Activation Of Nuclear Transcription Factor Research Articles

MiR-218 sensitizes glioma cells to apoptosis and inhibits tumorigenicity by regulating ECOP-mediated suppression of NF-κB activity

Malignant gliomas are the most common and deadly primary brain tumors in adults. Increasing evidence has indicated that microRNAs (miRNAs) have an influence on the regulation of apoptotic cell signaling. Downregulation of miRNA 218 (miR-218) has been indicated in human glioma specimens. Here, we investigate the function of miR-218 in apoptosis and tumor growth of glioma cells. The expression of miR-218 was detected by real-time quantitative reverse transcriptase PCR. The effects of miR-218 on glioma cell proliferation and tumorigenicity were investigated by in vitro clonogenicity and in vivo xenograft assay. Apoptosis was evaluated by flow cytometric analysis and assay by terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling. The downstream targets of miR-218 were identified by bioinformatics analysis and further validated by Western blot and luciferase reporter assay. Overexpression of miR-218 induces glioma cell apoptosis and inhibits glioma cell viability, proliferation, and tumorigenicity. Epidermal growth factor receptor-coamplified and overexpressed protein (ECOP) was identified as a functional downstream target of miR-218, which can regulate transcriptional activity of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and associated with apoptotic response. Ectopic expression of ECOP rescued the glioma cells from miR-218-induced apoptosis and increased NF-κB activity. These results suggest that miR-218 sensitizes glioma cells to apoptosis by regulating ECOP-mediated suppression of NF-κB activity, which may provide novel opportunities for glioma therapy.

2-Methoxyestradiol inhibits experimental autoimmune encephalomyelitis through suppression of immune cell activation

The endogenous metabolite of estradiol, 2-Methoxyestradiol (2ME2), is an antimitotic and antiangiogenic cancer drug candidate that also exhibits disease-modifying activity in animal models of rheumatoid arthritis (RA). We found that 2ME2 dramatically suppresses development of mouse experimental autoimmune encephalomyelitis (EAE), a rodent model of multiple sclerosis (MS). 2ME2 inhibits in vitro lymphocyte activation, cytokine production, and proliferation in a dose-dependent fashion. 2ME2 treatment of lymphocytes specifically reduced the nuclear translocation and transcriptional activity of nuclear factor of activated T-cells (NFAT) c1, whereas NF-κB and activator protein 1 (AP-1) activation were not adversely affected. We therefore propose that 2ME2 attenuates EAE through disruption of the NFAT pathway and subsequent lymphocyte activation. By extension, our findings provide a molecular rationale for the use of 2ME2 as a tolerable oral immunomodulatory agent for the treatment of autoimmune disorders such as MS in humans.

Transcutaneous Application of Carbon Dioxide (CO2) Induces Mitochondrial Apoptosis in Human Malignant Fibrous Histiocytoma In Vivo

Mitochondria play an essential role in cellular energy metabolism and apoptosis. Previous studies have demonstrated that decreased mitochondrial biogenesis is associated with cancer progression. In mitochondrial biogenesis, peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α) regulates the activities of multiple nuclear receptors and transcription factors involved in mitochondrial proliferation. Previously, we showed that overexpression of PGC-1α leads to mitochondrial proliferation and induces apoptosis in human malignant fibrous histiocytoma (MFH) cells in vitro. We also demonstrated that transcutaneous application of carbon dioxide (CO2) to rat skeletal muscle induces PGC-1α expression and causes an increase in mitochondrial proliferation. In this study, we utilized a murine model of human MFH to determine the effect of transcutaneous CO2 exposure on PGC-1α expression, mitochondrial proliferation and cellular apoptosis. PGC-1α expression was evaluated by quantitative real-time PCR, while mitochondrial proliferation was assessed by immunofluorescence staining and the relative copy number of mitochondrial DNA (mtDNA) was assessed by real-time PCR. Immunofluorescence staining and DNA fragmentation assays were used to examine mitochondrial apoptosis. We also evaluated the expression of mitochondrial apoptosis related proteins, such as caspases, cytochorome c and Bax, by immunoblot analysis. We show that transcutaneous application of CO2 induces PGC-1α expression, and increases mitochondrial proliferation and apoptosis of tumor cells, significantly reducing tumor volume. Proteins involved in the mitochondrial apoptotic cascade, including caspase 3 and caspase 9, were elevated in CO2 treated tumors compared to control. We also observed an enrichment of cytochrome c in the cytoplasmic fraction and Bax protein in the mitochondrial fraction of CO2 treated tumors, highlighting the involvement of mitochondria in apoptosis. These data indicate that transcutaneous application of CO2 may represent a novel therapeutic tool in the treatment of human MFH.

Orai3 is an estrogen receptor α‐regulated Ca 2+ channel that promotes tumorigenesis

Store-operated Ca(2+) entry (SOCE) encoded by Orai1 proteins is a ubiquitous Ca(2+)-selective conductance involved in cellular proliferation and migration. We recently described up-regulation of Orai3 channels that selectively mediate SOCE in estrogen receptor α-expressing (ERα(+)) breast cancer cells. However, the connection between ERα and Orai3 and the role of Orai3 in tumorigenesis remain unknown. Here, we show that ERα knockdown decreases Orai3 mRNA (by ∼63%) and protein (by ∼44%) with no effect on Orai1. ERα knockdown decreases Orai3-mediated SOCE (by ∼43%) and the corresponding Ca(2+) release-activated Ca(2+) (CRAC) current (by ∼42%) in ERα(+) MCF7 cells. The abrogation of SOCE in MCF7 cells on ERα knockdown can be rescued by ectopic expression of Orai3. ERα activation increased Orai3 expression and SOCE in MCF7 cells. Epidermal growth factor (EGF) and thrombin stimulate Ca(2+) influx into MCF7 cells through Orai3. Orai3 knockdown inhibited SOCE-dependent phosphorylation of extracellular signal-regulated kinase (ERK1/2; by ∼44%) and focal adhesion kinase (FAK; by ∼46%) as well as transcriptional activity of nuclear factor for activated T cells (NFAT; by ∼49%). Significantly, Orai3 knockdown selectively decreased anchorage-independent growth (by ∼58%) and Matrigel invasion (by ∼44%) of ERα(+) MCF7 cells with no effect on ERα(-) MDA-MB231 cells. Moreover, Orai3 knockdown inhibited ERα(+) cell tumorigenesis in immunodeficient mice (∼66% reduction in tumor volume). These data establish Orai3 as an ERα-regulated channel and a potential selective therapeutic target for ERα(+) breast cancers.

Nuclear factor of activated T cells (NFATc4) is required for BDNF-dependent survival of adult-born neurons and spatial memory formation in the hippocampus

New neurons generated in the adult dentate gyrus are constantly integrated into the hippocampal circuitry and activated during encoding and recall of new memories. Despite identification of extracellular signals that regulate survival and integration of adult-born neurons such as neurotrophins and neurotransmitters, the nature of the intracellular modulators required to transduce those signals remains elusive. Here, we provide evidence of the expression and transcriptional activity of nuclear factor of activated T cell c4 (NFATc4) in hippocampal progenitor cells. We show that NFATc4 calcineurin-dependent activity is required selectively for survival of adult-born neurons in response to BDNF signaling. Indeed, cyclosporin A injection and stereotaxic delivery of the BDNF scavenger TrkB-Fc in the mouse dentate gyrus reduce the survival of hippocampal adult-born neurons in wild-type but not in NFATc4(-/-) mice and do not affect the net rate of neural precursor proliferation and their fate commitment. Furthermore, associated with the reduced survival of adult-born neurons, the absence of NFATc4 leads to selective defects in LTP and in the encoding of hippocampal-dependent spatial memories. Thus, our data demonstrate that NFATc4 is essential in the regulation of adult hippocampal neurogenesis and identify NFATc4 as a central player of BDNF-driven prosurvival signaling in hippocampal adult-born neurons.

Abstract LB-207: Tissue transglutaminase (TG2) - the key driver in inflammation-induced progression of cancer*

Abstract It is becoming evident that chronic inflammation plays important roles during carcinogenesis, tumor invasion and metastasis. However, molecular mechanisms linking inflammation to cancer progression remains elusive. Here we report that aberrant expression of tissue transglutaminase (TG2), a stress-responsive gene whose expression is modulated by multiple inflammatory signals, plays a key role in reprogramming epithelial cancer cells to dedifferentiation pathway. TG2 expression initiated the developmental program of epithelial to mesenchymal transition (EMT) and conferred stem cell traits in non-transformed and transformed epithelial cells. At molecular level, TG2 expression resulted in constitutive activation of nuclear transcription factor, NF-κB via a non-canonical pathways. Thus, TG2 associates in complex with the inhibitory protein IκBα, resulting in its rapid degradation via non-proteasomal pathway. Hypoxia-induced factor (HIF-1α) was identified as one of the direct downstream targets for TG2-induced NF-κB. TG2/ NF-κB-induced increase in HIF-1α resulted in regulation of transcription repressors like Snail, Zeb-1, and Twist, the known inducers of EMT/CSC. Importantly, our studies suggested that TG2 in closed or compact conformation could promote the oncogenic functions while open or extended conformation is completely inactive in this regard. TG2-induced programming rendered epithelial cells resistant to stress-induced death (including chemo- and radiation-therapies) and increased their propensity to metastasize. Indeed, previous studies support this contention and suggested that inhibition of TG2 could increase the sensitivity of cancer cells to chemotherapy and inhibit their metastasis, both in vitro and in animal models. Moreover, high basal expression of TG2 in breast, ovarian and pancreatic tumor samples is associated with poor disease outcome. These data warrants further studies to develop selective inhibitors/modulators of TG2 to increase the sensitivity of cancer cells to chemotherapy and to prevent their progression to metastatic disease. *Supported by a grant from Susan G. Komen Foundation for Cure. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-207. doi:1538-7445.AM2012-LB-207

IKKβ–I-κBɛ–c-Rel/p50: a new axis of NF-κB activation in lung epithelial cells

Cigarette smoke (CS), a major risk factor for developing lung cancer, is known to activate transcriptional activator nuclear factor kappa B (NF-κB). However, the underlying mechanism of this activation remains unclear because of conflicting reports. As NF-κB has a pivotal role in the generation and maintenance of malignancies, efforts were targeted towards understanding its activation mechanism using both ex vivo and in vivo studies. The results show that CS-induced NF-κB activation mechanism is different from that of other pro-inflammatory signals such as lipopolysaccharide (LPS). The NF-κB dimer that translocates to the nucleus upon stimulation with CS is predominantly composed of c-Rel/p50 and this translocation involves degradation of I-κBɛ and not I-κBα. This degradation of I-κBɛ depends on IKKβ activity, which preferentially targets I-κBɛ. Consistently, CS-activated form of IKKβ was found to be different from that involved in LPS activation as neither Ser177 nor Ser181 of IKKβ is crucial for CS-induced NF-κB activation. Thus, unlike other pro-inflammatory stimulations where p65 and I-κBα have a central role, the predominantly active signaling cascade in CS-induced NF-κB activation in the lung epithelial cells comprises of IKKβ–I-κBɛ–c-Rel/p50. Thus, this study uncovers a new axis of NF-κB activation wherein I-κBɛ and c-Rel have the central role.

Double-edged sword effect of biochanin to inhibit nuclear factor kappaB: Suppression of serine/threonine and tyrosine kinases

Several protein tyrosine kinase (PTK) inhibitors predominantly isoflavones, such as genistein, erbstatin, quercetin, daidzein, present in red clover, cabbage and alfalfa, show apoptotic effect against cancer cells. In this study I found that biochanin, a methoxy form of genistein, inhibits IL-8-mediated activation of nuclear transcription factor kappaB (NF-κB) and activator protein 1 (AP-1) more potently than genistein as shown in Jurkat T-cell line. Both biochanin and genistein potently inhibited activity of Lck and Syk, but biochanin specifically inhibited activity of IKK. Biochanin inhibited completely NF-κB activation induced by PMA, LPS, pervanadate (PV), or H2O2, but only partially that induced by TNFα. Genistein was unable to inhibit IL-8-induced IKK activity, but it blocked PV-induced IKK activity. Biochanin inhibited activation of NF-κB by TRAF6 completely, but by TRAF2 partially. In silico data suggested that biochanin interacted strongly with serine/threonine kinase than genistein, though both equally interacted with PTK. The data show that both biochanin and genistein are potent inhibitors of PTK, but biochanin is a potent inhibitor of serine/threonine kinase too. Formononetin, having hydroxyl methoxy group is less potent to inhibit IKK than biochanin. Biochanin inhibits NF-κB activation not only by blocking the upstream IKK, but also PTK that phosphorylate tyrosine residues of IκBα. Thus, the double-edged sword effect of inhibition of NF-κB via inhibition of both serine/threonine kinase and PTK by biochanin might show useful therapeutic value against activities of cells that lead to tumorigenesis and inflammation.

Multi‐layered stochasticity and paracrine signal propagation shape the type‐I interferon response

The cellular recognition of viruses evokes the secretion of type-I interferons (IFNs) that induce an antiviral protective state. By live-cell imaging, we show that key steps of virus-induced signal transduction, IFN-β expression, and induction of IFN-stimulated genes (ISGs) are stochastic events in individual cells. The heterogeneity in IFN production is of cellular-and not viral-origin, and temporal unpredictability of IFN-β expression is largely due to cell-intrinsic noise generated both upstream and downstream of the activation of nuclear factor-κB and IFN regulatory factor transcription factors. Subsequent ISG induction occurs as a stochastic all-or-nothing switch, where the responding cells are protected against virus replication. Mathematical modelling and experimental validation show that reliable antiviral protection in the face of multi-layered cellular stochasticity is achieved by paracrine response amplification. Achieving coherent responses through intercellular communication is likely to be a more widely used strategy by mammalian cells to cope with pervasive stochasticity in signalling and gene expression.

Role of YAP/TAZ in mechanotransduction

Cells perceive their microenvironment not only through soluble signals but also through physical and mechanical cues, such as extracellular matrix (ECM) stiffness or confined adhesiveness. By mechanotransduction systems, cells translate these stimuli into biochemical signals controlling multiple aspects of cell behaviour, including growth, differentiation and cancer malignant progression, but how rigidity mechanosensing is ultimately linked to activity of nuclear transcription factors remains poorly understood. Here we report the identification of the Yorkie-homologues YAP (Yes-associated protein) and TAZ (transcriptional coactivator with PDZ-binding motif, also known as WWTR1) as nuclear relays of mechanical signals exerted by ECM rigidity and cell shape. This regulation requires Rho GTPase activity and tension of the actomyosin cytoskeleton, but is independent of the Hippo/LATS cascade. Crucially, YAP/TAZ are functionally required for differentiation of mesenchymal stem cells induced by ECM stiffness and for survival of endothelial cells regulated by cell geometry; conversely, expression of activated YAP overrules physical constraints in dictating cell behaviour. These findings identify YAP/TAZ as sensors and mediators of mechanical cues instructed by the cellular microenvironment.

NOD2 plays an important role in the inflammatory responses of macrophages to Staphylococcus aureus

Objective To investigate the effects of NOD2 on inflammatory responses of macrophages to Staphylococcus aureus. Methods Real-time RT-PCR detected NOD2 gene expression of macrophages infected by S. aureus. Synthesis of siRNA against NOD2 and interfere with macrophages, observed the effects of NOD2 gene silencing to phagocytosis of 5. aureus, cytokine secretion, activation of nuclear transcription factors, cell apoptosis of the macrophages infected by S. aureus using F.I .IS A, flow cytometry etc. Results S. aureus infection of macrophages can cause increased expression of intracellular NOD2. NOD2 gene silencing of macrophage lead to the decreased ability of phagocytosis with S. aureus, the lower levels of cytokines secretion, deficiencies of NF-κB activation. S. aureus can cause macrophage apoptosis, with the apoptosis rate increased with time. Conclusion The intracellular pattern recognition receptor NOD2 play a key role in pathogen recognition, signal transduction, activation of nuclear transcription factors in the process of macrophages infected by S. aureus. Key words: Staphylococcus aureus; Macrophages; NOD2 receptor; NF-κB

B cells lacking the tumor suppressor TNFAIP3/A20 display impaired differentiation and hyperactivation and cause inflammation and autoimmunity in aged mice

AbstractThe ubiquitin-editing enzyme A20/TNFAIP3 is essential for controlling signals inducing the activation of nuclear factor-κB transcription factors. Polymorphisms and mutations in the TNFAIP3 gene are linked to various human autoimmune conditions, and inactivation of A20 is a frequent event in human B-cell lymphomas characterized by constitutive nuclear factor-κB activity. Through B cell-specific ablation in the mouse, we show here that A20 is required for the normal differentiation of the marginal zone B and B1 cell subsets. However, loss of A20 in B cells lowers their activation threshold and enhances proliferation and survival in a gene-dose–dependent fashion. Through the expression of proinflammatory cytokines, most notably interleukin-6, A20-deficient B cells trigger a progressive inflammatory reaction in naive mice characterized by the expansion of myeloid cells, effector-type T cells, and regulatory T cells. This culminates in old mice in an autoimmune syndrome characterized by splenomegaly, plasma cell hyperplasia, and the presence of class-switched, tissue-specific autoantibodies.

Role of YAP/TAZ in mechanotransduction

Cells perceive their microenvironment not only through soluble signals but also in term of physical and mechanical cues, such as extracellular matrix (ECM) stiffness or confined adhesiveness. By mechanotransduction systems, cells translate these stimuli into biochemical signals controlling multiple aspects of cell behavior, including growth, differentiation and cancer malignant progression; but how rigidity mechanosensing is ultimately linked to activity of nuclear transcription factors remains poorly understood. Here we report the identification of the Yorkie-homologues YAP and TAZ as nuclear relays of mechanical signals exerted by ECM rigidity and cell-shape. This regulation requires Rho activity and tension of the acto-myosin cytoskeleton but is independent from the Hippo/LATS cascade. Crucially, YAP/TAZ are functionally required for differentiation of mesenchymal stem cells induced by ECM stiffness and for survival of endothelial cells regulated by cell geometry; conversely, expression of constitutive active YAP overrules physical constraints in dictating cell behavior. These findings identify YAP/TAZ as sensors and mediators of mechanical cues instructed by the cellular microenvironment.

The Expression and DNA-Binding Activity of NF-κB Nuclear Transcription Factor in the Tumors of Patients with Breast Cancer

The content and DNA-binding activity of NF-κB nuclear transcription factor subunit p65 (NF-κBp65) were evaluated by quantitative enzyme immunoassay in tumors and histologically intact tissues of 119 patients with breast cancer. DNA-binding activity of NF-κBp65 in the tumors was higher than in adjacent tissue in 97% cases. This elevation was paralleled by an increase in total protein content in the majority of cases. No significant relationship of the parameter with the disease stage, tumor size and histology, and degree of lymph node involvement was detected. However, the content of NF-κBp65 in tumors of malignancy degree III was significantly higher than in tumors of malignancy degree II. An increase in total expression of NF-κBp65 protein was found in HER-2+ tumors. This increase was not related to steroid hormone receptor status and was not paralleled by elevation of DNA-binding activity, which was maximum in tumors with the "triple negative" receptor status (RE-RP-HER-2-).

CADPE Inhibits PMA-Stimulated Gastric Carcinoma Cell Invasion and Matrix Metalloproteinase-9 Expression by FAK/MEK/ERK–Mediated AP-1 Activation

Metastasis is one of the main causes of death for patients with malignant tumors. Aberrant expression of matrix metalloproteinase-9 (MMP-9) has been implicated in the invasion and metastasis of various cancer cells. Here, we found that caffeic acid 3,4-dihydroxy-phenethyl ester (CADPE) could inhibit the migration and invasion of human gastric carcinoma cells in Transwell migration assays. To understand the underlying mechanism, we showed that CADPE significantly inhibited phorbol 12-myristate 13-acetate (PMA)-induced increases in MMP-9 expression and activity in a dose-dependent manner. The inhibitory effect of CADPE on MMP-9 expression correlated well with the suppression of MMP-9 promoter activity and the reduction of MMP-9 mRNA. Reporter gene assay and electrophoretic mobility shift assay showed that CADPE inhibited MMP-9 expression by suppressing the activation of the nuclear transcription factor activator protein-1 (AP-1) and c-Fos, but not NF-κB. Moreover, CADPE inhibited PMA-induced phosphorylation of protein kinases involved in AP-1 activation, such as focal adhesion kinase (FAK), mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase (MEK), and ERK1/2, whereas CADPE had little effect on the phosphorylation of p38 and c-jun NH(2)-terminal kinase. Taken together, our findings indicate that CADPE could be a unique antitumor agent that specifically inhibits MMP-9 activity by targeting the activation of FAK/MEK/ERK protein kinases and AP-1 transcription factor.

Anti-inflammatory Effect of Glycoprotein Isolated from Cudrania Tricuspidata Bureau: Involvement of MAPK/NF-κB Signaling

The aim of this study was to evaluate inhibitory effect of glycoprotein isolated from Cudrania tricuspidata Bureau (CTB glycoprotein) on allergic responses. We evaluated the activities of mitogen-activated protein kinase (MAPK), transcriptional factor, and production of immunoglobulin (Ig)E and interleukin (IL)-4 in RBL-2H3 cells and BALB/c mice. Our results showed that CTB glycoprotein inhibited the production of IgE and IL-4 in serum from ovalbumin (OVA)-treated BALB/c mice. We also found that CTB glycoprotein inhibited the phosphorylation of p38 MAPK, and transcriptional activation of nuclear factor (NF)-κB in RBL-2H3 cells. The activation of NF‐κB was effectively blocked by treatment with p38 MAPK inhibitor (SKF86002). The results from these experiments indicate that the CTB glycoprotein inhibits release of β-hexosaminidase, and production of IgE and IL-4 via down regulation of MAPK/ NF-κB on the stage of mast cell degranulation. In conclusion, we suggest that the CTB glycoprotein might be a potent preventive agent in allergic responses.

Cocoa polyphenols suppress TNF-α-induced vascular endothelial growth factor expression by inhibiting phosphoinositide 3-kinase (PI3K) and mitogen-activated protein kinase kinase-1 (MEK1) activities in mouse epidermal cells

Cocoa polyphenols have antioxidant and anti-inflammatory effects. TNF-α is a pro-inflammatory cytokine that has a vital role in the pathogenesis of inflammatory diseases such as cancer and psoriasis. Vascular endothelial growth factor (VEGF) expression is associated with tumorigenesis, CVD, rheumatoid arthritis and psoriasis. We tested whether cocoa polyphenol extract (CPE) inhibited TNF-α-induced VEGF expression in promotion-sensitive JB6 mouse epidermal cells. CPE significantly inhibited TNF-α-induced up-regulation of VEGF via reducing TNF-α-induced activation of the nuclear transcription factors activator protein-1 (AP-1) and NF-κB, which are key regulators of VEGF expression. CPE also inhibited TNF-α-induced phosphorylation of protein kinase B (Akt) and extracellular signal-regulated kinase. CPE blocked activation of their downstream kinases, p70 kDa ribosomal protein S6 kinase and p90 kDa ribosomal protein S6 kinase. CPE suppressed phosphoinositide 3-kinase (PI3K) activity via binding PI3K directly. CPE did not affect TNF-α-induced phosphorylation of mitogen-activated protein kinase kinase-1 (MEK1) but suppressed TNF-α-induced MEK1 activity. Collectively, these results indicate that CPE reduced TNF-α-induced up-regulation of VEGF by directly inhibiting PI3K and MEK1 activities, which may contribute to its chemopreventive potential.

Abstract 135: Apoptotic cell death triggering in the prostate carcinoma cell line PC3 by the anoikis effector Bit1

Abstract Highly aggressive cancer cells often are associated with resistance to caspase-dependent apoptosis, due, in part to loss in function or mutation in caspase effectors. Such apoptotic defects in caspase signaling pathway may provide a selective growth advantage for tumor cells and potentiate their aggressiveness and resistance to chemotherapeutic agents. Here, we test the responsiveness of the highly aggressive prostate cancer cell line PC3 cells to the apoptotic function of the novel, caspase-independent anoikis effector Bcl2 inhibitor of transcription (Bit1). Overexpression of the cytoplasmic localized Bit1 in PC3 cells results in significant induction of apoptosis as evidenced by the increased levels of histone-DNA fragments. Furthermore, PC3 cells transfected with the mitochondrial localized Bit1 exhibit significant apoptosis following loss of cell attachment as compared to vector treated cells, and such induction of anoikis is associated with increased levels of Bit1 in the cytosol. In contrast, both the control and Bit1 transfected PC3 cells show the same level of spontaneous apoptosis when grown attached to a culture dish. We also tested whether Bit1 alters the chemoresistant phenotype of PC3 cells. Consistent with the induction of anoikis sensitivity, overexpression of mitochondrial Bit1 also renders PC3 cells more sensitive to etoposide-induced apoptosis. Interestingly, the Bit1-mediated apoptosis in PC3 cells is correlated with increased NFkappa B activity as evidenced by increased nuclear translocation and transcription factor activity of the p65 subunit. Taken together, these findings indicate that activation of the caspase-independent Bit1 apoptotic pathway may serve as a preferred alternate approach in triggering apoptosis in aggressive prostate cancer cells. Bit1 may thus serve as a future therapeutic target for the treatment and alleviation of the chemoresistance characteristic of prostate cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 135.

Variable Expression of Nuclear Receptor Coactivator 4 (NcoA4) During Mouse Embryonic Development

Human nuclear receptor coactivator 4 (NcoA4) amplifies the activity of several ligand-activated nuclear transcription factors, including the aryl hydrocarbon receptor (AhR) and androgen receptor (AR). Because these receptors exert important regulatory effects during development, with AhR ubiquitously expressed after embryonic day 9.5 (E9.5) and AR expressed from E12 onward, we examined NcoA4 expression in mouse embryos from E9.5 to E17.5. Full-length NcoA4 transcript was detected by RT-PCR at all embryonic stages and in all adult mouse tissues examined, although a novel splice variant was also detected. Western blot analysis indicated the expression of full-length NcoA4 protein, which was more highly expressed at later (E15.5-E17.5) embryonic stages. NcoA4 protein was also present at varying levels in all adult mouse tissues examined. A dynamic expression profile for NcoA4 during early development was indicated by immunohistochemistry in cardiac, hepatic, and lung tissue. Unlike human NcoA4, murine NcoA4 lacks an LXXLL motif, which has been implicated in the interaction with AR. Overexpression of murine NcoA4 augmented the transcriptional activity of AhR by 5-fold and AR by only 1.5-fold in COS cells. These studies demonstrate ubiquitous NcoA4 expression throughout development and suggest that this coactivator may play a role in modulating nuclear receptor activity, particularly that of the AhR, during development.

Modulatory effects of phytoglycoprotein (75 kDa) on allergic inflammatory cytokines in Di(2‐ethylhexyl) phthalate (DEHP)‐stimulated RBL‐2H3 cells

This study investigated the inhibitory effect of a glycoprotein isolated from Cudrania tricuspidata Bureau (CTB glycoprotein) on di(2-ethylhexyl) phthalate (DEHP)-induced mast cell degranulation and related signaling cascade in RBL-2H3 cells. This experiment evaluated the intracellular Ca(2+) level, and the activities of protein kinase C (PKC), mitogen-activated protein kinase (MAPK), transcription factor, and the cytokines in DEHP-treated RBL-2H3 cells. Our results revealed that the CTB glycoprotein in the presence of DEHP inhibits the release of histamine and expression of interleukin (IL)-4, IL-6, and TNF-alpha in RBL-2H3 cells. We also found that the CTB glycoprotein inhibits the intracellular Ca(2+) level, translocation of PKC from cytosol to membrane and the phosphorylation of ERK1/2 in cells. Moreover, the CTB glycoprotein (100 microg/ml) has suppressive effects on transcriptional activation of nuclear factor (NF)-kappaB in DEHP-treated RBL-2H3 cells. The activation of NF-kappaB was collectively blocked by treatment with PKC inhibitor (staurosporine) as well as ERK1/2 inhibitor (PD98059), respectively. The results from these experiments indicated that the CTB glycoprotein inhibits release of histamine and expressions of IL-4, IL-6, and TNF-alpha via down regulations of PKC/MAPK and NF-kappaB on the stage of mast cell degranulation induced by DEHP. Moreover, oral administration of CTB glycoprotein (10-20 mg/kg) inhibited compound 48/80-mediated systemic reaction in mice. In conclusion, we speculated that the CTB glycoprotein might be one component for preparation of health supplements for prevention of allergic immune disorders.