASFV infection regulates the expression of the host NF-B signal pathway and related cytokines. Additionally, changes in the NF-κB signaling pathway and IL-1ß and IL-8 expression in porcine alveolar macrophages (PAMs) infected with ASFV were examined. Results show that ASFV infection activates the NF-κB signaling pathway and up-regulates the expression of IL-1ß and IL-8. The NF-κB inhibitor BAY11-7082 inhibited the expression profiles of phospho-NF-κB p65, p-IκB, and MyD88 proteins, and inhibited ASFV-induced NF-κB signaling pathway activation. Additionally, the results show that the NF-κB inhibitor BAY11-7082 can inhibit the replication of ASFV and can inhibit IL-1ß and, IL-8 expression. Overall, the findings of this study indicate that ASFV infection activates the NF-κB signaling pathway and up-regulates the expression of IL-1ß and IL-8, and inhibits the replication of ASFV by inhibiting the NF-κB signaling pathway and interleukin-1 beta and interleukin-8 production. These findings not only provide new insights into the molecular mechanism of the association between the NF-κB signaling pathway and ASFV infection, but also indicate that the NF-κB signaling pathway is a potential immunomodulatory pathway that controls ASF.

Pathway network of pyroptosis and its potential inhibitors in acute kidney injury.

Acute kidney injury (AKI) is a worldwide problem, and there is no effective drug to eliminate AKI. The death of renal cells is an important pathological basis of intrinsic AKI. At present, targeted therapy for TEC death is a research hotspot in AKI therapy. There are many ways of cell death involved in the occurrence and development of AKI, such as apoptosis, necrosis, ferroptosis, and pyroptosis. This article mainly focuses on the role of pyroptosis in AKI. The assembly and activation of NLRP3 inflammasome is a key event in the occurrence of pyroptosis, which is affected by many factors, such as the activation of the NF-κB signaling pathway, mitochondrial instability and excessive endoplasmic reticulum (ER) stress. The activation of NLRP3 inflammasome can trigger its downstream inflammatory cytokines, which will lead to pyroptosis and eventually induce AKI. In this paper, we reviewed the possible mechanism of pyroptosis in AKI and the potential effective inhibitors of various key targets in this process. It may provide potential therapeutic targets for novel intrinsic AKI therapies based on pyroptosis, so as to develop better therapeutic strategies.

Nuclear factor kappa B inhibitor suppresses experimental autoimmune neuritis in mice via declining macrophages polarization to M1 type.

Guillain-Barré syndrome (GBS) is an acute inflammatory and immune-mediated demyelinating disease of the peripheral nervous system (PNS). Macrophages play a central role in its animal model, experimental autoimmune neuritis (EAN), which has been well accepted. Additionally, nuclear factor (NF)-κB inhibitors have been used to treat cancers and have shown beneficial effects. Here, we investigated the therapeutic effect of M2 macrophage and the NF-κB pathway's correlation with macrophage activation in EAN in C57BL/6 mice. We demonstrate that M2 macrophage transfusion could alleviate the clinical symptoms of EAN by reducing the proportion of M1 macrophage in the peak period, inhibiting the phosphorylation of NF-κB p65. The NF-κB inhibitor (BAY-11-7082) could alleviate the clinical symptoms of EAN and shorten the duration of symptoms by reducing the proportion of M1 macrophages and the expression of proinflammatory cytokines. Consequently, BAY-11-7082 exhibits strong potential as a therapeutic strategy for ameliorating EAN by influencing the balance of M1/M2 macrophages and inflammatory cytokines.

Nuclear factor-κB inhibitor Bay11-7082 inhibits gastric cancer cell proliferation by inhibiting Gli1 expression.

Dysregulated nuclear factor (NF)-κB signaling pathway is involved in gastric carcinogenesis. The present study aimed to investigate the antitumor effects of the NF-κB inhibitor, Bay11-7082, on gastric cancer (GC) and elucidate its underlying molecular mechanisms. The MTT assay was performed to assess the effects of Bay11-7082 on the proliferation of HGC27 and MKN45 gastric cancer cells. In addition, the Transwell and wound healing assays were performed to determine cell migration and invasion, respectively. Reverse transcription-quantitative PCR and western blot analyses were performed to detect the mRNA and protein expression levels of the target genes. The results demonstrated that the half-maximal inhibitory concentration (IC50) of Bay11-7082 in HGC27 cells was 24.88, 6.72 and 4.23 nM at 24, 48 and 72 h, respectively. Furthermore, the IC50 of Bay11-7082 in MKN45 cells was 29.11, 11.22 and 5.88 nM at 24, 48 and 72 h, respectively. Treatment with Bay11-7082 significantly suppressed the cell migratory and invasive abilities compared with the control group. Notably, Bay11-7082 suppressed GLI Family Zinc Finger 1 (Gli1) mRNA and protein expression levels. Taken together, the results of the present study demonstrated that Bay11-7082 inhibited GC cell proliferation, at least in part through inhibition of Gli1.

BAY11-7082 inhibits the expression of tissue factor and plasminogen activator inhibitor-1 in type-II alveolar epithelial cells following TNF-α stimulation via the NF-κB pathway.

Pulmonary inflammation strongly promotes alveolar hypercoagulation and fibrinolytic inhibition. NF-κB signaling regulates the expression of molecules associated with coagulation and fibrinolytic inhibition in type-II alveolar epithelial cells (AECII) stimulated by lipopolysaccharide. However, whether TNF-α-induced alveolar hypercoagulation and fibrinolysis inhibition is also associated with the NF-κB pathway remains to be determined. The aim of the present study was to determine whether BAY11-7082, an inhibitor of the NF-κB pathway, inhibits the expressions of tissue factor (TF) and plasminogen activator inhibitor-1 (PAI-1) in AECⅡ in response to TNF-α. Rat AECII were treated with BAY11-7082 for 24 h and stimulated with TNF-α for 1 h. The expression of TF and PAI-1 were determined using western blotting and reverse transcription-quantitative PCR. The concentrations of TF and PAI-1 in culture supernatant were also measured by ELISA. Moreover, levels of NF-κB p65 (p65), phosphorylated (p)-p65 (p-p65), inhibitor of NF-κB α (IκBα) and p-IκBα were also evaluated. Immunofluorescence was used to detect p65 levels in cell nuclei. TNF-α significantly promoted TF and PAI-1 expression either at the mRNA or protein level in AECII cells. Concentrations of TF and PAI-1 in supernatant also significantly increased upon TNF-α stimulation. Furthermore, TNF-α upregulated the levels of p-IκBα, p65, and p-p65 in the cytoplasm. Immunofluorescence analysis indicated that TNF-α increased p65 translocation from the cytoplasm to the nucleus. However, AECII pre-treated with BAY11-7082 expressed lower levels of TF and PAI-1 following TNF-α treatment. Levels of p-IκBα, p65 and p-p65 in the cytoplasm also decreased, and translocation of p65 from cytoplasm into the nucleus was inhibited by BAY11-7082 pretreatment. These findings suggest that BAY11-7082 improves the hypercoagulation and fibrinolytic inhibition induced by TNF-α in alveolar epithelial cells via the NF-κB signaling pathway. BAY11-7082 might represent a therapeutic option for alveolar hypercoagulation and fibrinolytic inhibition in acute respiratory distress syndrome.

Berberine inhibits colorectal tumor growth by suppressing SHH secretion.

Hedgehog plays an important role in a wide range of physiological and pathological conditions. Paracrine activation of Hedgehog pathway in stromal cells increases the expression of VEGF, which promotes neovascularization in colorectal cancer and ultimately the growth of colorectal cancer. Berberine (BBR) has anticancer activity. In this study we investigated whether BBR inhibited the growth of colon cancer through suppressing the paracrine sonic hedgehog (SHH) signaling in vitro and in vivo. We showed that BBR (1-10 µM) dose-dependently inhibited the secretion and expression of SHH protein in HT-29 and SW480 cells. BBR did not influence the transcription of SHH, but promoted the degradation of SHH mRNA, thus decreased the SHH mRNA expression in the colorectal cancer cells. In nude mice bearing HT-29 xenograft, oral administration of BBR (100 mg · kg-1 · d-1) or a positive control drug GDC-0449 (100 mg · kg-1 · d-1) for 4 weeks markedly suppressed the growth of HT-29 tumor with BBR exhibiting a better antitumor efficacy. The tumor growth inhibition caused by BBR or GDC-0449 was comparable to their respective inhibitory effect on the mouse-specific Gli mRNA expression in the tumor. However, BBR (20 µM) did not affect the expression of human transcription factor Gli1 mRNA in HT-29 and SW480 cells. In conclusion, BBR promotes the degradation of SHH mRNA in colorectal cancer cells, interrupting the paracrine Hedgehog signaling pathway activity thus suppresses the colorectal cancer growth. This study reveals a novel molecular mechanism underlying the anticancer action of BBR.

Naringin Combined with NF-κB Inhibition and Endoplasmic Reticulum Stress Induces Apoptotic Cell Death via Oxidative Stress and the PERK/eIF2α/ATF4/CHOP Axis in HT29 Colon Cancer Cells.

Currently, combination therapy is considered the most effective solution for a selective chemotherapeutic effect in the treatment of colon cancer. This study investigated the death of both colon cancer HT29 cells and healthy vascular smooth muscle TG-Ha-VSMC cells (VSMCs) induced by naringin combined with endoplasmic reticulum (ER) stress and NF-κB inhibition. Naringin combined with tunicamycin and BAY 11-7082 suppressed the proliferation of HT29 cells in a dose-dependent manner and induced particularly apoptotic death without significantly affecting healthy VSMCs according to Annexin V/PI staining and AO/EB staining analyses. Insufficient antioxidant defense and heat shock response as well as excessive ROS generation were observed in HT29 cells following combination therapy. Quantitative real-time PCR and western blot analysis demonstrated that drug combination-induced mitochondrial apoptosis was activated through the ROS-mediated PERK/eIF2α/ATF4/CHOP pathway. Additionally, naringin combination significantly reduced the sXBP expression induced by tunicamycin+BAY 11-7082 in a dose-dependent manner. In conclusion, this study found that naringin combined with tunicamycin+BAY 11-7082 efficiently induced apoptotic cell death in HT29 colon cancer cells via oxidative stress and the PERK/eIF2α/ATF4/CHOP pathway, suggesting that naringin combined with tunicamycin plus BAY 11-7082 could be a new combination therapy strategy for effective colon cancer treatment with minimal side effects on healthy cells.

The temporal effects of topical NF-κB inhibition, in the in vivo prevention of bile-related oncogenic mRNA and miRNA phenotypes in murine hypopharyngeal mucosa: a preclinical model.

Supraesophageal bile reflux at strongly acidic pH can cause hypopharyngeal squamous cell cancer, through activation of the oncogenic NF-κB-related pathway. We hypothesize that topical pre- or post-application of pharmacologic NF-κB inhibitor, BAY 11-7082 (0.25 µmol), on murine (C57BL/6J) HM (twice a day for 10 days) can effectively inhibit acidic bile (10 mmol/l; pH 3.0) induced oncogenic molecular events, similar to prior in vitro findings. We demonstrate that the administration of BAY 11-7082, either before or after acidic bile, eliminates NF-κB activation, prevents overexpression of Bcl2, Rela, Stat3, Egfr, Tnf, Wnt5a, and deregulations of miR-192, miR-504, linked to bile reflux-related hypopharyngeal cancer. Pre- but not post-application of NF-κB inhibitor, significantly blocks overexpression of Il6 and prostaglandin H synthases 2 (Ptgs2), and reverses miR-21, miR-155, miR-99a phenotypes, supporting its early bile-induced pro-inflammatory effect. We thus provide novel evidence that topical administration of a pharmacological NF-κB inhibitor, either before or after acidic bile exposure can successfully prevent its oncogenic mRNA and miRNA phenotypes in HM, supporting the observation that co-administration of NF-κB inhibitor may not be essential in preventing early bile-related oncogenic events and encouraging a capacity for further translational exploration.

Role of PARP1 regulation in radiation-induced rescue effect.

Radiation-induced rescue effect (RIRE) in cells refers to the phenomenon where irradiated cells (IRCs) receive help from feedback signals produced by partnered bystander unirradiated cells (UIRCs) or from the conditioned medium (CM) that has previously conditioned the UIRCs. In the present work, we explored the role of poly (ADP-ribose) polymerase 1 (PARP1) regulation in RIRE and the positive feedback loop between PARP1 and nuclear factor-kappa-light-chain-enhancer of activated B cell (NF-κB) in RIRE using various cell lines, including HeLa, MCF7, CNE-2 and HCT116 cells. We first found that when the IRCs (irradiated with 2 Gy X-ray) were treated with CM, the relative mRNA expression levels of both tumor suppressor p53-binding protein 1 (53BP1) and PARP1, the co-localization factor between 53BP1 and γH2AX as well as the fluorescent intensity of PARP1 were reduced. We also found that IRCs treated with the PARP1 inhibitor, Olaparib (AZD2281) had a higher 53BP1 expression. These results illustrated that PARP1 was involved in RIRE transcriptionally and translationally. We further revealed that treatment of IRCs with CM together with Olaparib led to significantly lower mRNA expression levels and fluorescent intensities of NF-κB, while treatment of IRCs with CM together the NF-κB inhibitor BAY-11-7082 led to significantly lower mRNA expression levels as well as fluorescent intensities of PARP1. These results illustrated that PARP1 and NF-κB were involved in the positive feedback loop transcriptionally and translationally. Thus, the results supported the occurrence of a PARP1-NF-κB positive feedback loop in RIRE. The present work provided insights into potential exploitation of inhibition of PARP1 and/or the PARP1-NF-κB positive feedback loop in designing adjuncts to cancer radiotherapeutics.

Synergistic inhibition of lung cancer cells by EGCG and NF-κB inhibitor BAY11-7082.

Lung cancer has a poor 5-year survival rate and is the leading cause of cancer-related deaths worldwide. Thus, the development of more efficient therapeutic strategies is urgently needed. Many studies have shown that EGCG, a major polyphenol found in green tea, has potential anticancer effects. The present study aims to investigate the molecular mechanism of EGCG-mediated inhibition of proliferation in lung cancer cells and to explore the effects of combined treatment with EGCG and an NF-κB inhibitor, BAY11-7082, in A549 and H1299 cells both in vitro and in vivo. Our results showed that EGCG inhibits cell proliferation and migration and induces apoptosis in A549 and H1299 cells at relatively high concentrations (IC50=86.4 µM for A549 cells and 80.6 µM for H1299 cells). These effects are partially achieved via inhibition of the NF-κB signaling pathway. Combined treatment with EGCG and BAY11-7082, a potent NF-κB inhibitor, shows significant synergistic effects at relatively low concentrations. The inhibition rate reached approximately 50% in cells treated for 72 h with 20 µM EGCG and 5 µM (A549 cells) or 2.5 µM BAY11-7082 (H1299 cells). This synergistic anti-tumor effect was also observed in a xenograft model. These results indicated that EGCG inhibits lung cancer cell proliferation by suppressing NF-κB signaling. Coadministration of EGCG and BAY11-7082 has a synergistic effect both in vitro and in vivo and may serve as a novel therapeutic strategy for lung cancer.

[Bortezomib Inhibits Extranodal Natural Killer/T Cell Lymphoma, Nasal Type by Targeting NF-κB Signaling Pathway].

OBJECTIVE: To investigate the anti-tumor effect of bortezomib on extranodal natural killer/T cell lymphoma, nasal type (ENKTL). METHODS: SNK-6 cells were treated with different mass concentrations of bortezomib (0, 1, 2, 4, 5, 6 ng/mL) for 24, 48, 72 h, and different concentrations of nuclear factor-kappa B (NF-κB) signaling pathway inhibitor BAY11-7082 (0, 1, 2, 2.5, 5, 10, 20 µmol/L) for 24 h respectively, then the cell viability was measured by CCK8 kit and the half inhibitory concentration (IC 50) was calculated. SNK-6 cells were treated with 30µmol/L Z-VAD-FMK (Pan-caspase inhibitor)+3ng/mL bortezomib, and 5, 10 µmol/L BAY11-7082+3 ng/mL bortezomib for 24 h respectively, then the cell viability was measured by CCK8 kit. After treatment of SNK-6 cells with different mass concentrations of bortezomib for 24 h, apoptosis was detected by AnnexinⅤ/PI flow cytometry; the expression of apoptosis-related protein Caspase-3, poly ADP-ribose polymerase (PARP) and Bcl-2 and NF-κB signaling pathway key proteins P65 and P100/P52 were detected by Western blot. RESULTS: Bortezomib inhibited the proliferation of SNK-6 cells in a dose-dependent manner ( P<0.05), and IC 50( (2.87±0.06) ng/mL) at 24 h was lower than that at 48 h and 72 h ( P<0.05). BAY11-7082 also inhibited the proliferation of SNK-6 cells with an IC 50= (9.73±0.36) µmol/L at 24 h. The combination treatment indicated that Z-VAD-FMK could attenuate the inhibitory effect of bortezomib on the proliferation of SNK-6 cells ( P<0.05), while BAY11-7082 could enhance the inhibitory effect of bortezomib on the proliferation of SNK-6 cells ( P<0.05). After treatment of SNK-6 cells with bortezomib for 24 h, apoptosis-related protein Caspase-3 cleavage, PARP activation, and Bcl-2 cleavage; NF-κB signaling pathway-related protein P65 phosphorylation level decreased, and P52 decreased. CONCLUSION: Bortezomib inhibits ENKTL cells proliferation by inhibiting NF-κB signaling pathway and induces apoptosis of ENKTL cells via mitochondria-mediated caspase pathway.

Inflammasome as a promising therapeutic target for cancer.

Inflammasomes are the major mechanistic complexes that include members of the NOD-like receptor (NLRs) or AIM2-like receptors (ALRs) families, which are affiliated with the innate immune system. Once NLRs or ALRs are activated by pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs), the caspase-1 or -11 is activated by binding with NLRs or ALRs via its own unique cytosolic domains. As a result, caspase-1 or -11 enhances the production of IL-1ß and IL-18, which results in inflammation via the recruitment of immune cells, such as macrophages, and the promotion of programmed cell death mechanisms such as pyroptosis. In addition, the consistent cascades of inflammasomes would precede both minor and severe autoimmune diseases and cancers. The clinical relevance of inflammasomes in multiple forms of cancer highlights their therapeutic promise as molecular targets. To closely analyze the physiological roles of inflammasomes in cancers, here, we describe the fundamental knowledge regarding the current issues of inflammasomes in relevant cancers, and discuss possible therapeutic values in targeting these inflammasomes for the prevention and treatment of cancer.

The oncometabolite 2-hydroxyglutarate inhibits microglial activation via the AMPK/mTOR/NF-κB pathway.

Microglia, the brain-resident macrophage, is known as the innate immune cell type in the central nervous system. Microglia is also the major cellular component of tumor mass of gliomas that plays a key role in glioma development. Mutations of isocitrate dehydrogenases 1 and 2 (IDH1/2) frequently occur in gliomas, which leads to accumulation of oncometabolic product 2-hydroxyglutarate (2HG). Moreover, IDH1/2 mutations were found to correlate with better prognosis in glioma patients. In the present study, we investigated the effects of the 2HG on microglial inflammatory activation. We showed that the conditioned media (CM) from GL261 glioma cells stimulated the activation of BV-2 microglia cells, evidenced by markedly increased expression of interleukin-6 (IL-6), IL-1ß, tumor necrosis factor-α (TNF-α), CCL2 (C-C motif chemokine ligand 2) and CXCL10 (C-X-C motif chemokine 10). CM-induced expression of proinflammatory genes was significantly suppressed by pretreatment with a synthetic cell-permeable 2HG (1 mM) or a nuclear factor-κB (NF-κB) inhibitor BAY11-7082 (10 µM). In lipopolysaccharide (LPS)- or TNF-α-stimulated BV-2 microglia cells and primary microglia, pretreatment with 2HG (0.25-1 mM) dose-dependently suppressed the expression of proinflammatory genes. We further demonstrated that 2HG significantly suppressed LPS-induced phosphorylation of IκB kinase α/ß (IKKα/ß), IκBα and p65, IκB degradation, and nuclear translocation of p65 subunit of NF-κB, as well as NF-κB transcriptional activity. Similarly, ectopic expression of mutant isocitrate dehydrogenase 1 (IDH1) (R132H) significantly decreased TNF-α-induced activation of NF-κB signaling pathway. Finally, we revealed that activation of adenosine 5'-monophosphate-activated protein kinase (AMPK) and subsequent inhibition of mammalian target of rapamycin (mTOR) signaling contributed to the inhibitory effect of 2HG on NF-κB signaling pathway in BV-2 cells. Taken together, these results, for the first time, show that oncometabolite 2HG inhibits microglial activation through affecting AMPK/mTOR/NF-κB signaling pathway and provide evidence that oncometabolite 2HG may regulate glioma development via modulating microglial activation in tumor microenvironment.

NF-κB inhibitor, BAY11-7082, suppresses M2 tumor-associated macrophage induced EMT potential via miR-30a/NF-κB/Snail signaling in bladder cancer cells.

BACKGROUND: Chronic inflammatory microenvironment has been shown to play a key role in initiating tumorigenesis and facilitating malignant progression. Primary tumors surrounded with and infiltrated by tumor-associated macrophages (TAMs) significantly promote the epithelial-to-mesenchymal transition (EMT) and distant metastasis in urothelial bladder cancer. METHODS: In this study, we aimed to explore the potential of targeting TAMs for the treatment of malignant bladder cancer. RESULTS: First, we found a higher number of TAMs, CD68 (pan-macrophage marker), and clever-1 (M2 macrophage marker) was associated with a higher pT category and grade in a cohort of 108 patients. In vitro assays showed that the co-culture of TAMs promoted the metastatic potential in HTB-1 and T24 by up-regulating EMT markers including Snail, VEGF and Vimentin, as well as oncogenic markers such as ß-catenin and NF-κB. More importantly, M2 co-cultured HTB-1 and T24 showed an increased level of metastatic microRNA, miR-30. Silencing of miR-30 resulted in the reduced metastatic potential, migration/invasion, in association with the decreased expression of Twist1 and Vimentin. The addition of BAY11-7082 into the TAM/cancer co-culture system significantly reduced the M2 phenotype and tumorigenic properties. Coincidentally, miR-30a level was significantly lowered in the presence of BAY11-7082. CONCLUSION: Our study demonstrated that AMs promoted metastatic potential of bladder cancer cells via promoting EMT through the increase of miR-30a. BAY11-7082 treatment suppressed both oncogenic and metastatic potential in bladder cancer cells while preventing the M2 polarization of TAMs.

Bay11-7082 facilitates wound healing by antagonizing mechanical injury- and TNF-α-induced expression of MMPs in posterior cruciate ligament.

Purposes: To investigate the ability of synoviocytes (SCs) in regulating MMPs expression in the posterior cruciate ligament fibroblasts (PCLfs) after TNF-α treatment, to test whether a specific inflammation inhibitor Bay11-7082 can antagonize the expression of MMPs in PCLfs after injury. Methods: The microenvironment of knee joint cavity after PCL injury was mimicked in an in vitro co-culture system. The effects of TNF-α treatment on the expression of MMPs in PCL fibroblasts (PCLfs) were studied. The expression of MMPs mRNA and protein was detected by qRT-PCR and western blot. For the in vivo study, the Bay11-7082 inhibitor was injected into the knee joint cavity after injury, and then were performed on histological analysis. Results: In the mono-culture conditions, 6% mechanical injury upregulated the expression of MMP-2, whereas downregulated MMP-1 and -3, additionally 12% mechanical injury were upregulated all. However, in co-culture conditions, 6% and 12% both significantly increased MMPs expressions. Stretch injury and TNF-α treatment significantly upregulated expression of MMPs mRNA and protein levels in mono-cultured PCLfs. This effect was more significant in PCLfs Plus SCs co-culture system, in which the cells were treated by combination of stretch injury and TNF-α. In addition, Bay11-7082, a specific inflammation inhibitor, could significantly decrease the expression of MMPs induced by stretch injury and/or TNF-α treatment. Less infiltrated inflammatory cells and more integrated tissues were detected in injury PCL 2 weeks after Bay11-7082 treatment, compared to injury group. Immunofluorescent staining showed very low expression levels of MMPs in PCL of Bay11-7082-treated group, compared to the injury groups. Conclusions: SCs sever as the supporting cells that aggravate the TNF-α-induced MMPs accumulation in PCLfs. Inhibition of the expression of MMPs by Bay11-7082 is a promising way to facilitate the self-healing of PCL.

NF-KappaB Pathway Is Involved in Bone Marrow Stromal Cell-Produced Pain Relief.

Bone marrow stromal cells (BMSCs) produce long-lasting attenuation of pain hypersensitivity. This effect involves BMSC's ability to interact with the immune system and activation of the endogenous opioid receptors in the pain modulatory circuitry. The nuclear factor kappa B (NF-κB) protein complex is a key transcription factor that regulates gene expression involved in immunity. We tested the hypothesis that the NF-κB signaling plays a role in BMSC-induced pain relief. We focused on the rostral ventromedial medulla (RVM), a key structure in the descending pain modulatory pathway, that has been shown to play an important role in BMSC-produced antihyperalgesia. In Sprague-Dawley rats with a ligation injury of the masseter muscle tendon (TL), BMSCs (1.5 M/rat) from donor rats were infused i.v. at 1 week post-TL. P65 exhibited predominant neuronal localization in the RVM with scattered distribution in glial cells. At 1 week, but not 8 weeks after BMSC infusion, western blot and immunostaining showed that p65 of NF-κB was significantly increased in the RVM. Given that chemokine signaling is critical to BMSCs' pain-relieving effect, we further evaluated a role of chemokine signaling in p65 upregulation. Prior to infusion of BMSCs, we transduced BMSCs with Ccl4 shRNA, incubated BMSCs with RS 102895, a CCR2b antagonist, or maraviroc, a CCR5 antagonist. The antagonism of chemokines significantly reduced BMSC-induced upregulation of p65, suggesting that upregulation of p65 was related to BMSCs' pain-relieving effect. We then tested the effect of a selective NF-κB activation inhibitor, BAY 11-7082. The mechanical hyperalgesia of the rat was assessed with the von Frey method. In the pre-treatment experiment, BAY 11-7082 (2.5 and 25 pmol) was injected into the RVM at 2 h prior to BMSC infusion. Pretreatment with BAY 11-7082 attenuated BMSCs' antihyperalgesia, but post-treatment at 5 weeks post-BMSC was not effective. On the contrary, in TL rats receiving BAY 11-7082 without BMSCs, TL-induced hyperalgesia was attenuated, consistent with dual roles of NF-κB in pain hypersensitivity and BMSC-produced pain relief. These results indicate that the NF-κB signaling pathway in the descending circuitry is involved in initiation of BMSC-produced behavioral antihyperalgesia.