IκBα Degradation Research Articles

UCHL1 regulates inflammation via MAPK and NF-κB pathways in LPS-activated macrophages.

Inflammation is a common pathophysiological process as well as a clinical threat that occurs in various diseases worldwide. It is well-documented that nuclear factor-κB (NF-κB) and mitogen-activated protein kinase pathways are involved in inflammatory reactions to microbial infections in lipopolysaccharide (LPS)-activated macrophages. The deubiquitinase ubiquitin carboxyl-terminal hydrolase-L1 (UCHL1) has been reported as an oncoprotein to promote the growth and progression of cancer cells. However, the regulatory mechanism of UCHL1 in inflammation is currently unclear. Here, we aimed to assess the effects of UCHL1 on LPS-associated inflammatory response in vitro and in vivo by enzyme-linked immunosorbent assay, quantitative reverse-transcription polymerase chain reaction, and western blot analysis. This study identified that inhibition or knockdown of UCHL1 decreased the amounts of the key pro-inflammatory cytokines, including interleukin-6 and tumor necrosis factor-α in macrophages. Additionally, inhibition of UCHL1 suppressed LPS-induced extracellular signal-regulated protein kinase 1/2 phosphorylation and NF-κB translocation by regulating the inhibitor of NF-κB. Mechanically, UCHL1 interacts with IκBα protein in THP-1. Meanwhile, inhibition of UCHL1 blocked the LPS-induced degradation of IκBα through the ubiquitin-proteasome system. Moreover, in vivo assay showed that suppression of UCHL1 notably reduced the LPS-induced animal death and release of pro-inflammatory cytokines. Overall, the current findings uncover that UCHL1 functions as a crucial regulator for inflammatory response via reversing the degradation of IκBα, representing a potential target for the treatment of inflammatory diseases.

Benzoylaconine Modulates LPS-Induced Responses Through Inhibition of Toll-Like Receptor-Mediated NF-κB and MAPK Signaling in RAW264.7 Cells.

Previous studies have shown that benzoylaconine (BAC), a representative monoester alkaloid, has a potential anti-inflammatory effect. This study investigated the underlying molecular mechanisms using the mode of LPS-activated RAW264.7 macrophage cells. Our findings showed that BAC significantly suppressed the release of pro-inflammatory cytokines and mediators, including IL-6, TNF-α, IL-1β, ROS, NO, and PGE2. BAC treatment also effectively downregulated the elevated protein levels of iNOS and COX-2 induced by LPS in a dose-dependent manner. In this study, we found that BAC inhibited LPS-induced NF-κB activation by reducing the phosphorylation and degradation of IκBα by western blotting and blocking the nuclear translocation of p65 using an immunofluorescence assay. The elevated protein levels of JNK, p38, and ERK phosphorylation after LPS stimulation were restored effectively by BAC treatment. The protein expression of Toll-like receptor 4 (TLR4) and LPS-induced phosphorylation of TAK1, which is a crucial upstream regulatory factor of TLR-induced MAPK and NF-κB signaling, were inhibited by BAC in activated RAW264.7 macrophages. Moreover, BAC decreased the levels of TAK1 phosphorylation and pro-inflammatory cytokines and mediators associated with MAPK and NF-κB activation, similar to TLR4 inhibitor TAK-242. These findings demonstrated that BAC exhibited an anti-inflammatory effect by the inhibition of TLR-induced MAPK and NF-κB pathways, indicating that it could potentially be used for treating inflammatory diseases.

Human mesenchymal stromal cells small extracellular vesicles attenuate sepsis-induced acute lung injury in a mouse model: the role of oxidative stress and the mitogen-activated protein kinase/nuclear factor kappa B pathway

Background aimsAcute lung injury (ALI) secondary to sepsis is a complex disease associated with high morbidity and mortality. Mesenchymal stem cells (MSCs) and their conditioned medium have been demonstrated to reduce alveolar inflammation, improve lung endothelial barrier permeability and modulate oxidative stress in vivo and in vitro. Recently, MSCs have been found to release small extracellular vesicles (sEVs) that can deliver functionally active biomolecules into recipient cells. The authors’ study was designed to determine whether sEVs released by MSCs would be effective in sepsis-induced ALI mice and to identify the potential mechanisms. MethodsA total of 6 h after cercal ligation and puncture, the mice received saline, sEV-depleted conditioned medium (sEVD-CM) or MSC sEVs via the tail vein. ResultsThe administration of MSC sEVs improved pulmonary microvascular permeability and inhibited both histopathological changes and the infiltration of polymorphonuclear neutrophils into lung tissues. In addition, the activities of antioxidant enzymes were significantly increased in the group treated with sEVs compared with the saline and sEVD-CM groups, whereas lipid peroxidation was significantly decreased. Furthermore, sEVs were found to possibly inhibit phosphorylation of the mitogen-activated protein kinase/nuclear factor kappa B (MAPK/NF-κB) pathway and degradation of IκB but increase the activities of nuclear factor erythroid 2-related factor 2 and heme oxygenase 1. ConclusionsThese findings suggest that one of the effective therapeutic mechanisms of sEVs against sepsis-induced ALI may be associated with upregulation of anti-oxidative enzymes and inhibition of MAPK/NF-κB activation.

MCPIP1 Enhances TNF-α-Mediated Apoptosis through Downregulation of the NF-κB/cFLIP Axis

Simple SummaryTNF-α is considered to be a potential therapeutic drug for cancer, but the systemic toxicity problem still exists in clinical use. MCPIP1 plays a crucial role in anti-inflammatory and anti-viral contexts, as well as in the inhibition of cell growth. This study aims to investigate the roles of MCPIP1 in TNF-α-treated cells and their underlying molecular mechanisms. MCPIP1 was found to enhance TNF-α-induced apoptosis by activating the caspase cascade, and pan-caspase inhibitor and the caspase-1/-4 inhibitor could reverse TNF-α/MCPIP1-mediated apoptosis. MCPIP1 was first identified with cleavage site of caspase-1/-4 and could be cleaved during apoptosis. Downregulation of NF-κB activation and a caspase-8 inhibitor, cFLIP, were associated with TNF-α/MCPIP1-mediated apoptosis.Monocyte chemoattractant protein-1-induced protein 1 (MCPIP1) is rapidly produced under proinflammatory stimuli, thereby feeding back to downregulate excessive inflammation. In this study, we used the stable, inducible expressions of wild-type (WT) MCPIP1 and an MCPIP1-D141N mutant in T-REx-293 cells by means of a tetracycline on (Tet-on) system. We found that WT MCPIP1 but not MCPIP1-D141N mutant expression dramatically increased apoptosis, caspase-3, -7, -8, and -9 activation, and c-Jun N-terminal kinase (JNK) phosphorylation in TNF-α-treated cells. The pan-caspase inhibitor, z-VAD-fmk, and the caspase-1 inhibitor, z-YVAD-fmk, but not the JNK inhibitor, SP600125, significantly reversed apoptosis and caspase activation in TNF-α/MCPIP1-treated cells. Surprisingly, MCPIP1 itself was also cleaved, and the cleavage was suppressed by treatment with the pan-caspase inhibitor and caspase-1 inhibitor. Moreover, MCPIP1 was found to contain a caspase-1/-4 consensus recognition sequence located in residues 234~238. As expected, the WT MCPIP1 but not the MCPIP1-D141N mutant suppressed NF-κB activation, as evidenced by inhibition of IκB kinase (IKK) phosphorylation and IκB degradation using Western blotting, IKK activity using in vitro kinase activity, and NF-κB translocation to nuclei using an immunofluorescence assay. Interestingly, MCPIP1 also significantly inhibited importin α3 and importin α4 expressions, which are major nuclear transporter receptors for NF-κB. Inhibition of NF-κB activation further downregulated expression of the caspase-8 inhibitor, cFLIP. In summary, the results suggest that MCPIP1 could enhance the TNF-α-induced apoptotic pathway through decreasing NF-κB activation and cFLIP expression.

Comparison of the Potency of Pterostilbene with NF-κB Inhibitors in Platelet Activation: Mutual Activation by Akt-NF-κB Signaling in Human Platelets

Myocardial infarction and cerebral ischemic stroke are prominent causes of death worldwide. Platelets play major roles in these diseases, although they are anucleated cells, but also express the NF-κB. Pterostilbene (PTE) possesses some intriguing pharmacological properties, including the capacity to inhibit platelet activation. We investigated the inhibitory role of PTE in NF-κB-mediated signal events and compared the relative potency with that of classical NF-κB inhibitors. PTE and IκB kinase (IKK) inhibitor, BAY11-7082, and proteasome inhibitor, Ro106-9920, inhibited platelet aggregation; the activity of BAY11-7082 and PTE were similar, but Ro106-9920 was weak in this reaction. PTE and BAY11-7082 diminished NF-κB signaling molecules, including IKK, IκBα, and p65 phosphorylation, and reversed IκBα degradation. However, Ro106-9920 was only effective in diminishing p65 phosphorylation and reversing IκBα degradation. In investigating the role of Akt and NF-κB in cell signaling events, MK-2206 (an inhibitor of Akt) markedly abolished IKK and p65 phosphorylation; BAY11-7082 also reduced Akt phosphorylation. PTE exhibited more potent activity in vivo than did BAY11-7082 in acute pulmonary thromboembolism. In conclusion, we identified a distinctive activation pathway of NF-κB and Akt involved in PTE-mediated antiplatelet aggregation, and PTE demonstrated powerful activity as a prophylactic and as clinical therapy for cardiovascular diseases.

DAPK2 activates NF-κB through autophagy-dependent degradation of I-κBα during thyroid cancer development and progression.

BackgroundDeath-associated protein kinase 2 (DAPK2) is a serine/threonine kinase, which has been implicated in autophagy and apoptosis. DAPK2 functions as a tumor suppressor in various cancers. However, the role of DAPK2 in thyroid cancer (TC) is unclear.MethodsRNA sequencing of human TC samples was performed to identify differentially expressed genes that may play a role in TC development. The messenger RNA (mRNA) expression of DAPK2 was verified by quantitative real-time polymerase chain reaction (qRT-PCR). To investigate the role of DAPK2 in TC development, DAPK2 was knocked down and overexpressed in a TTA1 cell line. The effect of DAPK2 on cell proliferation, sensitization of TNF-related apoptosis-inducing ligand (TRAIL)-induced apoptosis and tumor growth was examined. The effect of DAPK2 on autophagy and NF-κB activation was investigated to address the underlying mechanism.ResultsDAPK2 was upregulated in TC. Knockdown of DAPK2 in TTA1 cells led to reduced cell proliferation, sensitization of TRAIL-induced apoptosis, and restricted tumor growth both in vitro and in vivo, while overexpression of DAPK2 exhibited the opposite effect. Mechanistically, DAPK2 promoted autophagy as demonstrated by the accumulation of microtubule-associated protein 1A/1B-light chain 3 (LC3)-II, which correlated with the level of nuclear factor-κB (NF-κB) activation. Knockdown of inhibitory-κBα (I-κBα) in short hairpin (sh) DAPK2 TTA1 cells restored the activity of NF-κB, suggesting DAPK2 activated NF-κB through autophagy-mediated I-κBα degradation.ConclusionsOur findings revealed a pivotal role of DAPK2 in thyroid carcinogenesis, being required for tumor growth and for resistance to TRAIL-induced apoptosis through autophagy-mediated I-κBα degradation. This result provides a novel target for the therapy of TC.

Abstract 3040: Inhibition of Exportin-1 as part of combinatorial treatment for patients with high-risk neuroblastoma

Abstract Background: In many adult cancers, upregulation of the nuclear-to-cytoplasmic transport protein Exportin-1 (XPO1) correlates with poor outcome and responsiveness to selinexor, an FDA-approved XPO1 inhibitor. Similar data are emerging in childhood cancers, for which selinexor is being evaluated in early phase clinical studies. Our aims are to identify patients most likely to benefit from selinexor combination therapy and define the cellular mechanism responsible this effect. We hypothesize that selinexor and the proteasome inhibitor, bortezomib, act synergistically to decrease NF-kB-mediated proliferation by halting excessive exportation and proteasome degradation of IkB, an XPO1 cargo protein. Methods: We performed ion intensity-based label-free semiquantitative proteomic profiling on primary tumor material from fifty patients with uniformly treated high-risk NB, half of whom were long term survivors and half of whom died <18 months from diagnosis from disease progression. We cross-referenced this dataset with gene expression profiling data available through the R2 database. A panel of neuroblastoma cell lines was screened for XPO1 expression using Western blotting, sensitivity to selinexor and bortezomib as single agents, and in combination using cellular viability assays. Localization and quantification of IkB after vehicle or selinexor treatment was done by confocal imaging, using immunofluorescent labeling and Volocity software. Flow cytometry was used to define treatment effect on cell cycle arrest and apoptosis using BrdU and Annexin-V labeling, respectively. siRNA-mediated knockdown of IkB was used to investigate the relationship between presence of IkB and drug effectiveness. NF-kB activity was measured with a luciferase assay. Results: High tumor protein and gene expression of XPO1 correlates with poor outcome. Neuroblastoma cell lines demonstrate variably high XPO1 expression compared to normal tissue, as well as sensitivity to selinexor (IC50 range 4-300nM). IkB is retained in the nuclear compartment after treatment with selinexor. When selinexor is used in combination with bortezomib the effect on cell proliferation is synergistic. Flow cytometry demonstrates that selinexor alone contributes to G1-0 arrest while combination treatment results in significantly enhanced apoptosis. Measurement of NF-kB transcriptional activity shows decrease in activity with single agents and the least amount of activity in combination and siRNA against IkB diminishes the effect of single and combination treatment. Conclusions: This work demonstrates that XPO1 expression is a biomarker of highly aggressive neuroblastoma and may define patients most likely to be responsive to treatment with selinexor-based combinatorial therapies. Synergistic treatment effect with selinexor and bortezomib is mediated, in part, through the presence of IkB to regulate NF-kB signaling. Citation Format: Basia Galinski, Yossef Landesman, Daniel Weiser. Inhibition of Exportin-1 as part of combinatorial treatment for patients with high-risk neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3040.

Betulinic Acid Ameliorates the Severity of Acute Pancreatitis via Inhibition of the NF-κB Signaling Pathway in Mice.

Acute pancreatitis (AP) is an inflammatory disorder, involving acinar cell death and the release of inflammatory cytokines. Currently, there are limited effective therapeutic agents for AP. Betulinic acid (BA) is a pentacyclic triterpenoid extracted from Betula platyphylla that has been shown to have anti-inflammatory effects. In this study, we aimed to investigate the effects of BA on AP and elucidate the potential underlying mechanisms. AP was induced in mice through six intraperitoneal injections of cerulein. After the last cerulein injection, the mice were sacrificed. Our results revealed that pre- and post-treatment with BA significantly reduced the severity of pancreatitis, as evidenced by a decrease in histological damage in the pancreas and lung, serum amylase and lipase activity and pancreatic myeloperoxidase activity. Furthermore, BA pretreatment reduced proinflammatory cytokine production, augmentation of chemokines, and infiltration of macrophages and neutrophils in the pancreas of AP mice. In addition, mice that were pretreated with BA showed a reduction in Iκ-Bα degradation and nuclear factor-kappa B (NF-κB) binding activity in the pancreas. Moreover, BA reduced the production of proinflammatory cytokines and NF-κB activation in pancreatic acinar cells (PACs). These findings suggest that BA may have prophylactic and therapeutic effects on AP via inhibition of the NF-κB signaling pathway.

MAP3K7 Is Recurrently Deleted in Pediatric T-Lymphoblastic Leukemia and Affects Cell Proliferation Independently of NF-κB

Deletions of 6q15-16.1 are recurrently found in pediatric T-cell acute lymphoblastic leukemia (T-ALL). This chromosomal region includes the mitogen-activated protein kinase kinase kinase 7 (MAP3K7) gene which is crucial for innate immune signaling. It is functionally and prognostically relevant both in hematologic malignancies and solid tumors. However, its distinct biological functions and relevance for different tumor entities are cell-type specific and remain controversial. Here, we show that MAP3K7 is recurrently deleted in pediatric T-ALL cases and that a residual expression of MAP3K7 is indispensable for T-ALL cell lines.We determined the frequency of MAP3K7 deletions in a cohort of 327 pediatric T-ALL patients by multiplex ligation-dependent probe amplification (MLPA). In a subgroup, we additionally employed targeted sequencing to identify point mutations. Deletions of MAP3K7 were identified in approximately 10% and point mutations in approximately 1% of children with T-ALL. All deletions of 6q15 were heterozygous and the adjacent CASP8AP2 gene was co-deleted in 32 of 33 cases. Clinically, the MAP3K7/CASP8AP2 deletions were significantly associated with a mature T-ALL immunophenotype, but not with any other patient characteristics or with the cumulative incidence of relapse and overall survival. Furthermore, deletions of MAP3K7/CASP8AP2 were preferentially associated with SIL-TAL1 fusions. Within the subgroup of SIL-TAL1 positive patients, those with MAP3K7/CASP8AP2 deletion showed a trend towards a higher risk of relapse compared to SIL-TAL1 -positive MAP3K7/CASP8AP2 -negative patients (25% vs 8%, p=0.13).We next analyzed the effects of shRNA-mediated MAP3K7 depletion on cell proliferation, apoptosis and NF-κB activation in three T-ALL cell lines (CCRF-CEM, Jurkat, MOLT4) that do not carry MAP3K7 deletions. We aimed at phenocopying a MAP3K7 deletion by using an Adeno-associated viral vector-mediated transduction system to efficiently deliver anti-MAP3K7 shRNA together with a GFP reporter to these cell lines. Transduction efficiency was above 80% for the cell lines CCRF-CEM and Jurkat. Depletion efficiency was uniformly in the range of 75%. Treatment with anti-MAP3K7 shRNA in comparison to non-silencing shRNA significantly slowed down proliferation in all three T-ALL cell lines and resulted in an increase of apoptotic cells up to 8-fold as measured by Annexin V staining.In order to assess the contribution of NF-κB signaling to the effects of MAP3K7 depletion, cells were treated with TNF-α and cell lysates analyzed for components of the NF-κB pathway by Western blotting and for expression of the NF-κB target genes BCL2, CMYC, FAS, PTEN and TNF-α by RT-PCR. Stimulation with TNF-α led to rapid and transient degradation of IκB and an accumulation of phospho-NF-κB p65 (Ser536), while other NF-κB proteins (p100, p105, p50) remained unaffected. Depletion of MAP3K7 neither changed IκB levels in unstimulated cells nor did it prevent the degradation of IκB after stimulation with TNF-α. Furthermore mRNA expression of none of the NF-κB target genes was consistently changed by more than 2-fold in any of the cell lines after MAP3K7 depletion. We conclude that in T-ALL cell lines, MAP3K7 is not required for the degradation of IκB and subsequent activation of NF-κB after stimulation with TNF-α.This study revealed that the recurrent deletion of MAP3K7/CASP8AP2 is associated with SIL-TAL1 fusions and a mature immunophenotype, but not with response to treatment and risk of relapse. Homozygous deletions of MAP3K7 were not observed, and efficient depletion of MAP3K7 interfered with viability of T-ALL cells, indicating that a residual expression of MAP3K7 is indispensable for T-lymphoblasts. DisclosuresSchrappe:Medac: Consultancy, Research Funding; Baxalta: Consultancy, Research Funding; JAZZ Pharma: Consultancy, Research Funding; SigmaTau: Consultancy, Research Funding; Novartis: Consultancy, Research Funding.

Immunostimulatory CpG Oligodeoxynucleotides Induce Sustained ERK2 Activation and Strong Proliferative Responses in CLL B-Cells from Patients with Progressive Disease.

Immunostimulatory DNA sequences that are present in microbial DNA, DNA-containing immune complexes and synthetic CpG-oligodeoxynucleotides (ODNs) can activate cells of the immune system via Toll-like receptor 9 (TLR9). Normal B-cells respond to such stimulation by upregulation of co-stimulatory molecules, proliferation and prolonged survival. The response of chronic lymphocytic leukemia B-cells to CpG ODN stimulation appears to be more variable, since both proliferation and induction of apoptosis have been reported. Because CLL B-cells with mutated and unmutated Ig VH genes show distinct clinical and biological behaviors, we compared their responsiveness to CpG ODN stimulation in terms of proliferation, survival, activation of TLR9 signaling pathways and expression of cell-cycle regulatory and anti-apoptotic proteins. A significant proliferative response was induced in most of the VH unmutated CLL samples (14/23), whereas 3H thymidine incorporation was low or absent in the majority of the VH mutated cases (22/27, P = 0.005). More strikingly, stratification of the CLL cases based on the clinical course showed strong proliferative responses in 14/21 cases with progressive disease and in only 5/29 cases with stable disease (P=0.001). The absence of a proliferative response to CpG ODN stimulation in the stable/VH mutated cases was not caused by different early signaling events, since phosphorylation of JNK, ERK1/2 and Akt, as well as degradation of IkB, was detected in almost all investigated samples (7/7 proliferating and 5/6 nonproliferating). However, a notable difference was observed in the duration of ERK2 activation, which was transient in the nonproliferating CLL B-cells, but lasted more than 24 hours in the proliferating cases. This difference was associated with dimished phosphorylation of the retinoblastoma protein (pRb) and less pronounced or absent induction of cyclin E and cyclin A in the nonproliferating cases, suggesting a block in the G1/S phase transition. On the other hand, cyclin D3 was induced and p27KIP1 was downregulated in all samples, indicating that CLL B-cells from both subgroups can enter the G1 phase of the cell cycle. Analysis of CLL B-cell survival by Annexin V/PI staining and PARP cleavage after 48 hours stimulation with CpG ODN showed significant levels of apoptosis in most of the nonproliferating cases. In contrast, proliferating CLL B-cells remained viable after CpG ODN stimulation, which was associated with upregulation of the anti-apoptotic proteins Mcl-1 and Bcl-xL. These data indicate that immunostimulatory CpG oligodeoxynucleotides can induce distinct responses in progressive/VH unmutated and stable/VH mutated CLL B-cells, which are characterized by proliferation and inhibition of spontaneous apoptosis in the former and induction of apoptosis in the latter group of cases. Although both subgroups show similar early activation of TLR9 signaling pathways and both enter into the G1 phase of the cell cycle, the absence of sustained ERK2 activation precludes cell cycle progression in most stable/VH mutated CLL B-cells. The greater capacity to traverse the cell cycle and proliferate in response to immunostimulatory CpG DNA may therefore be related to the more aggressive clinical course that is associated with CLL B-cells that express unmutated VH genes.

Inhibition of neuroinflammation by MIF inhibitor 3-({[4-(4-methoxyphenyl)-6-methyl-2-pyrimidinyl]thio}1methyl)benzoic acid (Z-312)

Microglial overactivation-mediated neuroinflammation contributes greatly to the pathogenesis of neurodegenerative diseases, such as Parkinson’s disease. Macrophage migration inhibitory factor (MIF) is a pleiotropic proinflammatory cytokine that is involved in the pathophysiology of various inflammatory diseases by inducing various proinflammatory cytokines. Compound 3-({[4-(4-methoxyphenyl)-6-methyl-2-pyrimidinyl]thio}methyl)benzoic acid (Z-312) is a novel small -molecule inhibitor of MIF tautomeric activity. In this study, we investigated the anti-inflammatory effects of Z-312 on liposaccharide (LPS)-induced neuroinflammation in vitro and in vivo. The results showed that Z-312 significantly decreased the production of nitric oxide (NO), interleukin (IL)-1β, tumor necrosis factor (TNF)-α and IL-6 in LPS-stimulated microglial cells. Mechanistically, nuclear translocation of the p65 subunit of nuclear factor (NF)-κB, degradation and phosphorylation of IκBα, NF-κB transcriptional activity and phosphorylation of p38 mitogen-activated protein kinase (MAPK) and JNK were markedly attenuated by pretreatment with Z-312 in BV-2 microglial cells. In addition, Z-312 suppressed the neurotoxic effects of cell culture medium of LPS-activated BV-2 microglia on cocultured mouse HT22 neuroblastoma cells. An in vivo study demonstrated that Z-312 markedly ameliorated microglial activation and subsequent DA neuron loss in an LPS-induced Parkinson’s disease (PD) mouse model. These results suggest that MIF inhibitor Z-312 may be a promising neuroprotective agent for the treatment of neuroinflammation-mediated neurological diseases.

In Vitro Antiviral Activity of Mentha cordifolia Plant Extract in HIV-1 Latently Infected Cells Using an Established Human Cell Line.

Emergence of drug resistance demands new therapeutic strategies against the human immunodeficiency virus (HIV). Currently, there is an increasing research focus on targeting gene expression-the crucial step wherein new viruses and new viral strains are amplified. Moreover, natural products are also being considered as potential candidates for new antivirals. We screened the extract obtained from a Philippine medicinal plant, Mentha cordifolia (Mc). In this study, we demonstrated that Mc ammonium sulfate extract has antiretroviral activity against HIV. HIV-1 latently infected cells (OM10.1) were pretreated with Mc extract and activated with TNFα. In treated cells, viral replication was inhibited in both cell culture supernatant and whole cell lysates. The level of viral production, as measured by the viral p24 protein concentration, was very much inhibited under noncytotoxic concentrations to the similar level without addition of TNFα. Luciferase assays, however, showed that Mc does not inhibit the HIV-1 long terminal repeat-driven gene expression. IκBα degradation and p65 nuclear translocation was also not affected as visualized through Western blot and immunofluorescence. These observations demonstrated that Mc possessed an antiviral component against HIV-1 and warrant further work to explore its target of action at a later step of gene expression. Our study introduces a potential source of a lead compound that targets steps in the HIV life cycle.

Sophoraflavanone M, a prenylated flavonoid from Sophora flavescens Ait., suppresses pro-inflammatory mediators through both NF-κB and JNK/AP-1 signaling pathways in LPS-primed macrophages

(2R)-3α,7,4′-trihydroxy-5-methoxy-8-(γ,γ-dimethylallyl)-flavanone is a prenylated flavonoid isolated from the anti-inflammatory herb Sophora flavescens Ait. We firstly named it sophoraflavanone M (SFM) in accordance with trivial names of related constitutes from this plant. Although various studies investigated the anti-inflammatory properties of prenylated flavonoids from Sophora flavescens Ait., that of SFM remains unclear and is yet to be determined. In the current study, we assessed the anti-inflammatory effects of SFM in LPS-induced in vivo and in vitro models. In the serum of endotoxemia mice, SFM significantly suppressed LPS-elevated inflammatory cytokines. Furthermore, at nontoxic concentrations, SFM reduced LPS-induced production of inflammatory mediators NO, IL-6, TNF-α, and MCP-1 in mouse primary peritoneal macrophages. Accordingly, in LPS-primed RAW264.7 cell line, it also inhibited these mediators' expression at both transcriptional and translational levels without cytotoxicity. Mechanistically, SFM is found to concurrently inhibit two important inflammatory signaling pathways, NF-κB and JNK/AP-1. SFM restrained phosphorylation and degradation of IκBα as well as the subsequent p65 translocation to dampen NF-κB activity. Meanwhile, it also suppressed JNK phosphorylation to inhibit the transcriptional activity of AP-1. These results provide material basis for traditional application of the anti-inflammatory herb Sophora flavescens Ait. and suggest SFM is a promising natural candidate for alleviating inflammatory conditions.

The ERβ‐CXCL19/CXCR4‐NFκB pathway is critical in mediating the E2-induced inflammation response in the orange-spotted grouper (Epinephelus coioides)

The main physiological function of 17β-estradiol (E2) in vertebrates is to regulate sexual development and reproduction. In fish, especially hermaphroditic fish, estrogen is often used to aid reproduction, but it also can trigger an inflammatory response. However, the molecular mechanism for this E2-induced inflammatory reaction is not clear. In this study, we found that the ERβ-CXCL19/CXCR4-NFκB cascade regulated the E2-induced inflammatory response in the orange-spotted grouper (Epinephelus coioides). Strikingly, E2 treatment resulted in significantly high expression of inflammatory cytokines and induced phosphorylation and degradation of IκBα and translocation of NFκB subunit p65 to the nucleus in grouper spleen cells. However, the E2-induced inflammatory response could be prevented by the broad estrogen receptor (ER) ligand ICI 182,780. Moreover, the luciferase assay showed that E2 induced the inflammatory response by activating the promotor of chemokine CXCL19 through ERβ1 and ERβ2. Knockdown of CXCL19 blocked the E2-induced inflammatory response and NFκB nucleus translocation. Additionally, knockdown of chemokines CXCR4a and CXCR4b together, but not alone, blocked the E2-induced inflammatory response. The immunofluorescence assay and co-immunoprecipitation analysis showed that CXCL19 mediated the E2-induced inflammatory response by activating CXCR4a or CXCR4b. Taken together, these results showed that the ERβ-CXCL19/CXCR4-NFκB pathway mediated the E2-induced inflammatory response in grouper. These findings are valuable for future comparative immunological studies and provide a theoretical basis for mitigating the adverse reactions that occur when using E2 to help fish reproduce.

Discovery of isoliquiritigenin analogues that reverse acute hepatitis by inhibiting macrophage polarization

Screening a natural product library of 850 compounds yield isoliquiritigenin as an effective anti-inflammatory agent by inhibiting the production of pro-inflammatory NO induced by Pam3CSK4, while its activity accompanied by toxicity. Further studies obtained the optimized isoliquiritigenin derivative SMU-B14, which can inhibit Pam3CSK4 triggered toll-like receptor 2 (TLR2) signaling with low toxicity and high potency. Preliminary mechanism studies indicated that SMU-B14 worked through TLR2/MyD88, phosphorylation of IKKα/β, leading to the reduce degradation of NF-κB related IKBα and p65 complex, then inhibited the production of inflammatory cytokines, such as TNF-α, IL-6, IL-1β both in human and murine cell lines. Subsequent polarization experiments showed SMU-B14 significant reversed the polarization of M1 phenotype primary macrophage activated by Pam3CSK4in vitro, and reduced the infiltration of neutrophil and polarization of M1-type macrophage, decreased serum alanine transaminase (ALT), as a result protected liver from being injured in vivo. In summary, we obtained an optimized lead compound SMU-B14 and found it functionally blocked TLR2/MyD88/NF-κB signaling pathway to down-regulate the production of inflammatory cytokines resulted significant liver protection property.

Upregulation of Rpn10 promotes tumor progression via activation of the NF-κB pathway in clear cell renal cell carcinoma.

The ubiquitin-proteasome system (UPS) plays a central role in regulating protein homeostasis in tumor progression. The proteasome subunit Rpn10 is associated with the progression of several tumor types. However, little is known regarding the role of Rpn10 in clear cell renal cell carcinoma (ccRCC). In this study, we found that overexpression of Rpn10 increased ccRCC cell proliferation, migration, and invasion. Silencing Rpn10 expression resulted in decreased cell proli-feration, migration, and invasion in ccRCC cells. Knockdown of Rpn10 inhibits tumor growth and cell proliferation in vivo. Furthermore, we demonstrated that Rpn10 increased cell proliferation, migration, and invasion via regulation of the nuclear factor kappa B (NF-κB) pathway. Rpn10 directly promoted inhibitor of nuclear factor-kappa B alpha (IκBα) degradation through the UPS. Moreover, we observed that upregulation of Rpn10 or downregulation of IκBα in ccRCC was associated with poor prognosis. We found that the combination of these two parameters was a more powerful predictor of poor prognosis than either parameter alone. Collectively, these findings provide evidence that Rpn10 promotes the progression of ccRCC by regulation of the NF-κB pathways and is a prognostic indicator for patients with ccRCC.

PKCδ/MAPKs and NF-κB Pathways are Involved in the Regulation of Ingenane-Type Diterpenoids from Euphorbia neriifolia on Macrophage Function.

PurposeEuphorbia neriifolia Linn. has important medicinal value in the treatment of ulcers, tumors, inflammation, chronic respiratory troubles, and so on. Although many ingredients with anti-inflammatory activity have been discovered and isolated from the Euphorbia neriifolia, the current research still cannot explain its multivariate effects on the immune response. This article aims to introduce two Ingenane-type diterpenoids from Euphorbia neriifolia with macrophage regulatory effects and to investigate the mechanism of their action.MethodsThe stem bark of E. neriifolia was extracted with various separation methods to obtain ingenane-type diterpenoids. The RAW264.7 cells were treated with lipopolysaccharide (LPS, 1 μg/mL) to establish an inflammatory cell model. The cell viability was detected by MTT assay. The secretion of PGE2, TNF-α, IL-1β, and IL-6 was tested with ELISA. The levels of iNOS, COX-2, IκBα, JNK, ERK, p38, p-IκBα, p-JNK, p-ERK, and p-p38 in cells were detected by Western blotting. The translocation of nuclear factor-kappa B (NF-κB)/p65 subunit were evaluated by Immunofluorescence staining.ResultsIngenane-type diterpenoids, eurifoloid A (Euri A) and a new compound euphorneroid E (Euph E), were isolated from the EtOAc fraction of E. neriifolia stem bark extracts. Euph E and Euri A exhibited significant inhibition on the levels of pro-inflammatory mediators NO, IL-1β, IL-6, and iNOS on LPS-induced macrophage RAW264.7. Cellular signaling pathway studies showed that they prevented the degradation of IκBα and the translocation of NF-κB/p65 subunit. Furthermore, the production of PGE2, TNFα, and COX-2 was dramatically increased under the influence of the compounds, which were closely related to the phosphorylation of protein kinase C δ (PKCδ) and activation of mitogen-activated protein kinase (MAPKs) signaling pathway.ConclusionThese results demonstrated that Euph E and Euri A exhibited multidirectional regulation on cytokines and immune function of macrophages, in addition to a good anti-inflammatory activity, and which was closely related to the regulation of PKCδ/MAPKs and NF-κB signal pathways.

ERα-dependent estrogen-TNFα signaling crosstalk increases cisplatin tolerance and migration of lung adenocarcinoma cells

Lung adenocarcinoma is the most common type of lung cancer in women. Our previous studies demonstrated that 17β-estradiol (E2) promoted lung adenocarcinoma cell proliferation and tumor growth through estrogen receptor ERα. Transcriptomic analysis suggested that E2 potentiated TNFα-NFκB signaling in ERα-expressing lung adenocarcinoma cells. This study further demonstrated that E2 increased TNFα receptor expression and TNFα-triggered NFκB activity in ERα-expressing cells. E2-activated ERα had no physical association with NFκB p65/p50 heterodimer but facilitated TNFα-initiated IκBα degradation, NFκB nuclear translocation, and S468/S536 phosphorylation of p65 essential for NFκB activity. While knockdown of ERα prevented E2 from boosting NFκB activity, antiestrogen ICI 182,780 stimulated NFκB activity like E2. Inhibition of GSK3β hampered E2:ERα-promoted NFκB activity and abolished S468 phosphorylation of p65, suggesting that GSK3β played a role in the E2-TNFα signaling crosstalk. In ERα-expressing cells, E2 and TNFα synergistically regulated many genes that were not typically responsive to either E2 or TNFα. Functional analysis of microarray data inferred that E2/TNFα-induced transcriptomic changes improved cell survival and movement. Viability and colony formation assays validated that E2 and TNFα together increased cisplatin tolerance of ERα-expressing cells. Wound healing assays also confirmed that E2/TNFα cotreatment increased cell migration in an ERα-dependent manner. E2/TNFα-induced dysregulation of genes such as cell survival and movement-associated genes, proto-oncogenes, metallothioneins and histone core genes was correlated with poor overall survival in patients. In summary, E2 and TNFα engaged in an ERα-dependent positive crosstalk in lung adenocarcinoma cells, consequently increasing NFκB activation, cisplatin tolerance and cell migration and worsening prognosis.

Nargenicin A1 attenuates lipopolysaccharide-induced inflammatory and oxidative response by blocking the NF-κB signaling pathway.

Inflammation caused by the excessive production of pro-inflammatory mediators and cytokines in abnormally activated macrophages promotes the initiation and progression of many diseases along with oxidative stress. Previous studies have suggested that nargenicin A1, an antibacterial macrolide isolated from Nocardia sp. may be a potential treatment for inflammatory responses and oxidative stress, but the detailed mechanisms are still not well studied. In this study, we investigated the inhibitory effect of nargenicin A1 on inflammatory and oxidative stress in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages and zebrafish (Danio rerio) models. Our results indicated that nargenicin A1 treatment significantly inhibited LPS-induced release of pro-inflammatory mediators including nitric oxide (NO) and prostaglandin E2, which was associated with decreased inducible NO synthase and cyclooxygenase-2 expression. In addition, nargenicin A1 attenuated the LPS-induced expression of pro-inflammatory cytokines, such as tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, and monocyte chemotactic protein-1, reducing their extracellular secretion. Nargenicin A1 also suppressed LPS-induced generation of reactive oxygen species. Moreover, nargenicin A1 abolished the LPS-mediated nuclear translocation of nuclear factor-kappa B (NF-κB) and the degradation of inhibitor IκB-α, indicating that nargenicin A1 exhibited anti-inflammatory effects by inhibiting the NF-κB signaling pathway. Furthermore, nargenicin A1 showed strong protective effects against NO and ROS production in LPS-injected zebrafish larvae. In conclusion, our findings suggest that nargenicin A1 ameliorates LPS-induced anti-inflammatory and antioxidant effects by downregulating the NF-κB signaling pathway, and that nargenicin A1 can be a potential functional agent to prevent inflammatory- and oxidative-mediated damage.

Low-dose mono(2-ethylhexyl) phthalate promotes ovarian cancer development through PPARα-dependent PI3K/Akt/NF-κB pathway

The plasticizer di(2-ethylhexyl) phthalate (DEHP) and its hydrolysate mono(2-ethylhexyl) phthalate (MEHP) are major toxicants from plastics, but their association with hormone-dependent cancers has been controversial. We treated the human ovarian cancer cell lines SKOV3 and A2780 with low concentrations of DEHP/MEHP, and found that although no significant effect on cell proliferation was observed, ovarian cancer cell migration, invasion, and epithelial-mesenchymal transition (EMT) were promoted by submicromolar MEHP but not DEHP. Next, ovarian cancer patient data from The Cancer Genome Atlas (TCGA) were obtained and subjected to Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) supported enrichment and Kaplan-Meier survival analyses, which identified PI3K/Akt pathway as a pivotal signaling pathway in ovarian cancer. We found that 500 nM MEHP treatment significantly increased PIK3CA expression, which could be reversed by the knockdown of peroxisome proliferator-activated receptor alpha (PPARα). Silencing PIK3CA significantly suppressed the MEHP-induced migration, invasion and EMT. In addition, we validated that MEHP treatment promoted phosphorylation of Akt and degradation of IκB-α, thereby activating NF-κB and enhancing NF-κB nuclear translocation. In nude mice, MEHP exposure significantly promoted the metastasis of ovarian cancer xenografts, which could be suppressed by the treatment of PPARα inhibitor GW6471. Our findings showed that low-dose MEHP promoted ovarian cancer progression through activating PI3K/Akt/NF-κB pathway, in a PPARα-dependent manner.