IkappaBalpha Kinase Research Articles

Abstract 5835: Functional and mechanistic characterization of ΔNp63α in esophageal squamous cell carcinoma

Abstract Esophageal squamous cell carcinoma (ESCC) is highly prevalent in Asia with a poor prognosis and a high mortality rate [1, 2]. The p63 protein, encoded by TP63, is a master regulator involved in many cell events including cell cycle, DNA damage, cell proliferation, stem cell maintenance, and cell death in different cancer types. ΔNp63α is the dominantly expressed isoform of p63 in squamous cell carcinomas, playing a critical role in tumorigenesis [3, 4]. However, the functional roles of ΔNp63α in ESCC have not been fully elucidated. Our results have revealed that ΔNp63α is highly expressed and up-regulated in ESCC. We have shown that ΔNp63α protein expression plays an oncogenic role in ESCC cells, depletion of which inhibits in vitro cell proliferation and colony formation and greatly suppresses in vivo tumor growth, in a panel of ESCC cell lines and our latest patient-derived organoid cultures, potentially in a differentiation-dependent manner. Glycolysis pathway and two novel downstream signaling pathways of ΔNp63α, MYC/4EBP1 and AKT/NDRG1, are significantly suppressed upon ΔNp63α depletion in ESCC cell lines. Notably, xenografts of ΔNp63α-depleted human ESCC cells show elevated infiltrating cancer-associated fibroblasts (CAFs), which may contribute to tumor suppression by ΔNp63α depletion. These data not only shed light on the ΔNp63α functions and how they contribute to the ESCC tumorigenesis, but also unravel potential therapeutic benefits for ESCC patients in the future. Acknowledgements: We acknowledge DSMZ (German Collection of Microorganisms and Cell Culture) for the KYSE cell lines. We acknowledge the Research Grants Council Theme-Based Research Scheme grant T12-701/17-R to MLL.

Cantharidin-induced acute hepatotoxicity: the role of TNF-α, IKK-α, Bcl-2, Bax and caspase3.

Cantharidin is of high medicinal value but has strong toxicity. Nowadays, multiple research has focused on the mechanism of its antitumor activity while research on toxicological profiles associated with cantharidin poisoning is still limited. Its hepatotoxicity has attracted attention recently for the crucial role of the liver in detoxification. Here, we aim to find a potential mechanism for cantharidin-induced acute hepatotoxicity with a view to assisting subsequent research or clinical use or detoxification. Twenty-one male Sprague-Dawley rats were randomly divided into control, low-dose (1.34 mg/kg) and high-dose (2.67 mg/kg) cantharidin exposure groups. We used hematoxylin-eosin to observe pathological changes and used immunofluorescent staining, western blotting and real-time quantitative polymerase chain reaction to detect the expression of the markers. The main pathological changes in livers of cantharidin-treated rats were necrosis, inflammatory infiltration and hemorrhage. We found coexpression of tumor necrosis factor alpha (TNF-α), IkappaB kinase-alpha (IKK-α) and caspase3 by immunofluorescent staining in livers of cantharidin-treated rats. Compared with the control, the levels of TNF-α, IKK-α and caspase3 increased significantly in the experimental groups (P < .05). The ratio of B-cell lymphoma-2 (Bcl-2)/Bax increased in the low-dose group but decreased in the high-dose group (P < .05). Cantharidin exposure raised IKK-α mRNA and caspase3 mRNA levels (P < .05). In conclusion, the participation of TNF-α, IKK-α, Bcl-2, Bax and caspase3 uncovered a novel mechanism underlying cantharidin-induced acute hepatotoxicity, and the mechanism needs to be studied further.

Analysis of Streptococcus agalactiae ‐induced liver injury in tilapia ( Oreochromis niloticus )

Streptococcosis, a bacterial disease for multiple fish species, especially tilapia, caused more disruption to the fish culture industry. However, the underlying mechanisms for the occurrence of streptococcosis remain unclear. The aim of this study was to study the effects of streptococcus on the liver of tilapia. In this study, tilapia were injected streptococcal solution (0.05 ml/10 g body weight) and collected blood and liver at 4, 8, 24, 48, 72, 96, 120 and 144 hr after injection. The results showed that the activities of glutamate oxalate transaminase (GOT), glutamate pyruvate transaminase (GPT), alkaline phosphatase (AKP) and superoxide dismutase (SOD) in the serum of tilapia exhibited irregular changes: increase at first, then decrease and increase again. The levels of total antioxidant capacity (T-AOC), glutathione (GSH) and malondialdehyde (MDA) showed a decreasing tendency in serum after injection, except GSH at 4 hr. The activity of catalase (CAT) decreased in serum at 4, 24, 120 and 144 hr after injection of streptococcal solution, while the content TP enhanced in serum at 48, 72 and 120 hr after injection. Observations of pathological sections showed obvious damage to the liver tissue structure in response to streptococcal infection. Western blotting revealed an increase in ikkbeta (Ikkβ) protein with prolonged infection time and a decrease in NF-kappa-B inhibitor alpha (IkB-α) and ikappab kinase alpha (Ikkα) proteins. The gene expression of cytochrome P4501A (CYP1A) was downregulated in liver at 24 and 48 after injection, while the mRNA levels of heat stress protein 70 (HSP70) and tumour necrosis factor alpha (TNF-α) increased at 48 and/or 72 hr. Taken together, our findings confirmed that streptococcus can cause serious damage to the liver of tilapia.

Exome Sequencing Identifies a Novel MAP3K14 Mutation in Recessive Atypical Combined Immunodeficiency.

Primary immunodeficiency disorders (PIDs) render patients vulnerable to infection with a wide range of microorganisms and thus provide good in vivo models for the assessment of immune responses during infectious challenges. Priming of the immune system, especially in infancy, depends on different environmental exposures and medical practices. This may determine the timing and phenotype of clinical appearance of immune deficits as exemplified with early exposure to Bacillus Calmette-Guérin (BCG) vaccination and dissemination in combined immunodeficiencies. Varied phenotype expression poses a challenge to identification of the putative immune deficit. Without the availability of genomic diagnosis and data analysis resources and with limited capacity for functional definition of immune pathways, it is difficult to establish a definitive diagnosis and to decide on appropriate treatment. This study describes the use of exome sequencing to identify a homozygous recessive variant in MAP3K14, NIKVal345Met, in a patient with combined immunodeficiency, disseminated BCG-osis, and paradoxically elevated lymphocytes. Laboratory testing confirmed hypogammaglobulinemia with normal CD19, but failed to confirm a definitive diagnosis for targeted treatment decisions. NIKVal345Met is predicted to be deleterious and pathogenic by two in silico prediction tools and is situated in a gene crucial for effective functioning of the non-canonical nuclear factor-kappa B signaling pathway. Functional analysis of NIKVal345Met- versus NIKWT-transfected human embryonic kidney-293T cells showed that this mutation significantly affects the kinase activity of NIK leading to decreased levels of phosphorylated IkappaB kinase-alpha (IKKα), the target of NIK. BCG-stimulated RAW264.7 cells transfected with NIKVal345Met also presented with reduced levels of phosphorylated IKKα, significantly increased p100 levels and significantly decreased p52 levels compared to cells transfected with NIKWT. Ideally, these experiments would have been conducted in patient-derived immune cells, but we were unable to source these cells from the patient. The functional analysis described in this paper supports previous illustrations of the importance of NIK in human immune responses and demonstrates the involvement of function-altering mutations in MAP3K14 in PIDs. The genomic approach used for this patient demonstrates its value in the diagnosis of an unusual PID and as a tool for detecting rarer mutations to help guide treatment approaches.

Immunodeficiency and disseminated mycobacterial infection associated with homozygous nonsense mutation of IKKβ

A, Genealogical tree for the kindred. Arrow highlights index case. Asterisks denote family members who died within the first 2 months of life with febrile illnesses. B, Histology showing granuloma in skin by hematoxylin and eosin staining.

Increase in IkappaB kinase alpha expression suppresses the tumor progression and improves the prognosis for nasopharyngeal carcinoma.

Recent studies have suggested that the action of IkappaB kinase alpha (IKKα) as a tumor suppressor is crucial in the development of skin carcinoma, but its role in nasopharyngeal carcinoma (NPC) remains unknown. We examined the IKKα expression in specimens from 157 NPC patients by immunohistochemistry and analyzed the effect of IKKα on prognosis. The functional significance of IKKα expression in NPC cell lines was investigated by IKKα overexpression or downregulation in in vitro studies. The in vitro assays revealed that the IKKα expression was negatively correlated with the invasiveness, migration, and angiogenesis of NPC cells. Overexpression or downregulation of IKKα could significantly repress or enhance the above characteristics, respectively, and these effects were independent of IKKα kinase or EBNA1. In 157 NPC cases, IKKα was differentially expressed in NPC tissues. High expression of IKKα was associated significantly with a high disease-free survival (DFS; P = 0.002) or overall survival (OS; P = 0.014). Multivariate analyses showed that the IKKα expression was an independent risk factor for DFS (HR, 2.302; P = 0.011) and OS (HR, 3.578; P = 0.006). Our findings indicated that IKKα plays a crucial role as a tumor suppressor that suppresses the invasion, metastasis, and angiogenesis of NPC cells in vitro and correlates with the survival in NPC patients. Therefore, IKKα is not only a novel independent prognostic indicator in NPC, but also targeting IKKα expression may provide a potential therapeutic strategy for NPC.

Abstract B34: IKKalpha regulation of mTORC1 and modulation of prostate tumorigenesis through phosphorylation of mTOR

Abstract The mammalian target of rapamycin (mTOR) is a critical mediator of cell growth, cell proliferation, survival, and energy metabolism. mTOR is activated downstream of growth factors, insulin, and Akt-dependent signaling associated with oncoprotein expression or loss of the tumor-suppressor PTEN. In this regard, mTOR activity is associated with cancer cell growth and survival. Here, we have explored an involvement of the I kappa B kinase (IKK) pathway, associated with nuclear factor-kappaB activation, in controlling mTOR activity. The experiments show that IKK alpha controls mTOR activity in Akt-active, PTEN-null prostate cancer cells by directly phosphorylation of mTOR at Serine 1415. In these cells, IKK alpha associates with mTORC1 and weaken the association between mTOR and Raptor, an important component of mTORC1, to increase mTORC1 kinase activity by phosphorylation in an Akt-dependent manner. Additionally, IKKalpha phosphorylation of mTOR is required for efficient induction of cell proliferation downstream of constitutively active Akt in these cells. The results indicate a novel role for IKKalpha in controlling mTOR function in protate cancer cells with loss of PTEN and constitutive Akt activity. Citation Format: Hancai Dan, Albert S. Baldwin. IKKalpha regulation of mTORC1 and modulation of prostate tumorigenesis through phosphorylation of mTOR [abstract]. In: Proceedings of the AACR Special Conference on Advances in Prostate Cancer Research; 2012 Feb 6-9; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(4 Suppl):Abstract nr B34.

Abstract 1389: Cyr61 increases cell migration in human oral cancer cells through FAK, ERK and NF-κB pathways

Abstract Cysteine-rich 61 (Cyr61), from the CCN gene family, is a secreted and matrix-associated protein, which is involved in many cellular activities such as growth and differentiation. Most of CCN family can regulate the cell motility, division and adhesion. However, the effect of Cyr61 on migration activity in human oral squamous carcinoma cells is mostly unknown. Here, we found that Cyr61 increased the migration in human oral squamous carcinoma cells (SCC-4 cells). Activations of focal adhesion kinase (FAK), extracellular signal-regulated kinase (ERK) and NF-κB pathways after Cyr61 treatment was demonstrated, and Cyr61-induced migration activity was inhibited by specific inhibitor of ERK and NF-κB cascades. Transfection of cells with FAK, ERK, IKKα(IkappaB kinase alpha) and IKKβ mutant also reduced Cyr61-mediated cancer migration. Taken together, our results suggest that Cyr61 acts through FAK/ERK, which in turn activates NF-κB, contributing to the migration of human oral squamous carcinoma cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1389. doi:10.1158/1538-7445.AM2011-1389

Abstract 1396: Bradykinin enhances cell migration in human prostate carcinoma cells via bradykinin B2 receptor, PKCdelta, c-Src and NF-kappaB signaling pathways

Abstract Bradykinin (BK) is an inflammatory mediator, and shows elevated levels in regions of severe injury and inflammatory diseases. In addition, bradykinin has been found in many tumor cell lines and has been shown to be involved in carcinogenesis and cancer progression. Prostate cancer is the most common cancer excluding skin cancer in men and usually metastasizes to the particular organs in the late stages of cancer processes. The matrix metalloproteinase (MMP) play an important role in tissue remodeling associated with various physiological and pathological processes such as morphogenesis, angiogenesis, tissue repair, cirrhosis, arthritis and metastasis. In this study, we found that bradykinin increased the migration and the expression of MMP-9 in human prostate cancer cells (PC3,DU145 and LNCaP). Bradykinin-mediated migration and matrix MMP-9 up-regulation was attenuated by bradykinin B2 receptor antagonist (HOE140), protein kinase Cdelta (PKCdelta) inhibitor (Rottlerin), c-Src inhibitor (PP2), NF-kappaB inhibitor (PDTC), and IkappaB protease inhibitor (TPCK). Transfection of cells with bradykinin B2 receptor and protein kinase Cdelta (PKCdelta), siRNA or dominant-negative mutant of c-Src, IkappaBalpha kinase alpha(IKK alpha) and IkappaBalpha kinase beta(IKK beta), also inhibited the potentiating action of bradykinin. Stimulation of PC3 cells with bradykinin induced PKCdelta, c-Src, IKK alpha/beta, IkappaB and p65 phosphorylation, and kappaB-luciferase activity. In addition, BKR2 PCKdelta and c-Src pathways are involved in bradykinin-induced NFkappaB activation. Taken together, our results suggest that bradykinin enhances the migration of prostate carcinoma cells by increasing MMP9 expression through the bradykinin B2 receptor /PKCdelta/c-Src/NF-kappaB signal transduction pathways. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1396. doi:10.1158/1538-7445.AM2011-1396

Respiratory Syncytial Virus Pathophysiology and Affect of Palivizumab in Special Populations: Cystic Fibrosis and Immunosuppression

monary disease in patients with cystic fibrosis (CF). This appears to be due to an exaggerated pulmonary inflammatory response, particularly seen with Respiratory Syncytial Virus lowerrespiratory-tract infection (RSV LRTI). In the normal mouse, infection with RSV leads to increased lymphocytes, neutrophils, and inflammatory cytokines recovered from bronchoalveolar lavage and chronic pulmonary inflammatory infiltrates may last up to 154 days after inoculation. In CF mice, clearance of RSV is further impaired and a much exaggerated inflammatory response is seen compared to non CF mice. As observed using in vitro and in vivo models, RSV causes an exaggerated nuclear factor (NF)kappaB activation and subsequent elevated cytokine and chemokine production. NF-kappaB dimers exist as inactive complexes in the cytoplasm of unstimulated cells by virtue of their interaction with inhibitory proteins (IjBs). These inhibitory proteins are phosphorylated by cellular kinase complexes. This seems to be regulated through activation of the IkappaB kinase alpha (IKK-a) in lung tissue. RSV infection activates IKK-a and IKK-b. In the CF human airway gland cells this exaggerated response of NF-KappaB also can be caused by elevated sodium chloride content. Exposure of non-CF cells to hypotonic sodium chloride solution, compared to isotonic or hypertonic sodium chloride solutions, inhibited activated IKK-a and IL-8 levels. While interleukin (IL) 8 (IL8) levels were inhibited by hypotonic saline solution in CF cells, marked inhibition of the IKK-a system did not occur. Abnormally high sodium chloride levels within the lung also act to inactivate human b-defensin-1, a naturally occurring antibiotic, and change the antimicrobial characteristics of epithelial surface fluid. Respiratory viruses also can cause induction of inflammatory cytokines in human bronchial cells by activation of the p38 mitogenactivated protein kinase (MAPK) signaling pathway. RSV infection inhibits granulocyte apoptosis via kinase and NF-kappaB dependent mechanisms. Lastly, in the mouse model, RSV infection increases expression of inducible nitric oxide synthase and nitric oxide production adding to the inflammatory burden. Palivizumab exhibits both neutralizing and fusion inhibitory activity against RSV. This inhibits RSV replication and appears to decrease the severity of LRTI in high risk human populations. When given in a non-CF murine model 24 hours before RSV inoculation, significant decreases in the number of RSV recovered in bronchoalveolar lavage and histopathological inflammatory scores are observed. Airway obstruction, mucus production and airway hyper-responsiveness also are less than in controls. Palivizumab also has been shown to decrease neurogenic RSV induced inflammation even when given 72 hours after virus inoculation in this same model. Further, Piedra and co-workers have demonstrated that a purified fusion protein vaccine can increase RSV neutralizAddress correspondence to: Michael E. Speer, MD, 6621 Fannin Street, WT 6 – 104, Houston, TX 77030, email: mspeer@bcm.tmc.edu 2011 Pediatric Pharmacy Advocacy Group

Phosphoproteomic Analysis of Signaling Pathways in Head and Neck Squamous Cell Carcinoma Patient Samples

Molecular targeted therapy represents a promising new strategy for treating cancers because many small-molecule inhibitors targeting protein kinases have recently become available. Reverse-phase protein microarrays (RPPAs) are a useful platform for identifying dysregulated signaling pathways in tumors and can provide insight into patient-specific differences. In the present study, RPPAs were used to examine 60 protein end points (predominantly phosphoproteins) in matched tumor and nonmalignant biopsy specimens from 23 patients with head and neck squamous cell carcinoma to characterize the cancer phosphoproteome. RPPA identified 18 of 60 analytes globally elevated in tumors versus healthy tissue and 17 of 60 analytes that were decreased. The most significantly elevated analytes in tumor were checkpoint kinase (Chk) 1 serine 345 (S345), Chk 2 S33/35, eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) S65, protein kinase C (PKC) ζ/ι threonine 410/412 (T410/T412), LKB1 S334, inhibitor of kappaB alpha (IκB-α) S32, eukaryotic translation initiation factor 4E (eIF4E) S209, Smad2 S465/67, insulin receptor substrate 1 (IRS-1) S612, mitogen-activated ERK kinase 1/2 (MEK1/2) S217/221, and total PKC ι. To our knowledge, this is the first report of elevated PKC ι in head and neck squamous cell carcinoma that may have significance because PKC ι is an oncogene in several other tumor types, including lung cancer. The feasibility of using RPPA for developing theranostic tests to guide personalized therapy is discussed in the context of these data.

TNF-alpha mediated NF-kappaB activation is constantly extended by transglutaminase 2

Increased levels of transglutaminase 2 (TGase 2) expression have been reported in many inflammatory diseases, as well as in drug resistant cancer cells. Previous reports have shown that TGase 2 is capable of inducing nuclear factor-kappaB (NF-kappaB) activation via depletion of inhibitor of kappaB (I-kappaB)alpha through polymerization in the absence of I-kappaBalpha kinase activation. This raises the question of whether increased expression of TGase 2 can extend NF-kappaB activation mediated by a canonical activation pathway. In the TGase 2-inducible EcR23/TG cell line, TGase 2 over-expression resulted in sustained activation of NF-kappa B in the presence of TNF-alpha, for up to 24 hrs, while in the absence of TGase 2 induction, NF-kappaB activity was restored to basal levels within 6 hrs of TNF-alpha treatment. In mice injected with an adenovirus vector expressing TGase 2, NF-kappaB was constitutively activated for up to 5 days, whereas Adeno/GFP-injected mice exhibited attenuated activation of NF-kappaB in response to TNF-alpha stress. Thus, the presence of increased levels of TGase 2 may exacerbate NF-kappa B activation in inflammatory states.

CARD9 Mediates Dectin-2-induced IκBα Kinase Ubiquitination Leading to Activation of NF-κB in Response to Stimulation by the Hyphal Form of Candida albicans

The scaffold protein CARD9 plays an essential role in anti-fungus immunity and is implicated in mediating Dectin-1/Syk-induced NF-kappaB activation in response to Candida albicans infection. However, the molecular mechanism by which CARD9 mediates C. albicans-induced NF-kappaB activation is not fully characterized. Here we demonstrate that CARD9 is involved in mediating NF-kappaB activation induced by the hyphal form of C. albicans hyphae (Hyphae) but not by its heat-inactivated unicellular form. Our data show that inhibiting Dectin-2 expression selectively blocked Hyphae-induced NF-kappaB, whereas inhibiting Dectin-1 mainly suppressed zymosan-induced NF-kappaB, indicating that Hyphae-induced NF-kappaB activation is mainly through Dectin-2 and not Dectin-1. Consistently, we find that the hyphae stimulation induces CARD9 association with Bcl10, an adaptor protein that functions downstream of CARD9 and is also involved in C. albicans-induced NF-kappaB activation. This association is dependent on Dectin-2 but not Dectin-1 following the hyphae stimulation. Finally, we find that although both CARD9 and Syk are required for Hyphae-induced NF-kappaB activation, they regulate different signaling events in which CARD9 mediates IkappaBalpha kinase ubiquitination, whereas Syk regulates IkappaBalpha kinase phosphorylation. Together, our data demonstrated that CARD9 is selectively involved in Dectin-2-induced NF-kappaB activation in response to C. albicans hyphae challenging.

Crotepoxide Chemosensitizes Tumor Cells through Inhibition of Expression of Proliferation, Invasion, and Angiogenic Proteins Linked to Proinflammatory Pathway

Crotepoxide (a substituted cyclohexane diepoxide), isolated from Kaempferia pulchra (peacock ginger), although linked to antitumor and anti-inflammatory activities, the mechanism by which it exhibits these activities, is not yet understood. Because nuclear factor kappaB (NF-kappaB) plays a critical role in these signaling pathways, we investigated the effects of crotepoxide on NF-kappaB-mediated cellular responses in human cancer cells. We found that crotepoxide potentiated tumor necrosis factor (TNF), and chemotherapeutic agents induced apoptosis and inhibited the expression of NF-kappaB-regulated gene products involved in anti-apoptosis (Bcl-2, Bcl-xL, IAP1,(2) MCl-1, survivin, and TRAF1), apoptosis (Bax, Bid), inflammation (COX-2), proliferation (cyclin D1 and c-myc), invasion (ICAM-1 and MMP-9), and angiogenesis (VEGF). We also found that crotepoxide inhibited both inducible and constitutive NF-kappaB activation. Crotepoxide inhibition of NF-kappaB was not inducer-specific; it inhibited NF-kappaB activation induced by TNF, phorbol 12-myristate 13-acetate, lipopolysaccharide, and cigarette smoke. Crotepoxide suppression of NF-kappaB was not cell type-specific because NF-kappaB activation was inhibited in myeloid, leukemia, and epithelial cells. Furthermore, we found that crotepoxide inhibited TAK1 activation, which led to suppression of IkappaBalpha kinase, abrogation of IkappaBalpha phosphorylation and degradation, nuclear translocation of p65, and suppression of NF-kappaB-dependent reporter gene expression. Overall, our results indicate that crotepoxide sensitizes tumor cells to cytokines and chemotherapeutic agents through inhibition of NF-kappaB and NF-kappaB-regulated gene products, and this may provide the molecular basis for crotepoxide ability to suppress inflammation and carcinogenesis.

Phytoestrogen bavachin mediates anti-inflammation targeting IκB kinase-IκBα-NF-κB signaling pathway in chondrocytes in vitro

The pro-inflammatory cytokine interleukin-1 beta (IL-1 beta) plays critical roles in pathogenesis of osteoarthritis. Although estrogen is protective for cartilage in osteoarthritis patients, it also potentially increases the risk of stroke and cancer. Phytoestrogens acting as natural estrogen receptor modulators may serve as alternatives. This study aimed to identify medicinal phytoestrogens that preserve anti-inflammatory property and may function as potential chondro-protective compounds. Both human chondrocytes and chondrocytic cell line CHON-002 were used for this study. Protein concentrations or expressions were measured by ELISA or Western blot, respectively. The DNA-binding activity and transcriptional activity of transcription factors were evaluated by electrophoretic mobility shift assay and dual-luciferase reporter assay, respectively. Cell migration was analyzed by chemotaxis assays. We found that among screened phytoestrogens, bavachin could potently decrease IL-1 beta-induced nuclear factor-kappa B (NF-kappaB) but not activator protein-1 (AP-1) DNA-binding activity. Bavachin also inhibited I kappaB alpha degradation, increased nuclear translocation of p65 and p50 as well as decreased I kappaB alpha kinase (IKK) activity. Furthermore, bavachin inhibited IL-1 beta-induced chemokine production that resulted in reduced migration of THP-1 monocytic cells. Our results suggest that through decreasing IL-1 beta-induced activation of IKK-I kappaB alpha-NF-kappaB signaling pathway, bavachin potentially protects cartilage from inflammation-mediated damage in joints of osteoarthritis patients.

Nuclear factor κB transcription factors are coexpressed and convey a poor outcome in ovarian cancer

Recent work has suggested a role for nuclear factor kappaB (NF-kappaB) in the propagation of ovarian cancer cell lines, but the significance and mechanism of NF-kappaB in ovarian cancer is unknown. The authors hypothesized that the NF-kappaB pathway is over activated in aggressive ovarian cancers. The levels of 3 NF-kappaB transcription factors, the activating inhibitors of NF-kappaB (IkappaB) kinases, and the NF-kappaB target matrix metalloproteinase 9 (MMP9) were assessed by immunohistochemistry in specimens of ovarian cancer that were obtained at diagnosis from a cohort of 33 patients who subsequently received combined paclitaxel, cisplatin, and cyclophosphamide. Associations were made between NF-kappaB pathway proteins and outcome. The validation of coexpression was performed at the gene level in 2 independently collected cohorts of 185 and 153 ovarian cancers. The presence of NF-kappaB proteins in newly diagnosed advanced ovarian cancers was established, and a potential association with overall survival was identified. Transcription factors p65 and v-rel reticuloendotheliosis viral oncogene homolog B (RelB) were coexpressed with IkappaB kinase alpha, 1 component of a key trimolecular regulatory complex. Coexpression of the NF-kappaB machinery suggested activity of NF-kappaB signaling in these ovarian tumors. A significant association of p50 with poor overall survival was observed (P = .02). MMP9 expression had the opposite association, in which patients who had tumors without MMP9 staining had the poorest prognosis (P = .01), and this association held true at the gene expression level in an independently collected cohort of 185 ovarian cancers. The deregulation of NF-kappaB activity may influence outcome in women who receive standard therapy for advanced ovarian cancer. Modification of the NF-kappaB pathway may present an opportunity to improve outcome in the subset of women who have pathway activity.

The pivotal role of the alternative NF-κB pathway in maintenance of basal bone homeostasis and osteoclastogenesis

The alternative NF-kappaB pathway consists predominantly of NF-kappaB-inducing kinase (NIK), IkappaB kinase alpha (IKKalpha), p100/p52, and RelB. The hallmark of the alternative NF-kappaB signaling is the processing of p100 into p52 through NIK, thus allowing the binding of p52 and RelB. The physiologic relevance of alternative NF-kappaB activation in bone biology, however, is not well understood. To elucidate the role of the alternative pathway in bone homeostasis, we first analyzed alymphoplasic (aly/aly) mice, which have a defective NIK and are unable to process p100, resulting in the absence of p52. We observed increased bone mineral density (BMD) and bone volume, indicating an osteopetrotic phenotype. These mice also have a significant defect in RANKL-induced osteoclastogenesis in vitro and in vivo. NF-kappaB DNA-binding assays revealed reduced activity of RelA, RelB, and p50 and no binding activity of p52 in aly/aly osteoclast nuclear extracts after RANKL stimulation. To determine the role of p100 itself without the influence of a concomitant lack of p52, we used p100(-/-) mice, which specifically lack the p100 inhibitor but still express p52. p100(-/-) mice have an osteopenic phenotype owing to the increased osteoclast and decreased osteoblast numbers that was rescued by the deletion of one allele of the relB gene. Deletion of both allele of relB resulted in a significantly increased bone mass owing to decreased osteoclast activity and increased osteoblast numbers compared with wild-type (WT) controls, revealing a hitherto unknown role for RelB in bone formation. Our data suggest a pivotal role of the alternative NF-kappaB pathway, especially of the inhibitory role of p100, in both basal and stimulated osteoclastogenesis and the importance of RelB in both bone formation and resorption.

Azadirachtin Interacts with the Tumor Necrosis Factor (TNF) Binding Domain of Its Receptors and Inhibits TNF-induced Biological Responses

The role of azadirachtin, an active component of a medicinal plant Neem (Azadirachta indica), on TNF-induced cell signaling in human cell lines was investigated. Azadirachtin blocks TNF-induced activation of nuclear factor kappaB (NF-kappaB) and also expression of NF-kappaB-dependent genes such as adhesion molecules and cyclooxygenase 2. Azadirachtin inhibits the inhibitory subunit of NF-kappaB (IkappaB alpha) phosphorylation and thereby its degradation and RelA (p65) nuclear translocation. It blocks IkappaB alpha kinase (IKK) activity ex vivo, but not in vitro. Surprisingly, azadirachtin blocks NF-kappaB DNA binding activity in transfected cells with TNF receptor-associated factor (TRAF)2, TNF receptor-associated death domain (TRADD), IKK, or p65, but not with TNFR, suggesting its effect is at the TNFR level. Azadirachtin blocks binding of TNF, but not IL-1, IL-4, IL-8, or TNF-related apoptosis-inducing ligand (TRAIL) with its respective receptors. Anti-TNFR antibody or TNF protects azadirachtin-mediated down-regulation of TNFRs. Further, in silico data suggest that azadirachtin strongly binds in the TNF binding site of TNFR. Overall, our data suggest that azadirachtin modulates cell surface TNFRs thereby decreasing TNF-induced biological responses. Thus, azadirachtin exerts an anti-inflammatory response by a novel pathway, which may be beneficial for anti-inflammatory therapy.

B-cell CLL/Lymphoma 10 (BCL10) Is Required for NF-κB Production by Both Canonical and Noncanonical Pathways and for NF-κB-inducing Kinase (NIK) Phosphorylation

B-cell CLL/lymphoma 10 (BCL10), the caspase recruitment domain (CARD)-containing protein involved in the etiology of the mucosa-associated lymphoid tissue (MALT) lymphomas, has been implicated in inflammatory processes in epithelial cells, as well as in immune cells. Experiments in this report indicate that BCL10 is required for activation of nuclear factor (NF)-kappaB by both canonical and noncanonical pathways, following stimulation by the sulfated polysaccharide carrageenan (CGN). In wild type and IkappaB-kinase (IKK)alpha(-/-) mouse embryonic fibroblasts, increases in phospho-IkappaBalpha, nuclear NF-kappaB p65 (RelA) and p50, and KC, the mouse analog of human interleukin-8, were markedly reduced by silencing BCL10 or by exposure to the free radical scavenger Tempol. In IKKbeta(-/-) cells, BCL10 silencing, but not Tempol, reduced the CGN-induced increases in KC, phospho-NF-kappaB-inducing kinase (NIK), cytoplasmic NF-kappaB p100, and nuclear NF-kappaB p52 and RelB, suggesting a BCL10 requirement for activation of the noncanonical pathway. In NCM460 cells, derived from normal, human colonic epithelium, the CGN-induced increases in NF-kappaB family members, p65, p50, p52, and RelB, were inhibited by BCL10 silencing. Although enzyme-linked immunosorbent assay and confocal images demonstrated no change in total NIK following CGN, increases in phospho-NIK in the wild type, IKKbeta(-/-) and IKKalpha(-/-) cells were inhibited by silencing BCL10. These findings indicate an upstream signaling role for BCL10, in addition to its effects on IKKgamma, the regulatory component of the IKK signalosome, and a requirement for BCL10 in both canonical and noncanonical pathways of NF-kappaB activation. Also, the commonly used food additive carrageenan can be added to the short list of known activators of both pathways.

Berbamine, a novel nuclear factor κB inhibitor, inhibits growth and induces apoptosis in human myeloma cells

We sought to investigate the effect of berbamine on the growth of human multiple myeloma cell line KM3 and elucidate the mechanism of its action. MTT assay was used to determine the inhibitory effect of berbamine alone or combined with chemotherapeutic drugs. Flow cytometry was performed to characterize cell cycle profile in response to berbamine treatment. Western blot was used to measure the protein levels of p65, IkappaB Kinase alpha (IKKalpha), TNFAIP3 (A20), IkappaBalpha, p-IkappaBalpha, cyclinD1, Bcl-2, BAX, Bcl-x(L), Bid, and survivin. Berbamine inhibits the proliferation of KM3 cells in a dose- and time-dependent manner. Combination of berbamine with dexamethasone (Dex), doxorubicin (Dox) or arsenic trioxide (ATO) resulted in enhanced inhibition of cell growth. Flow cytometric analysis revealed that KM3 cells were arrested at G(1) phase and apoptotic cells increased from 0.54% to 51.83% for 36 h. Morphological changes of cells undergoing apoptosis were observed under light microscope. Berbamine treatment led to increased expression of A20, down-regulation of IKKalpha, p-IkappaBalpha, and followed by inhibition of p65 nuclear localization. As a result, NF-kappaB downstream targets such as cyclinD1, Bcl-x(L), Bid and survivin were down-regulated. Berbamine inhibits the growth of KM3 cells by inducing G(1) arrest as well as apoptosis. Berbamine blocks NF-kappaB signaling pathway through up-regulating A20, down-regulating IKKalpha, p-IkappaBalpha, and then inhibiting p65 nuclear translocation, and resulting in decreased expression of the downstream targets of NF-kappaB. Our results suggest that berbamine is a novel inhibitor of NF-kappaB activity with remarkable anti-myeloma efficacy.