NF-kappaB Protein Research Articles

Pyrrolidine dithiocarbamate attenuate shock wave induced MDCK cells injury via inhibiting nuclear factor-kappa B activation

Shock wave lithotripsy (SWL)-induced renal damage appears to be multifactorial. Recent data indicated that the mechanism of renal tissue damage secondary to SWL is similar to that of ischemia reperfusion injury. Nuclear factor-kappa B (NFkappaB) and its target genes, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), have been demonstrated to play a very important role in a variety of cells or tissues ischemia reperfusion injuries. Thus in the present study, using an in vitro model MDCK cells, we investigated the role of NFkappaB and its target cytotoxic enzyme in shock wave-induced renal cellular damage. We also examined whether inhibition this pathway by pyrrolidine dithiocarbamate (PDTC) is contributed to alleviate SWL-caused cell damage. Suspensions of MDCK cells were placed in containers for shock wave exposure. Three groups of six containers each were examined: control group, no shock wave treatment and SWL group, which received 100 shocks at 18 kV; 3 SWL + PDTC group. PDTC were added to the suspensions before shock wave exposure. After shock wave 0, 2, 4, 6 and 8 h, respectively, the cell supernatants were detected for the level of MDA and release of LDH. At post-shock wave 8 h, cells were harvested to detect the nuclear translocation of NFkappaBp65 by immunofluorescence staining. Degradation of IkappaB-a (an inhibitor protein of NFkappaB) and expression of iNOS and COX-2 were also examined by western blotting. Our results indicated that shock wave initiated the apparent activation of NFkappaB, which in turn induced high expression of iNOS and COX-2. Blocking degradation of IkappaB-a by PDTC was contributed to decrease the expression of iNOS. And the level of MDA and the release of LDH were also significantly reduced by using PDTC. However, the degree of COX-2 expression does not differ significantly between SWL and SWL + PDTC groups. Activation of NFkappaB and subsequent expression of its target cytotoxic enzyme have been demonstrated to be a potential and crucial mechanism in SWL-induced renal cell damage. Blocking this pathway by PDTC is contributed to protect against cellular damage from shock wave.

Repression of Dmp1 and Arf transcription by anthracyclins: critical roles of the NF-κB subunit p65

Both genotoxic and oncogenic stress activates the nuclear factor-kappa B (NF-kappaB) and p53 proteins; however, the p53 activity is antagonized by NF-kappaB signaling. Dmp1 is a Myb-like transcription factor that activates the Arf-p53 pathway. The Dmp1 promoter was activated by a classical NF-kappaB activator tumor necrosis factor alpha, but repressed by treatment of cells with non-classical NF-kappaB activators, anthracyclins and UV-C. p65 and other subsets of NF-kappaB proteins were bound to the Dmp1 promoter following anthracyclin/UV-C treatment of rodent fibroblasts. This resulted in the downregulation of Dmp1 mRNA and protein. Repression of the Dmp1 transcription by anthracyclins depended on the unique NF-kappaB site on the promoter. Downregulation of p65 significantly attenuated the repression of the Dmp1 promoter by anthracyclins/UV-C. The amount of Dmp1 bound to the Arf promoter decreased significantly upon anthracyclin treatment; this, in turn, downregulated the Arf levels. Repression of the Arf promoter by p65 or anthracyclins depended on Dmp1, which was significantly attenuated in Dmp1(-/-) cells. Both Dmp1(-/-)and Arf(-/-)cells showed resistance to anthracyclin-induced cell death compared to wild-type cells; non-immortalized p65-knockdown cells were much more sensitive. Thus, the Dmp1-Arf pathway is repressed by p65 in response to genotoxic stress, which implicates a novel mechanism of p53 inactivation by NF-kappaB.

Cisplatin Cytotoxicity of Auditory Cells Requires Secretions of Proinflammatory Cytokines via Activation of ERK and NF-κB

The ototoxicity of cisplatin, a widely used chemotherapeutic agent, involves a number of mechanisms, including perturbation of redox status, increase in lipid peroxidation, and formation of DNA adducts. In this study, we demonstrate that cisplatin increased the early immediate release and de novo synthesis of proinflammatory cytokines, including TNF-alpha, IL-1beta, and IL-6, through the activation of ERK and NF-kappaB in HEI-OC1 cells, which are conditionally immortalized cochlear cells that express hair cell markers. Both neutralization of proinflammatory cytokines and pharmacologic inhibition of ERK significantly attenuated the death of HEI-OC1 auditory cells caused by cisplatin and proinflammatory cytokines. We also observed a significant increase in the protein and mRNA levels of proinflammatory cytokines in both serum and cochleae of cisplatin-injected rats, which was suppressed by intraperitoneal injection of etanercept, an inhibitor of TNF-alpha. Immunohistochemical studies revealed that TNF-alpha expression was mainly located in the spiral ligament, spiral limbus, and the organ of Corti in the cochleae of cisplatin-injected rats. NF-kappaB protein expression, which overlapped with terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labeling-positive signal, was very strong in specific regions of the cochleae, including the organ of Corti, spiral ligament, and stria vascularis. These results indicate that proinflammatory cytokines, especially TNF-alpha, play a central role in the pathophysiology of sensory hair cell damage caused by cisplatin.

Beneficial effects of losartan on vascular injury induced by advanced glycosylation end products and their receptors in spontaneous hypertension rats

This study was designed to explore the role of losartan, an angiotensin II receptor blocker, in hypertensive injuries of blood vessels and the potential mechanisms related to the vascular advanced glycosylation end product (AGE)/receptor (RAGE) system, oxidative stress and endothelial proinflammatory factors. Spontaneously hypertensive rats (SHR) were employed for our study, and age-matched Wistar-Kyoto rats (WKY) were used for control experiments. After losartan treatment for 12 weeks, we observed by immunofluorescence that the vascular AGE level in the losartan group was significantly lower than that of the SHR group and that the vascular mRNA expression of RAGE, NF-kappaB, NADPH oxidase p47phox and ET-1, as detected by RT-PCR, was significantly lower in losartan group than in the SHR group. Meanwhile, we found that the expression of RAGE and NF-kappaB proteins in the losartan group and the WKY group was remarkably lower than that of the SHR group. Compared with the SHR group, the activities of plasma superoxide dismutase (SOD) and glutathione peroxidase (GSH-PX) and the NO level were robustly increased, while the plasma malondialdehyde (MDA) and ET-1 were substantially reduced. These findings suggest that losartan decreases the vascular AGE level, suppresses RAGE and NF-kappaB activation, and enhances the antioxidant capacity thereby improving the endothelial function, which induce hypertensive vascular remodeling.

Sesquiterpene lactone parthenolide suppresses tumor growth in a xenograft model of renal cell carcinoma by inhibiting the activation of NF‐κB

The transcription factor nuclear factor-kappaB (NF-kappaB) has been shown to be constitutively activated in various human malignancies, including leukemia, lymphoma and a number of solid tumors. NF-kappaB regulates the transcriptional of genes important for tumor invasion, metastasis and chemoresistance. The sesquiterpene lactone parthenolide, an inhibition of NF-kappaB, has been used conventionally to treat migraines and inflammation. In this study, renal cancer cell lines OUR-10 and ACHN were used for in vitro experiments to evaluate growth-inhibitory effects of parthenolide. An OUR-10 xenograft model in nude mice was also used to investigate the in vivo growth-inhibitory effects of parthenolide. Apoptosis in response to treatment of OUR-10 cells with parthenolide was confirmed. Localization of NF-kappaB in response to parthenolide treatment was examined of by immunofluorostaining of OUR-10 cells with antibody against NF-kappaB p65 and by Western blot analysis of OUR-10 cell and tumor nuclear and cytosol fraction. Parthenolide effectively inhibited proliferation of cultured OUR-10 cells and triggered apoptosis in vitro. Subcutaneous injection or oral administration of parthenolide showed significant tumor growth inhibition in the xenograft model via decreased production of interleukin-8 (IL-8) or vascular endothelial growth factor (VEGF). Immunohistochemistry and Western blot analysis showed decreased nuclear localization of NF-kappaB and phosphorylated NF-kappaB protein and subsequently expression of MMP-9, Bcl-xL and Cox-2 in response to parthenolide treatment. These results indicate that parthenolide is a useful in the treatment of renal cell carcinoma and acts via inhibition of NF-kappaB.

NFkappaB is persistently activated in continuously stimulated human neutrophils.

Increased activation of the transcription factor NFkappaB in the neutrophils has been associated with the pathogenesis of sepsis, acute lung injury (ALI), bronchopulmonary dysplasia (BPD), and other neutrophil-mediated inflammatory disorders. Despite recent progress in analyzing early NFkappaB activation in human neutrophils, activation of NFkappaB in persistently stimulated neutrophils has not been previously studied. Because it is the persistent NFkappaB activation that is thought to be involved in the host response to sepsis and the pathogenesis of ALI and BPD, we hypothesized that continuously stimulated human neutrophils may exhibit a late phase of NFkappaB activity. The goal of this study was to analyze the NFkappaB activation and expression of IkappaB and NFkappaB proteins during neutrophil stimulation with inflammatory signals for prolonged times. We demonstrate that neutrophil stimulation with lipopolysaccharide (LPS) and tumor necrosis factor-alpha (TNFalpha) induces, in addition to the early activation at 30-60 min, a previously unrecognized late phase of NFkappaB activation. In LPS-stimulated neutrophils, this NFkappaB activity typically had a biphasic character, whereas TNFalpha-stimulated neutrophils exhibited a continuous NFkappaB activity peaking around 9 h after stimulation. In contrast to the early NFkappaB activation that inversely correlates to the nuclear levels of IkappaBalpha, however, in continuously stimulated neutrophils, NFkappaB is persistently activated despite considerable levels of IkappaBalpha present in the nucleus. Our data suggest that NFkappaB is persistently activated in human neutrophils during neutrophil-mediated inflammatory disorders, and this persistent NFkappaB activity may represent one of the underlying mechanisms for the continuous production of proinflammatory mediators.

THE RON RECEPTOR TYROSINE KINASE REGULATES ACUTE LUNG INJURY AND SUPPRESSES NUCLEAR FACTOR κB ACTIVATION

Emerging information implies that the Ron receptor tyrosine kinase may play a role in the inflammatory response. However, the manner in which this receptor contributes to the response is not well understood. In the present studies, we investigated the role of the Ron receptor in the acute lung inflammatory response. Wild-type and mutant mice lacking the tyrosine kinase domain of Ron (Ron TK-/-) were subjected to acute lung injury induced by intranasal administration of bacterial lipopolysaccharide (LPS). Wild-type mice showed increased lung injury after LPS administration, as determined by the leakage of albumin into the lung and by histopathological changes. Ron TK-/- mice had more than twice the amount of albumin leak and much greater thickening of the alveolar septae. Lipopolysaccharide administration caused neutrophil recruitment into the lungs, as measured by myeloperoxidase. However, Ron TK-/- mice had much higher baseline levels of myeloperoxidase, which did not increase further after LPS. Lung injury in wild-type mice occurred with activation of the transcription factor, nuclear factor kappaB (NF-kappaB), and subsequent increases in intrapulmonary generation of tumor necrosis factor alpha. In TK-/- mice, there was far less IkappaB-alpha and IkappaB-beta protein and greater activation of NF-kappaB. This was associated with substantially increased production of tumor necrosis factor alpha and the nitric oxide (NO) by-product, nitrite. The data suggest that the Ron receptor tyrosine kinase plays an important regulatory role in acute inflammatory lung injury by suppressing signals leading to activation of NF-kappaB.

Mechanisms of antiprostate cancer by gum mastic: NF-?B signal as target

To study the effect of gum mastic, a natural resin, on the proliferation of androgen-independent prostate cancer PC-3 cells, and further investigate the mechanisms involved in this regulatory system, taking nuclear factor kappaB (NF-kappaB) signal as the target. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and a flow cytometer were used to detect the effect of gum mastic on the proliferation of PC-3 cells. Then, reporter gene assay, RT-PCR, and Western blotting were carried out to study the effects of gum mastic on the NF-kappaB protein level and the NF-kappaB signal pathway. The expression of genes involved in the NF-kappaB signal pathway, including cyclin D1, inhibitors of kappaBs (I kappaB alpha), and phosphorylated Akt (p-AKT), were measured. In addition, transient transfection assays with the 5X NF-kappaB consensus sequence promoter was also used to test the effects of gum mastic. Gum mastic inhibited PC-3 cell growth and blocked the PC-3 cell cycle in the G1 phase. Gum mastic also suppressed NF-kappaB activity in the PC-3 cells. The expression of cyclin D1, a crucial cell cycle regulator and an NF-kappaB downstream target gene, was reduced as well. Moreover, gum mastic decreased the p-AKT protein level and increased the I kappa B alpha protein level. Gum mastic inhibited the proliferation and blocked the cell cycle progression in PC-3 cells by suppressing NF-kappaB activity and the NF-kappaB signal pathway.

Rel homology domain-containing transcription factors in the cnidarian Nematostella vectensis

The Rel/NF-kappaB and NFAT families of transcription factors are related through an N-terminal DNA-binding domain called the Rel Homology domain (RHD). Neither the RHD nor the NF-kappaB pathway has been identified in a basal (i.e., nonbilaterian) animal phylum. Using genomic and cDNA databases, we have identified two RHD domain-containing proteins from the cnidarian Nematostella vectensis: an NF-kappaB-like protein (Nv-NF-kappaB) and an NFAT-like protein (Nv-NFAT). The gene structure and RHD predicted amino acid sequence of Nv-nfkb are similar to those of the vertebrate NF-kappaB p50/p52 proteins, whereas the sequence of Nv-NFAT allows only ambiguous assignment to the NFAT family. Nv-NF-kappaB lacks the C-terminal IkappaB-like sequences present in all other NF-kappaB proteins. There are, however, two IkappaB-like genes in Nematostella encoded by loci distinct from Nv-nfkb. The separate nfkb and ikb genes of Nematostella may reflect the ancestral metazoan condition, suggesting that a gene fusion event created the nfkb genes in Drosophila and vertebrates. Nematostella also has genes that encode upstream and downstream components of the vertebrate NF-kappaB signaling pathway. Upstream components include Toll- and tumor necrosis-like receptors and ligands, adaptor proteins (Trafs, Myd88), caspases, and a TBK-like kinase. Downstream components include the NF-kappaB coactivator protein Bcl-3 and several NF-kappaB target genes. These results demonstrate that RHD-containing transcription factors and associated pathways are evolutionarily more ancient than previously known. Moreover, they suggest models for the evolutionary diversification of the insect and vertebrate Rel/NF-kappaB/IkappaB and NFAT gene families and suggest that cnidarians possess an NF-kappaB-regulated developmental or stress response pathway.

Nuclear factor-kappa B pathways in Drosophila

The nuclear factor kappa B (NF-kappaB) pathways in Drosophila are multi-component pathways, as in vertebrates, that regulate the expression of many genes responsible for the formation of dorsal-ventral polarity in the early embryo, the innate immune response to infection with Gram- negative and positive bacteria and fungi, the cellular immune response and hematopoiesis. Overactivation of the fly pathway can result in developmental defects, overproliferation of hemocytes and the formation of melanotic tumors or nodules. The extracellular events leading to the maturation of the ligand for initiation of the Drosophila NF-kappaB pathway is not conserved between flies and vertebrates, but the Toll receptor and downstream events are remarkably similar. NF-kappaB proteins have been identified in mollusks, and arthropods such as horseshoe crabs and beetles, indicating that this pathway has been established more than 500 million years ago. The fly NF-kappaB pathways are less complex than those in vertebrates, with the involvement of fewer proteins, but they are, nonetheless, just as important as their vertebrate counterparts for the life of the fly.

Differential expression and activation of NF‐κB family proteins during oral carcinogenesis: Role of high risk human papillomavirus infection

Oral cancer is one of the most common cancers in India and south-east Asian region consisting of more than 50% of all malignant tumors. Along with many known risk factors, infection of Human Papillomavirus (HPV) has been associated with the development of oral cancer and is suggested to modulate host cell transcription. Reciprocally, cellular transcription factors, such as NF-kappaB and AP-1 are known to modulate the expression of viral and other genes involved in the development of cancer. In the absence of data on NF-kappaB in relation to HPV in oral cancer, we studied the DNA binding activity and expression pattern of NF-kappaB family of proteins in different stages of oral cancer and correlated with HPV infection that has been associated with better prognosis of the disease. A total of 110 fresh oral tissue biopsies were collected comprising 10 normal controls, 34 precancer and 66 oral cancer lesions prior to chemotherapy/radiotherapy. Diagnosis of HPV was done by both consensus and type-specific PCR. Electrophoretic mobility shift assays, western blots and immunohistochemical analysis were performed to assess the binding activity and expression pattern of NF-kappaB family of proteins (p50, p65, p52, c-Rel, RelB and Bcl-3) in oral tissue biopsies. Twenty seven percent (18/66) of the oral cancer biopsies showed the presence of HPV infection exclusively of high risk HPV type 16, which was primarily associated with the well differentiated squamous cell carcinomas (WDSCC). We observed a high constitutive activation of NF-kappaB with concomitant upregulated expression of all the NF-kappaB members in oral cancer tissues. Expression of NF-kappaB components gradually increased as the severity of lesion increased from precancer to invasive cancer. NF-kappaB p50 was found to be the major DNA binding component, which is indicative of homodimerization of p50 subunits. Interestingly, in HPV16 infected oral cancers although p50 showed high binding activity, p65 also showed a partial involvement as evidenced in supershift assay. Both by western blotting and immunohistochemistry, a differential overexpression and nuclear localization of p50, p65 and partially of Bcl-3 were observed in HPV16 positive oral cancer patients that also showed an over-expression of p21. We therefore, demonstrate a constitutive activation and differential expression of NF-kappaB proteins, which change as a function of severity of oral lesions during development of oral cancer. The NF-kappaB DNA binding is primarily due to homodimerization of p50 but infection of high risk HPV promotes participation of p65 in NF-kappaB complex formation, leading to heterodimerization of p50/p65. We propose that the involvement of p65 in HPV infected oral cancer may be linked to improved differentiation and better prognosis of the disease when treated.

Elevated expression of NF-κB in oral submucous fibrosis – Evidence for NF-κB induction by safrole in human buccal mucosal fibroblasts

Nuclear factor-kappa B (NF-kappaB) is considered to be important in many inflammatory and immune responses. The aim of this study was to compare NF-kappaB expression in normal human buccal mucosa and oral submucous fibrosis (OSF) specimens and further explore the potential mechanism that may lead to induction of NF-kappaB expression. Seventeen OSF and six normal buccal mucosa specimens were examined by immunohistochemistry. Primary human buccal mucosal fibroblasts (BMFs) were established and challenged with safrole, a major polyphenolic compound in the influorescence of Piper betel, by cytotoxicity and western blot assays. Furthermore, glutathione precursor N-acetyl-L-cysteine (NAC), extracellular signal-regulated protein kinase (ERK) inhibitor PD98059, cyclooxygenase-2 (COX-2) inhibitor NS-398, dexamethasone, and cyclosporin A were added to find the possible mechanism. NF-kappaB expression was significantly higher in OSF specimens and expressed mainly by fibroblasts, endothelial cells, and inflammatory cells. Safrole was cytotoxic to BMFs in a dose-dependent manner (p<0.05). Western blot demonstrated highly elevated NF-kappaB protein expression in BMFs stimulated by safrole (p<0.05). In addition, pretreatment with pharmacological agents markedly inhibited the safrole induced-NF-kappaB expression (p<0.05). The result suggests that chewing areca quid may activate NF-kappaB expression that may be involved in the pathogenesis of OSF. NF-kappaB expression induced by safrole in fibroblasts may be mediated by ERK activation and COX-2 signal transduction pathway.

Protective effects of propofol on lipopolysaccharide-activated endothelial cell barrier dysfunction

Propofol has been widely used in intravenous anesthesia. It possesses antioxidant and immunomodulating effects. This study aimed to investigate whether propofol may attenuate lipopolysaccharide (LPS)-induced endothelial cell barrier dysfunction and the possible mechanisms of such modulation. Cultured human umbilical vein endothelial cells (HUVECs) were used to assess the following treatments: (i) no additives (negative control), (ii) LPS alone (1 and 10 microg/ml), (iii) propofol alone (20 microg/ml), (iv) intralipid (a solvent of propofol) alone (20 microg/ml), (v) LPS (10 microg/ml) combination with propofol (4 and 20 microg/ml) and (vi) LPS (10 microg/ml) combination with intralipid (20 microg/ml). Changes of cell permeability and filamentous actin (F-actin) were determined. Expression of nitrotyrosine proteins and activity of nuclear factor kappaB (NF-kappaB) were analyzed by Western blot and immunocytochemistry. Expression of endothelial nitric-oxide synthase (eNOS) and inducible nitric-oxide synthase (iNOS) were analyzed by reverse transcriptase-polymerase chain reaction. LPS markedly increased the permeability of endothelial cells, the formation of peroxynitrite and depolymerization of F-actin in HUVECs. LPS also significantly increased mRNA of iNOS, protein level of NF-kappaB and decreased mRNA of eNOS (P < 0.05). Propofol at both concentrations (4 and 20 microg/ml) significantly inhibited the LPS-induced increase in cell permeability and alteration in F-actin organization. Propofol also reduced the LPS-enhanced iNOS mRNA and NF-kappaB protein levels whilst it increased eNOS mRNA expression (P < 0.05). This study demonstrated that propofol, both at therapeutic concentrations and 5 times therapeutic concentrations, inhibited NF-kappaB activation in LPS-stimulated endothelial cells and was found to protect endothelial cells against LPS-induced barrier dysfunction.

Basic Science for the Clinician 39

Were there to be a crossroads through which all inflammatory signaling passed, controlling that junction would provide the ultimate therapeutic target for rheumatoid arthritis and many, if not all, autoimmune diseases. It now seems likely that no single cytokine or cytokine family represents such a crucial nexus. However, there is reason to believe that there may be an intracellular bottleneck that does: the family of NF-kappaB proteins. This family of proteins allows cytokine-receptor signals to enter the nucleus and either enhance or suppress the transcription of many genes involved in inflammation and in cellular survival itself. The same set of proteins is also involved in apoptosis and likely in carcinogenesis. The delicate choreography of control systems, balancing the effects of NF-kappaB proteins on the multiple DNA sites that are targeted, is also a prime target for specific therapies. Moreover, the NF-kappaB system interdigitates with other intracellular systems, eg, kinases, ubiquitin-associated protein degradation, that are critical to the normal function of cells, involved in homeostasis and inflammation, in autoimmune diseases and malignancy.

Magnetocapture of abalone transcription factor NF-κB: A new strategy for isolation and detection of NF-κB both in vitro and in vivo

Rel\NF-kappaB plays an important role in the immune response. Recently, a homologue of Rel\NF-kappaB transcription factor was identified from a gastropod abalone, Haliotis diversicolor supertexta, named Ab-Rel. We present here a new strategy, so called magnetocapture, based on magnetic separation that was developed to isolate and detect this abalone transcriptional factor. With this method, Ab-Rel was successfully isolated from abalone haemocytes and the recombinant Ab-Rel protein from an insect cell line. Western blot analysis was performed to verify the effectiveness of this magnetocapture assay. Since only the activated NF-kappaB can be captured and separated from the whole cell extracts, this method was simultaneously used to detect the NF-kappaB activity in vivo, as well as the biological activity of the recombinant protein in vitro. In addition, several proteins which might interact with the abalone NF-kappaB were together isolated, suggesting that the magnetocapture method would be useful for identifying interaction molecules. These results illustrated that the magnetocapture strategy is rapid, reliable and versatile, providing a promising tool for studying Rel\NF-kappaB protein.

Nuclear Factor Kappa B is a Promising Therapeutic Target in Inflammatory Lung Disease

Nuclear factor kappa B (NF-kappaB) regulates the transcription of a wide array of gene products that are involved in the molecular pathobiology of the lung. Three lung cell types, epithelial cells, macrophages and neutrophils, have been shown to be involved in the generation of lung inflammation through signaling mechanisms that are dependent on activation of the NF-kappaB pathway. The basic molecular biology of the NF-kappaB activation pathway is well described, and approaches to modify this axis have involved inhibition of various components of the classical activation pathway, including ubiquitination and proteosomal degradation of IkappaB. Recently, there have been detailed characterizations of molecular mechanisms that involve reversible post-translational modification of RelA, including phosphorylation and acetylation that might be amenable to therapeutic interdiction. Alternately DNA decoy, antisense and siRNA technologies that interfere with NF-kappaB binding and inhibition of gene expression, respectively, of NF-kappaB proteins have been employed in experimental settings, but this has not been practically or effectively applied in human disease. A very promising approach, in our view, is inhibition of inhibitory kappa B kinases (IKK) since these appear to be highly specific for the NF-kappaB activation pathway and amenable to conventional small molecule pharmaceutical approaches.

Nuclear Translocation During the Cross-Talk Between Cellular Stress, Cell Cycle and Anticancer Agents

The function of many endogenous molecules in all eukaryotic cells depends on their subcellular localisation, being active when localized in one cellular compartment and inactive in another. Translocation or re-localization of mislocalized components in the optimal subcellular site may contribute to the development of novel cancer therapies and to the re-evaluation of conventional treatment. For instance, various agents are able to entrap cytoplasmic anti-apoptotic pathways to the nucleus, thus activating apoptosis. Moreover, amongst the factors identified so far, the optimal location of the tumor suppressor p53 for promoting cell arrest and apoptosis seems to be the nucleus, while the nuclear factor kappa B (NFkappaB) is desirable to stay in the cytoplasm. Thus, the mechanisms of nuclear translocation of endogenous signaling components, like p53, NFkappaB and various heat shock proteins (HSPs), may serve as targets for pharmacological intervention, without excluding the possible role of uptake and active transport into the nucleus of extracellular proteins.

Dissection of the NF-κB signalling cascade in transgenic and knockout mice

Studies in transgenic and knockout mice have made a major contribution to our current understanding of the physiological functions of the NF-kappaB signalling cascade. The generation and analysis of mice with targeted modifications of individual components of the NF-kappaB pathway tremendously advanced our knowledge of the roles of the NF-kappaB proteins themselves, and also of the many activators and negative regulators of NF-kappaB. These studies have highlighted the complexity of the NF-kappaB system, by revealing the multiple interactions, redundancies, but also diverse functions, performed by the different molecules participating in the regulation of NF-kappaB signalling. Furthermore, inhibition or enforced activation of NF-kappaB in transgenic mice has uncovered the critical roles that NF-kappaB plays in the pathogenesis of various diseases such as liver failure, diabetes and cancer.

Effects of 17β-estradiol on the expression of IL-6, IL-11 and NF-κB in human MG-63 osteoblast-like cell line

In order to characterize the effects of 17beta-estradiol (17beta-E2) on the expression of IL-6, IL-11 and NF-kappaB in the human MG-63 osteoblast-like cell line, the expression of IL-6 was detected by RT-PCR, Northern blot and Western blot. The expression of IL-11 was determined by RT-PCR, and NF-kappaB by Western blot. The results showed that 17beta-E2 down-regulated the expression of IL-6 mRNA and protein, IL-11 mRNA and NF-kappaB protein in MG-63 cells. It was suggested that the expression of NF-kappaB, IL-6 and IL-11 in MG-63 cells could be suppressed by 17beta-E2, and this might lend support to estrogen replacement therapy in postmenopausal women.

Role of transforming growth factor-beta1-smad signal transduction pathway in patients with hepatocellular carcinoma

To explore the role of transforming growth factor-beta1 (TGF-beta1)-smad signal transduction pathway in patients with hepatocellular carcinoma. Thirty-six hepatocellular carcinoma specimens were obtained from Qidong Liver Cancer Institute and Department of Pathology of the Second Affiliated Hospital of Nanjing Medical University. All primary antibodies (polyclonal antibodies) to TGF-beta1, type II Transforming growth factor-beta receptor (TbetaR-II), nuclear factor-kappaB (NF-kappaB), CD34, smad4 and smad7,secondary antibodies and immunohistochemical kit were purchased from Zhongshan Biotechnology Limited Company (Beijing, China). The expressions of TGF-beta1, TbetaR-II, NF-kappaB, smad4 and smad7 proteins in 36 specimens of hepatocellular carcinoma (HCC) and its adjacent tissue were separately detected by immunohistochemistry to observe the relationship between TGF-beta1 and TbetaR-II, between NF-kappaB and TGF-beta1, between smad4 and smad7 and between TGF-beta1 or TbetaR-IIand microvessel density (MVD). MVD was determined by labelling the vessel endothelial cells with CD34. The expression of TGF-beta1, smad7 and MVD was higher in HCC tissue than in adjacent HCC tissue (P<0.01, P<0.05, P<0.01 respectively). The expression of TbetaR-IIand smad4 was lower in HCC tissue than in its adjacent tissue (P<0.01, P<0.05 respectively). The expression of TGF-beta1 protein and NF-kappaB protein was consistent in HCC tissue. The expression of TGF-beta1 and MVD was also consistent in HCC tissue. The expression of TbetaR-IIwas negatively correlated with that of MVD in HCC tissue. The expressions of TGF-beta1, TbetaR-II, NF-kappaB, smad4 and smad7 in HCC tissue, which are major up and down stream factors of TGF-beta1-smad signal transduction pathway , are abnormal. These factors are closely related with MVD and may play an important role in HCC angiogenesis. The inhibitory action of TGF-beta1 is weakened in hepatic carcinoma cells because of abnormality of TGF-beta1 receptors (such as TbetaR-II) and postreceptors (such as smad4 and smad7). NF-kappaB may cause activation and production of TGF-beta1.