NF-kappaB Inhibitor Research Articles

Chromium dinicocysteinate supplementation can lower blood glucose, CRP, MCP‐1, ICAM‐1, creatinine, apparently mediated by elevated blood vitamin C and adiponectin and inhibition of NFκB, Akt, and Glut‐2 in livers of zucker diabetic fatty rats

Chromium and cysteine supplementation can improve glucose metabolism in animal studies. This study examined the hypothesis that a cysteinate complex of chromium is significantly beneficial than either of them in lowering blood glucose and vascular inflammation markers in Zucker diabetic fatty (ZDF) rats. Starting at the age of 6 wk, ZDF rats were supplemented orally (daily gavages for 8 more weeks) with saline-placebo (D) or chromium (400 microg Cr/Kg body weight) as chromium dinicocysteinate (CDNC), chromium dinicotinate (CDN) or chromium picolinate (CP) or equimolar L-cysteine (LC, img/Kg body weight), and fed Purina 5008 diet for 8 wk. ZDF rats of 6 wk age before any supplementations and onset of diabetes were considered as baseline. D rats showed elevated levels of fasting blood glucose, HbA(1), CRP, MCP-1, ICAM-1 and oxidative stress (lipid peroxidation) and lower adiponectin and vitamin C, when compared with baseline rats. In comparison to D group, CDNC group had significantly lower blood glucose, HbA(1), CRP, MCP-1, ICAM-1 and lipid peroxidation and increased vitamin C and adiponectin levels. CDN, CP or LC showed significantly less or no effect on these biomarkers. Only CDNC lowered blood creatinine levels in comparison to D. While CDN and CP had no effect, activation of NFkappaB, Akt and glucose transporter-2 levels were decreased, insulin receptor substrate 1 (IRS-1) activation increased in livers of CDNC-rats. CDNC effect on glycemia, NFkappaB, Akt and IRS-1 in liver was significantly greater compared with LC. Blood chromium levels did not differ between Cr-groups. Exogenous vitamin C supplementation significantly inhibited MCP-1 secretion in U937 monocytes cultured in high-glucose-medium. CDNC is a potent hypoglycemic compound with anti-inflammatory activity apparently mediated by elevated blood vitamin C and adiponectin and inhibition of NFkappaB, Akt, and Glut-2 and increased IRS-1 activation in livers of type 2 diabetic rats.

Angiotensin II-induced upregulation of AT(1) receptor expression: sequential activation of NF-kappaB and Elk-1 in neurons.

It has been clearly established that increased circulating angiotensin II (ANG II) with concurrent upregulation of brain and peripheral ANG II type 1 receptors (AT(1)R) are important mediators in the pathophysiology of several diseases characterized by sympatho-excitation. In an effort to further understand the regulation of AT(1)R expression in neurons, we determined the role of sequential activation of the transcription factors nuclear factor-kappaB (NF-kappaB) and Ets-like protein 1 (Elk-1) in AT(1)R upregulation. We used CATH.a neurons as our neuronal cell model. Cells were treated with ANG II (100 nM) over a preset time course. Following ANG II activation, there was a temporal increase in the p65 subunit of NF-kappaB that was observed at 30 min, peaked at 1 h, and was sustained up to 24 h. There was a concomitant decrease of IkappaB and increased IkappaK expression. We also observed an increase in AT(1)R expression which followed the temporal increase of NF-kappaB. The activation of NF-kappaB was blocked by using the inhibitors parthenolide or p65 small interfering RNA (siRNA) which both led to a decrease in AT(1)R expression. The expression of Elk-1 was upregulated over a time period following ANG II activation and was decreased following NF-kappaB inhibition. p65-DNA binding was assessed using electrophoretic mobility shift assay, and it was shown that there was a time-dependent increased binding that was inhibited by means of parthenolide pretreatment or siRNA-mediated p65 gene silencing. Therefore, our results suggest a combined role for the transcription factors NF-kappaB and Elk-1 in the upregulation of AT(1)R in the CATH.a cell neuronal model. These data imply a positive feedback mechanism that may impact neuronal discharge sensitivity in response to ANG II.

Effect of nuclear factor kappa B inhibition on L-NAME-induced hypertension and cardiovascular remodelling

We aimed to analyze effects of nuclear factor-kappa B (NF-kappaB) inhibition on blood pressure (BP) regulation and cardiovascular remodelling. Adult 12-week-old male Wistar Kyoto rats (WKY) were treated with the nitric oxide synthase (NOS) inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME, 40 mg/kg/day) for seven weeks. From the fourth week of L-NAME treatment, the NF-kappaB inhibitor lactacystin (1 microg/kg) was applied once a week. Furthermore, age-matched WKY received L-NAME or lactacystin alone for 7 or 3 weeks, respectively. Total NOS activity was determined in the left ventricle (LV) and aorta. The concentration of conjugated dienes, fibrosis, and collagen I and III levels were determined in the LV. The cross-sectional area (CSA) and wall thickness to internal diameter ratio (WT/ID) were measured in the aorta. L-NAME treatment increased BP significantly (145 +/- 2 mmHg vs. 110 +/- 1 mmHg in controls). The addition of lactacystin resulted in further significant increase in BP (161 +/- 3 mmHg). Similarly, lactacystin potentiated the increased conjugated dienes concentration induced by L-NAME. Whereas L-NAME alone did not affect NOS activity, the addition of lactacystin decreased it in both tissues investigated. The addition of lactacystin did not affect LV hypertrophy, fibrosis, and collagen I and III, already increased by L-NAME; however, it further amplified CSA in the aorta increased by L-NAME alone. WT/ID increased significantly only after the addition of lactacystin. Decreased NOS activity along with increased oxidative load may be responsible for decreased NO bio-availability and further BP increase after NF-kappaB inhibition in L-NAME-induced hypertension. Increased CSA and WT/ID could contribute to this hypertensive process.

The role of nuclear factor kappa B and nitric oxide interaction in heart remodelling

Hypertrophy of the myocardium is induced by haemodynamic and humoral alterations, which in turn stimulate the production of factors triggering protein synthesis, thus initiating myocardial growth. It has recently been documented that nuclear factor kappa B (NF-kappaB) represents a key factor that mediates both neurohormonal and proinflammatory signals leading finally to hypertrophic and/or fibrotic processes in the cardiovascular system. It was suggested that blockade of NF-kappaB may exert a protective effect against pathological myocardial growth. Recent data indicate that increased activity of NF-kappaB, besides its deleterious effects, leads to the increased production of nitric oxide, which as a result of its hypotensive and antigrowth activity exerts protection against the hypertrophic and proliferative processes. This ambivalent nature of NF-kappaB might limit the use of NF-kappaB inhibitors for therapeutic purposes and should evoke further studies to clarify unequivocally the nature of NF-kappaB inhibition in the haemodynamically overloaded circulation.

Involvement of integrin up‐regulation in RANKL/RANK pathway of chondrosarcomas migration

Invasion of tumor cells is the primary cause of therapeutic failure in malignant chondrosarcomas treatment. Receptor activator of nuclear factor-kappaB ligand (RANKL) and its receptor, RANK, play a key roles in osteoclastogenesis and tumor metastasis. We found that the RANKL and RANK expression in human chondrosarcoma tissues was higher than that in normal cartilage. We also found that RANKL directed the migration and increased cell surface expression of beta1 integrin in human chondrosarcoma cells (JJ012 cells). Pretreatment of JJ012 cells with MAPK kinase (MEK) inhibitors, PD98059 or U0126, inhibited the RANKL-induced migration and integrin expression. Stimulation of cells with RANKL increased the phosphorylation of MEK and extracellular signal-regulating kinase (ERK). In addition, NF-kappaB inhibitor (PDTC) or IkappaB protease inhibitor (TPCK) also inhibited RANKL-induced cells migration and integrin up-regulation. Taken together, these results suggest that the RANKL acts through MEK/ERK, which in turn activates IKKalpha/beta and NF-kappaB, resulting in the activation of beta1 integrin and contributing to the migration of human chondrosarcoma cells.

Oncostatin M synergistically induces CXCL10 and ICAM‐1 expression in IL‐1β‐stimulated‐human gingival fibroblasts

Periodontitis is a chronic bacterial infection of tooth-supporting structures. T-helper type 1 (Th1) cells are related to the exacerbation of periodontal disease. Human gingival fibroblasts (HGFs), the major cell type in periodontal connective tissues, are involved in immunological response in periodontal tissues. However, it is uncertain whether HGFs are related to Th1 response. Chemokine (C-X-C motif) ligand 10 (CXCL10) is a cytokine, that is related to Th1 cells migration. Intercellular adhesion molecule (ICAM)-1 is involved in Th1 cells retention and activation in inflamed tissue. The aim of this study is to examine the effect of oncostatin M (OSM) on CXCL10 and ICAM-1 expression in HGFs. OSM stimulation induced CXCL10 and ICAM-1 expression in HGFs. Moreover, the synergistic effects of CXCL10 release and ICAM-1 expression in HGFs were observed with combined stimulation of interleukin (IL)-1beta and OSM. OSM increased type 1 IL-1 receptor (IL-1R1) expression, and IL-1beta enhanced OSMRbeta expression on HGFs. IL-1beta + OSM stimulation enhanced the phosphorylation of inhibitor of nuclear factor kappaB (IkappaB)-alpha, signal transducer and activator of transcription (STAT)3, c-Jun N terminal kinase (JNK), and protein kinase B (Akt) compared to OSM or IL-1beta stimulation. CXCL10 production from OSM + IL-1beta stimulated HGFs was suppressed by nuclear factor (NF)-kappaB, STAT3, JNK, and phosphoinositide-3-kinase (PI3K) inhibitors. On the other hand, only NF-kappaB and STAT3 inhibitors suppressed ICAM-1 expression enhanced by OSM + IL-1beta treatment. These effects of OSM and IL-1beta may promote Th1 cells infiltration and retention in periodontally diseased tissues and be related to exacerbation of periodontal disease.

Inverse expression of estrogen receptor‐β and nuclear factor‐κB in urinary bladder carcinogenesis

To investigate the expression of nuclear factor-kappaB (NF-kappaB) and estrogen receptor-beta (ER-beta) signalling pathways in bladder urothelial carcinoma according to clinicopathological features, in order to elucidate their role during carcinogenesis. Immunohistochemical methodology was carried out on formalin-fixed, paraffin-embedded sections from urinary bladder carcinomas of 140 patients (94 males and 46 females) who underwent transurethral resection of bladder neoplasms. Correlations between ER-beta and NF-kappaB, and tumor grade and T-stage were evaluated, along with demographic data, sex and age. A significant decrease in ER-beta expression in the nucleus of bladder cells during loss of cell differentiation (r(s) = -0.61, P-value < 0.001, test of trend P-value = 0.003) and in muscle invasive carcinomas (T2-T4; test of trend P-value < 0.001) was found. p65 Subunit of NF-kappaB was expressed in the nucleus and in the cytoplasm of bladder epithelial cells. A strong positive association between tumor grade and nuclear expression of NF-kappaB was shown. No correlation between NF-kappaB, nuclear or cytoplasmic staining, with T-stage was observed. An inverse correlation between ER-beta and nuclear p65 immunoreactivity was observed (r(s) = -0.45, P-value < 0.001). There was no correlation with demographic data. Our immunohistochemical study suggests the possible inverse regulation of NF-kappaB and ER-beta transcription factor during bladder carcinogenesis. Selective ER-beta agonists and agents, inhibitors of NF-kappaB, might represent a possible new treatment strategy for bladder urothelial tumors.

Nitric oxide regulates stretch-induced proliferation in C2C12 myoblasts

Mechanical stretch of skeletal muscle activates nitric oxide (NO) production and is an important stimulator of satellite cell proliferation. Further, cyclooxygenase (COX) activity has been shown to promote satellite cell proliferation in response to stretch. Since COX-2 expression in skeletal muscle can be regulated by NO we sought to determine if NO is required for stretch-induced myoblast proliferation and whether supplemental NO can counter the effects of COX-2 and NF-kappaB inhibitors. C2C12 myoblasts were cultured for 24 h, then switched to medium containing either the NOS inhibitor, L-NAME (200 microM), the COX-2 specific inhibitor NS-398 (100 microM), the NF-kappaB inhibiting antioxidant, PDTC (5 mM), the nitric oxide donor, DETA-NONOate (10-100 microM) or no supplement (control) for 24 h. Subgroups of each treatment were exposed to 1 h of 15% cyclic stretch (1 Hz), and were then allowed to proliferate for 24 h before fixing. Proliferation was measured by BrdU incorporation during the last hour before fixing, and DAPI stain. Stretch induced a twofold increase in nuclear number compared to control, and this effect was completely inhibited by L-NAME, NS-398 or PDTC (P < 0.05). Although DETA-NONOate (10 microM) did not affect basal proliferation, the NO-donor augmented the stretch-induced increase in proliferation and rescued stretch-induced proliferation in NS-398-treated cells, but not in PDTC-treated cells. In conclusion, NO, COX-2, and NF-kappaB are necessary for stretch-induced proliferation of myoblasts. Although COX-2 and NF-kappaB are both involved in basal proliferation, NO does not affect basal growth. Thus, NO requires the synergistic effect of stretch in order to induce muscle cell proliferation.

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.

Inhibition of NF-κB Signaling Reduces Virus Load and Gammaherpesvirus-Induced Pulmonary Fibrosis

Idiopathic pulmonary fibrosis (IPF) is a chronic progressive lung disorder of unknown etiology. Several studies have demonstrated an association between pulmonary infection with a herpesvirus and IPF. Based on those observations, we have developed a mouse model in which interferon (IFN)gammaR(-/-) mice infected intranasally with murine gammaherpesvirus 68 (MHV68) develop lung fibrosis. We hypothesize that viral load was a critical factor for the development of fibrosis. Because nuclear factor (NF)-kappaB signaling is required to efficiently establish gammaherpesvirus, latency we infected IFNgammaR(-/-) mice with a MHV68 virus that expresses a mutant dominant inhibitor of the NF-kappaB signaling pathway, called IkappaBalphaM. Striking differences were observed at the onset of the chronic infection, which correlated with a decreased virus load in mice infected with MHV68-IkappaBalphaM compared with mice infected with control MHV68 (MHV68-MR). IFNgammaR(-/-) mice infected with MHV68-IkappaBalphaM lacked vasculitis and fibrosis 15 to 120 days post infection. Inhibition of NF-kappaB in MHV68-infected cells of the lungs diminished the expression of the fibrocyte recruiting chemokines monocyte chemoattractant protein 1 (MCP-1) and CXCL12, ameliorated macrophage expression of markers of alternative activation, and failed to increase expression of the integrin alphavbeta6, which is implicated in the activation of the profibrotic factor TGF-beta. Thus, the inhibition of NF-kappaB signaling in the infected lung cells of IFNgammaR(-/-) mice reduces virus persistence and ameliorates profibrotic events. Host determinants of latency might therefore represent new therapeutic targets for gammaherpesvirus-associated pulmonary fibrosis.

Inhibition of NFκB and Pancreatic Cancer Cell and Tumor Growth by Curcumin Is Dependent on Specificity Protein Down-regulation

Curcumin activates diverse anticancer activities that lead to inhibition of cancer cell and tumor growth, induction of apoptosis, and antiangiogenic responses. In this study, we observed that curcumin inhibits Panc28 and L3.6pL pancreatic cancer cell and tumor growth in nude mice bearing L3.6pL cells as xenografts. In addition, curcumin decreased expression of p50 and p65 proteins and NFkappaB-dependent transactivation and also decreased Sp1, Sp3, and Sp4 transcription factors that are overexpressed in pancreatic cancer cells. Because both Sp transcription factors and NFkappaB regulate several common genes such as cyclin D1, survivin, and vascular endothelial growth factor that contribute to the cancer phenotype, we also investigated interactions between Sp and NFkappaB transcription factors. Results of Sp1, Sp3, and Sp4 knockdown by RNA interference demonstrate that both p50 and p65 are Sp-regulated genes and that inhibition of constitutive or tumor necrosis factor-induced NFkappaB by curcumin is dependent on down-regulation of Sp1, Sp3, and Sp4 proteins by this compound. Curcumin also decreased mitochondrial membrane potential and induced reactive oxygen species in pancreatic cancer cells, and this pathway is required for down-regulation of Sp proteins in these cells, demonstrating that the mitochondriotoxic effects of curcumin are important for its anticancer activities.

A20 suppresses inflammatory responses and bone destruction in human fibroblast‐like synoviocytes and in mice with collagen‐induced arthritis

Nuclear factor-kappaB (NF-kappaB) has been implicated as a therapeutic target for the treatment of rheumatoid arthritis (RA). The purpose of this study was to determine whether A20, a universal inhibitor of NF-kappaB, might have antiarthritic effects. An adenovirus containing A20 complementary DNA (AdA20) was used to deliver A20 to human rheumatoid fibroblast-like synoviocytes (FLS) in vitro as well as to mice with collagen-induced arthritis (CIA) in vivo via intraarticular injection into the ankle joints bilaterally. In vitro experiments demonstrated that AdA20 suppressed NF-kappaB activation, chemokine production, and matrix metalloproteinase secretion induced by tumor necrosis factor alpha in FLS. Mice with CIA that were treated with AdA20 had a lower cumulative disease incidence and severity of arthritis, based on hind paw thickness, radiologic and histopathologic findings, and inflammatory cytokine levels, than did control virus-injected mice. The protective effects of AdA20 were mediated by the inhibition of the NF-kappaB signaling pathway. The severity of arthritis was also significantly decreased in the untreated front paws, indicating a beneficial systemic effect of local suppression of NF-kappaB. Surprisingly, mice treated with AdA20 after the onset of CIA had significantly decreased arthritis severity from the onset of clinical signs to the end of the study. These results suggest that using A20 to block the NF-kappaB pathway in rheumatoid joints reduces both the inflammatory response and the tissue destruction. The development of an immunoregulatory strategy based on A20 may therefore have therapeutic potential in the treatment of RA.

Role of nuclear factor-κB in volatile anaesthetic preconditioning with sevoflurane during myocardial ischaemia/reperfusion

Anaesthetic preconditioning (APC) protects against myocardial ischaemia/reperfusion injury. Nuclear factor-kappaB (NF-kappaB) has been implicated in APC-induced myocardial protection in vitro. Our study tested the hypothesis that in-vivo APC with sevoflurane is triggered by NF-kappaB through downregulation of inflammatory mediators and upregulation of antiapoptosis factors to prevent myocardial injury during ischaemia/reperfusion. In this in-vivo study, rats were anaesthetized and maintained with sodium pentobarbital throughout the experiment. Rats were exposed to 30 min of 2.5% sevoflurane followed by 15 min washout (APC group) or no inhalation anaesthetics (ischaemia/reperfusion group) before ischaemia/reperfusion. In the sevoflurane group, rats were exposed to 30 min sevoflurane followed by a 165 min washout period. The NF-kappaB inhibitor parthenolide (PTN) was used before or after exposure to sevoflurane (PTN+APC group and APC+PTN group). Left ventricular samples were obtained to measure infarct size, pro-inflammation and apoptosis. A P value less than 0.05 was considered significant. APC reduced infarct size (34 +/- 6%) compared with ischaemia/reperfusion (53 +/- 6%, P<0.05). PTN administered before or after APC abolished the cardioprotection (53 +/- 5 and 52 +/- 7%, respectively, P < 0.05). APC decreased the myocardium apoptosis compared with the ischaemia/reperfusion only group (6 +/- 1 vs.19 +/- 3%, P < 0.05); PTN administered before or after sevoflurane preconditioning abolished this effect. APC induced upregulation of NF-kappaB p50/p65 before ischaemia (51 +/- 4/26 +/- 3% vs. 15 +/- 1/11 +/- 1% in the control group, P < 0.05). After reperfusion, NF-kappaB was upregulated in the ischaemia/reperfusion and APC groups, but it was lower in the APC group than in the ischaemia/reperfusion group. PTN administered before and after APC inhibited the expressions. Before ischaemia, Bcl-2 was increased in the APC and sevoflurane groups (94 +/- 3 and 102 +/- 4%, respectively) compared with the control group (68 +/- 2%, P < 0.05). After reperfusion, intercellular adhesion molecule-1, tumour necrosis factor-alpha and caspase-3 expressions were significantly increased in the ischaemia/reperfusion group (92 +/- 5, 115 +/- 4 and 65 +/- 2% compared with the control group, P < 0.05); these increases were blunted in the APC group. APC with sevoflurane produced myocardial protection against ischaemia/reperfusion in vivo. NF-kappaB acted not only as a trigger but also as a mediator that played an important role in APC through upregulation of NF-kappaB and the antiapoptosis protein Bcl-2 during the preconditioning period and then through downregulation of the inflammatory proteins intercellular adhesion molecule-1 and tumour necrosis factor-alpha during reperfusion, ultimately decreasing caspase-3 expression and apoptosis.

Astragalus saponins modulate mTOR and ERK signaling to promote apoptosis through the extrinsic pathway in HT-29 colon cancer cells

We have previously demonstrated that the total saponins of Astragalus membranaceus (AST) possess potential anti-tumorigenic effects in human colon cancer cells and tumor xenografts. In the present study, the proapoptotic effects of AST were investigated in native and cytokine-induced HT-29 cells to further unveil its mechanism of action. Growth-inhibitory action of AST (60 microg/ml) was demonstrated in native HT-29 cells, which was exaggerated in tumor necrosis factor (TNF) (5 ng/ml)-induced cells. These were accompanied by caspase 3 activation, cleavage of poly(ADP-ribose) polymerase and a subsequent increase in apoptotic cell numbers. Furthermore, activation of procaspase 8 indicates that the extrinsic apoptotic pathway was involved, while cleavage of Bid into t-Bid implicates cross-talk with the intrinsic apoptotic pathway. Alternatively, AST caused S and G2/M phase arrest, while in cytokine-induced cells S phase arrest was predominant. Further adding to our recent suggestion on its correlation with phosphatidylinositol 3-kinase (PI3K)-Akt signaling, we have now revealed that AST caused overexpression of PTEN and down-regulation of mammalian target of rapamycin (mTOR) expression. Nevertheless, these events were preceded by a decrease in nuclear factor-kappaB (NF-kappaB)/DNA binding activity with continuous ERK 1/2 activation. Some of these effects became more intense in cytokine-induced cells. Our findings in this study suggest that AST induces the extrinsic apoptotic cascade and causes cell cycle arrest in HT-29 cells by modulation of both mTOR and ERK signaling pathways, of which inhibition of NF-kappaB is important in the latter mechanism. Most of the above processes are more pronounced in cytokine-induced cells.

Activation of toll‐like receptors 2, 3 or 5 induces matrix metalloproteinase‐1 and ‐9 expression with the involvement of MAPKs and NF‐κB in human epidermal keratinocytes

Toll-like receptors (TLRs) on epidermal keratinocytes are the first line of defense against microbe invasion, and matrix metalloproteases (MMPs) regulate inflammation, cell migration and wound healing. In this study, we demonstrate that the mRNA and protein expressions of MMP-1 and MMP-9 in human epidermal keratinocytes are induced by ligands for TLR2, TLR3 and TLR5 [Pam3CSK4, Poly(I:C) and flagellin, respectively] in a dose-dependent manner. We also found that the ligands for TLR2, TLR3 and TLR5 activate the MAP kinases, JNK and p38 MAPK, but not ERK1/2. Furthermore, treatment with the ligands for TLR2, TLR3 and TLR5 also induced the degradation of IkappaB-alpha and activated the nuclear translocation of NF-kappaB. MMP-1 induction by the ligands for TLR2, TLR3 and TLR5 was inhibited by pretreatment with BAY11-7082 (NF-kappaB inhibitor) or SP600125 (JNK inhibitor), whereas MMP-9 expression was inhibited by pretreatment with BAY11-7082, SP600125 or SB203580. These findings demonstrate that the activation of TLR2, TLR3 or TLR5 induces the expression of MMP-1 and MMP-9 in human epidermal keratinocytes. In addition, NF-kappaB or JNK mediated the MMP-1 expression induced by TLR2, TLR3 and TLR5, whereas NF-kappaB, JNK or p38 MAPK mediated the MMP-9 expression induced by TLR2, TLR3 and TLR5.

Differential Inhibitory Effects of the Polyphenol Ellagic Acid on Inflammatory Mediators NF‐κB, iNOS, COX‐2, TNF‐α, and IL‐6 in 1,2‐Dimethylhydrazine‐Induced Rat Colon Carcinogenesis

Dietary polyphenols have been found to possess preventive and therapeutic potential against several types of cancers. We investigated the effect of ellagic acid on colon cancer induced by 1,2-dimethylhydrazine in rats. Male Wistar albino rats were divided into four groups. Group 1 served as control, group 2 rats received ellagic acid 60 mg/kg bodyweight/every day p.o. throughout the experiment. Rats from groups 3 and 4 were given subcutaneous (s.c.) injections of 1,2-dimethylhydrazine (20 mg/kg body weight) once a week for the first 15 weeks; rats in group 4 received ellagic acid as in group 2 after the last injection of 1,2-dimethylhydrazine and continued till the end of the experimental period of 30 weeks. 1,2-dimethylhydrazine-induced rats exhibited alterations in cancer tumour markers [5'-nucleotidase (5'-ND), gamma glutamyl transpeptidase (gamma-GT), carcinoembryonic antigen (CEA), alphafetoprotein (AFP) and cathepsin-D (CD)]; pathophysiological markers [alkaline phosphatase (ALP) and lactate dehydrogenase (LDH)] and oral administration of ellagic acid restored the levels of these marker enzymes. Nuclear factor-kappa B (NF-kappaB) actively involved in the regulation of both pro-inflammatory proteins [inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2)] and pro-inflammatory cytokines [tumour necrosis factor (TNF)-alpha and interleukin (IL)-6] and in our study 1,2-dimethylhydrazine-induced group exhibited elevated expressions of all these inflammatory proteins. Ellagic acid administration reduced the expressions of NF-kappaB, COX-2, iNOS, TNF-alpha and IL-6 as confirmed by immunohistochemical, immunoblot and immunofluorescence analysis during 1,2-dimethylhydrazine-induced colon carcinogenesis. In conclusion, ellagic acid demonstrates anti-inflammatory property by iNOS, COX-2, TNF-alpha and IL-6 down-regulation due to inhibition of NF-kappaB and exerts its chemopreventive effect on colon carcinogenesis.

Methoxychalcone induces cell‐cycle arrest and apoptosis in human hormone‐resistant prostate cancer cells through PI 3‐kinase‐independent inhibition of mTOR pathways

Chalcones are contained in fruits and vegetables, and have been suggested to display anticancer activities. In this study, the anticancer mechanism of WJ9708011 (a methoxychalcone derivative) was delineated in human prostate cancer cells. Cell proliferation was examined by sulforhodamine B and clonogenic assays. Cell-cycle progression and mitochondrial membrane potential (DeltaPsi(m)) were detected by flow cytometric analysis. Expressions of protein and mRNA were detected by Western blot and RT-PCR technique, respectively. The protein synthesis was examined by [(3)H]leucine incorporation assay. The overexpression or knockdown techniques for specific target protein were also used in this study. WJ9708011 induced time- and concentration-dependent G1 arrest of the cell cycle and subsequent apoptosis in human prostate cancer cells. The G1-arrest effect was confirmed by down-regulated expressions of several G1-phase regulators, including cyclin D1, cyclin E, cyclin-dependent kinase (Cdk)-4, Cdk2, phospho-RB, E2F-1, and Cdc25A. The mRNA expressions of cyclin D1 and cyclin E were also inhibited through the suppression of NF-kappaB. WJ9708011 blocked the protein synthesis and inhibited mammalian target of rapamycin (mTOR) signaling pathways. The suppression of mTOR pathways were irrespective of Akt- and AMPK-activated protein kinase (AMPK), but were attributed to mitochondrial stress, in which the down-regulation of survivin protein level may play a crucial role. The data suggest that WJ9708011 induces transcriptional and translational suppression of cell-cycle regulators that might be through Akt- and AMPK-independent loss of DeltaPsi(m) and inhibition of mTOR signaling pathway, leading to G1 arrest of the cell cycle and subsequent apoptotic cell death.

Novel Derivative of Benzofuran Induces Cell Death Mostly by G2/M Cell Cycle Arrest through p53-dependent Pathway but Partially by Inhibition of NF-κB

The Dracaena resin is widely used in traditional medicine as an anticancer agent, and benzofuran lignan is the active component. In this report, we provide evidence that the synthetic derivative of benzofuran lignan (Benfur) showed antitumor activities. It induced apoptosis in p53-positive cells. Though it inhibited endotoxin-induced nuclear factor kappaB (NF-kappaB) activation in both p53-positive and -negative cells, the activation of caspase 3 was observed in p53-positive cells. It showed partial cell death effect in both p53-positive and -negative cells through inhibition of NF-kappaB. Cell cycle analysis using flow cytometry showed that treatment with this novel benozofuran lignan derivative to Jurkat T-cells, but not U-937 cells, resulted in a G2/M arrest in a dose- and time-dependent manner. It increased amounts of p21, p27, and cyclin B, but not phospho-Rb through p53 nuclear translocation in Jurkat T-cells, but not in U-937 cells. It inhibited amounts of MDM2 (murine double minute 2) by repressing the transcription factor Sp1, which was also proved in silico. It induced cell death in tumor cells, but not in primary T-cells. Overall, our data suggest that Benfur-mediated cell death is partially dependent upon NF-kappaB, but predominantly dependent on p53. Thus, this novel benzofuran lignan derivative can be effective chemopreventive or chemotherapeutic agent against malignant T-cells.

Oncolytic targeting of renal cell carcinoma via encephalomyocarditis virus

Apoptosis is a fundamental host defence mechanism against invading microbes. Inactivation of NF-κB attenuates encephalomyocarditis virus (EMCV) virulence by triggering rapid apoptosis of infected cells, thereby pre-emptively limiting viral replication. Recent evidence has shown that hypoxia-inducible factor (HIF) increases NF-κB-mediated anti-apoptotic response in clear-cell renal cell carcinoma (CCRCC) that commonly exhibit hyperactivation of HIF due to the loss of its principal negative regulator, von Hippel–Lindau (VHL) tumour suppressor protein. Here, we show that EMCV challenge induces a strong NF-κB-dependent gene expression profile concomitant with a lack of interferon-mediated anti-viral response in VHL-null CCRCC, and that multiple established CCRCC cell lines, as well as early-passage primary CCRCC cultured cells, are acutely susceptible to EMCV replication and virulence. Functional restoration of VHL or molecular suppression of HIF or NF-κB dramatically reverses CCRCC cellular susceptibility to EMCV-induced killing. Notably, intratumoural EMCV treatment of CCRCC in a murine xenograft model rapidly regresses tumour growth. These findings provide compelling pre-clinical evidence for the usage of EMCV in the treatment of CCRCC and potentially other tumours with elevated HIF/NF-κB-survival signature.

15-DEOXY-Δ12,14 PROSTAGLANDIN GJ2 BUT NOT ROSIGLITAZONE REGULATES METALLOPROTEINASE 9, NOS-2, AND CYCLOOXYGENASE 2 EXPRESSION AND FUNCTIONS BY PEROXISOME PROLIFERATORYACTIVATED RECEPTOR γ-DEPENDENT AND -INDEPENDENT MECHANISMS IN CARDIAC CELLS

Sepsis or endotoxemia produced by LPS followed by hypotension and multiorganic failure may lead to cardiac dysfunction contributing to mortality. Cardiac failure is usually associated to activation of nuclear factor kappaB (NF-kappaB) and mitogen-activated protein kinase (MAPK), which play an important role in proinflammatory enzymes expression. It has been shown that 15-deoxy-Delta12,14 prostaglandin J2 (15dPGJ2) can repress the inflammatory response by means of peroxisome proliferator-activated receptor gamma (PPARgamma)-dependent and -independent mechanisms. However, its precise role in heart is poorly understood. In the present study, mouse neonatal cardiomyocytes were isolated and stimulated with LPS to investigate the role of PPARgamma-specific ligands 15dPGJ2 and rosiglitazone on cardiac inflammatory response. Inducible NO synthase, cyclooxygenase 2, and metalloproteinase 9 mRNA levels, protein expression, and activity were inhibited with 15dPGJ2 but not by rosiglitazone. Peroxisome proliferator-activated receptor gamma antagonist, GW9662, prevented all these 15dPGJ2 actions. To go inside the mechanisms by which 15dPGJ2 exerts inhibitory effects, cells were preincubated with specific chemical inhibitors of NF-kappaB and p38 MAPK, and we found that these signaling cascades are implicated in 15dPGJ2 action as well as PPARgamma. These results suggest that only the natural PPARgamma ligand, 15dPGJ2, but not the synthetic one, rosiglitazone, regulates the inflammatory response by inhibition of inducible NO synthase, cyclooxygenase 2, and metalloproteinase 9 expression. Moreover, our results offer an additional 15dPGJ2 mechanism of action, despite PPARgamma, showing NF-kappaB and p38 MAPK participation.