NF-kappaB Transcriptional Activity Research Articles

Glucocorticoid Receptor α and β Variant Expression Is Associated with ASF/SF2 Splicing Factor Upregulation in HT-29 Colon Cancer and MCF-7 Breast Carcinoma Cells

Transcriptional activity of NF-kappaB is inhibited by the liganded glucocorticoid receptor (GR), which exists mainly in two splice variants as functional GRalpha and nonfunctional GRbeta. We investigated the effect of 5-aza-2'-deoxycytidine (5-dAzaC), trichostatin A (TSA), and sodium butyrate (NaBu) on GRalpha,GRbeta and ASF/SF2 splicing factor expression in HT-29 colon and MCF-7 breast carcinoma cells. HT-29 and MCF-7 cells were cultured in the absence or in the presence of 5-dAzaC, TSA, and NaBu, followed by RNA and protein isolation. The transcript and protein levels of GRalpha, GRbeta ASF/SF2 were determined by reverse transcription, real-time quantitative PCR and Western blot analysis. We found that 5-dAzaC, TSA, and NaBu lead to an increase in GRalpha and ASF/SF2 transcript levels and a decrease in GRbeta transcript levels in HT-29 and MCF-7 cells. The 5-dAzaC, TSA, and NaBu resulted in increased GRalpha and ASF/SF2 protein levels and GRbeta protein downregulation in HT-29 cells. The most increased GRalpha protein expression in MCF-7 cells was observed with NaBu. However, all of these compounds inhibited GRbeta protein expression in MCF-7 cells. The MCF-7 cells treated with NaBu demonstrated a remarkable increase in ASF/SF2 protein expression. Because NF-kappaB is considered to be a factor in the augmentation of malignant properties of cells, treatment of tumors with 5-dAzaC, TSA, and NaBu may provide a novel approach to the enhancement of therapeutic effects of glucocorticoids in epithelial carcinomas.

Termination of NF-κB activity through a gammaherpesvirus protein that assembles an EC5S ubiquitin-ligase

Host colonisation by lymphotropic gammaherpesviruses depends critically on the expansion of viral genomes in germinal centre (GC) B cells. Yet, host and virus molecular mechanisms involved in driving such proliferation remain largely unknown. Here, we show that the ORF73 protein encoded by the murid herpesvirus-4 (MuHV-4) inhibits host nuclear factor-kappa B (NF-κB) transcriptional activity through poly-ubiquitination and subsequent proteasomal-dependent nuclear degradation of the NF-κB family member p65/RelA. The mechanism involves the assembly of an ElonginC/Cullin5/SOCS (suppressors of cytokine signalling)-like complex, mediated by an unconventional viral SOCS-box motif present in ORF73. Functional deletion of this SOCS-box motif ablated NF-κB inhibitory effect of ORF73, suppressed MuHV-4 expansion in GC B cells and prevented MuHV-4 persistent infection in mice. These findings demonstrate that viral inhibition of NF-κB activity in latently infected GC centroblasts is critical for the establishment of a gammaherpesvirus persistent infection, underscoring the physiological importance of proteasomal degradation of RelA/NF-κB as a regulatory mechanism of this signalling pathway.

Curcuminoids Suppress the Growth of Pharynx and Nasopharyngeal Carcinoma Cells through Induced Apoptosis

Nasopharyngeal carcinoma (NPC) is one of the common malignant cancers in China, and radiotherapy or chemotherapy is the main therapy method for NPC. Curcuminoids (or curcumin), natural antioxidants, have been recently shown to produce a potent chemopreventive action against several types of cancer. They have also displayed antioxidant and anti-inflammatory activities. In the present study, the antiproliferation and induced apoptosis effects of curcuminoids have been investigated in Detroit 562 cells (human pharynx carcinoma) and HONE-1 (human nasopharyngeal carcinoma) cells. Results indicated that curcuminoids have produced an inhibition of cell proliferation as well as the activation of apoptosis in these cancer cells. Both effects were observed to increase in proportion with the dose of curcuminoids. The DNA fragmentation, caspase-3 activation,and NF-kappaB transcriptional factor activity have been studied. By these approaches, apoptosis was induced by curcuminoids in the pharynx and nasopharyngeal cancer cells via caspase-3-dependent pathway. However, a different dependency has been observed, whereas proliferation inhibition and apoptosis depend upon the amount of curcuminoid treatment in the cancer cells.

Angiotensin II Activates IκB Kinase Phosphorylation of RelA at Ser536 to Promote Myofibroblast Survival and Liver Fibrosis

The transcription factor nuclear factor-kappaB (NF)-kappaB promotes survival of hepatic myofibroblasts and fibrogenesis through poorly defined mechanisms. We investigated the activities of angiotensin II and I kappaB kinase (IKK) in regulation of NF-kappaB activity and the role of these proteins in liver fibrosis in rodents and humans. Phosphorylation of the NF-kappaB subunit RelA at serine 536 (P-Ser(536)-RelA) was detected by immunoblot and immunohistochemical analyses. P-Ser(536)-RelA function was assessed using vectors that expressed mutant forms of RelA, cell-permeable blocking peptides, and assays for RelA nuclear transport and apoptosis. Levels of P-Ser(536)-RelA were compared with degree of fibrosis in liver sections from chronically injured rats and patients with hepatitis C virus-mediated fibrosis who had been treated with the AT1 antagonist losartan. Constitutive P-Ser(536)-RelA is a feature of human hepatic myofibroblasts, both in vitro and in situ in diseased livers. Autocrine angiotensin II stimulated IKK-mediated phosphorylation of RelA at Ser(536), which was required for nuclear transport and transcriptional activity of NF-kappaB. Inhibition of angiotensin II, the angiotensin II receptor type 1 (AT1), or IKK blocked Ser(536) phosphorylation and stimulated myofibroblast apoptosis. Treatment of fibrotic rodent liver with the angiotensin converting enzyme (ACE) inhibitor captopril or the IKK inhibitor sulphasalazine resulted in loss of P-Ser(536)-RelA-positive myofibroblasts and fibrosis regression. In human liver samples, increased numbers of P-Ser(536)-RelA-positive cells were associated with fibrosis that regressed following exposure to losartan. An autocrine pathway that includes angiotensin II, IKK, and P-Ser(536)-RelA regulates myofibroblast survival and can be targeted to stimulate therapeutic regression of liver fibrosis.

SiRNA knocks down Hsp27 and increases angiotensin II-Induced phosphorylated NF-κB p65 levels in aortic smooth muscle cells

Angiotensin II (Ang II) induces the proinflammatory nuclear factor kappaB (NF-kappaB) in the vasculature. Heat shock and elevated levels of heat shock proteins (Hsps) decrease Ang II-induced NF-kappaB transcriptional activity and inflammation, but little is known about the role of specific Hsps. Here we used small interfering RNA (siRNA) technology to examine the role of Hsp27 in the Ang II-induced NF-kappaB signaling pathway. Hsp27 siRNA was transfected into rat aortic vascular smooth muscle (A10) and 48 hrs later, the cells were stressed with 100 nM of Ang II for up to 24 hrs. Hsp27 levels were determined by immunofluorescence microscopy and Western analysis and inhibitor kappaB-alpha (I kappaB-alpha), the p65 subunit of NF-kappaB, and I kappaB kinase (IKK) levels were determined by Western analysis. When Hsp27 was specifically knocked down with Hsp27 siRNA in A10 cells there was a trend toward an increase in Ang II-induced phosphorylated p65. I kappaB-alpha and IKK-beta levels were not changed by the knockdown of Hsp27. Hsp27 may regulate the phosphorylation of the p65 subunit of NF-kappaB in the Ang II-induced signaling pathway of NF-kappaB in A10 cells. The proinflammatory effects of Ang II on NF-kappaB in vascular smooth muscle cells may be through a non-canonical pathway and be dependent on p65 phosphorylation.

Tomato Lycopene Extract Prevents Lipopolysaccharide-Induced NF-κB Signaling but Worsens Dextran Sulfate Sodium-Induced Colitis in NF-κBEGFP Mice

BackgroundThe impact of tomato lycopene extract (TLE) on intestinal inflammation is currently unknown. We investigated the effect of TLE on lipopolysaccharide (LPS)-induced innate signaling and experimental colitis.Methodology/Principal FindingsMice were fed a diet containing 0.5 and 2% TLE or isoflavone free control (AIN-76). The therapeutic efficacy of TLE diet was assessed using dextran sulfate sodium (DSS) exposed mice and IL-10−/−;NF-κBEGFP mice, representing an acute and spontaneous chronic colitis model respectively. A mini-endoscope was used to determine the extent of macroscopic mucosal lesions. Murine splenocytes and intestinal epithelial cells were used to determine the in vitro impact of TLE on LPS-induced NF-κB signaling. In vitro, TLE blocked LPS-induced IκBα degradation, RelA translocation, NF-κB transcriptional activity and MIP-2 mRNA accumulation in IEC-18 cells. Moreover, LPS-induced IL-12p40 gene expression was dose-dependently inhibited in TLE-treated splenocytes. Interestingly, DSS-induced acute colitis worsened in TLE-fed NF-κBEGFP mice compared to control diet as measured by weight loss, colonoscopic analysis and histological scores. In contrast, TLE-fed IL-10−/−;NF-κBEGFP mice displayed decreased colonic EGFP expression compared to control diet. IL-6, TNFα, and MCP-1 mRNA expression were increased in the colon of TLE-fed, DSS-exposed NF-κBEGFP mice compared to the control diet. Additionally, caspase-3 activation and TUNEL positive cells were enhanced in TLE diet-fed, DSS-exposed mice as compared to DSS control mice.Conclusions/ SignificanceThese results indicate that TLE prevents LPS-induced proinflammatory gene expression by blocking of NF-κB signaling, but aggravates DSS-induced colitis by enhancing epithelial cell apoptosis.

Radiation‐induced HIF‐1α cell survival pathway is inhibited by soy isoflavones in prostate cancer cells

We previously showed that treatment of prostate cancer cells with soy isoflavones and radiation resulted in greater cell killing in vitro, and caused downregulation of NF-kappaB and APE1/Ref-1. APE1/Ref-1 functions as a redox activator of transcription factors, including NF-kappaB and HIF-1alpha. These molecules are upregulated by radiation and implicated in radioresistance of cancer cells. We extended our studies to investigate the role of HIF-1alpha survival pathway and its upstream Src and STAT3 molecules in isoflavones and radiation interaction. Radiation induced phosphorylation of Src and STAT3 leading to induction of HIF-1alpha. Genistein, daidzein or a mixture of soy isoflavones did not activate this pathway. These data were observed both in PC-3 (AR-) and C4-2B (AR+) androgen-independent cell lines. Pretreatment with isoflavones inhibited Src/STAT3/HIF-1alpha activation by radiation and nuclear translocation of HIF-1alpha. These findings correlated with decreased expression of APE1/Ref-1 and DNA binding activity of HIF-1alpha and NF-kappaB. In APE1/Ref-1 cDNA transfected cells, radiation caused a greater increase in HIF-1alpha and NF-kappaB activities but this effect was inhibited by pretreatment with soy prior to radiation. Transfection experiments indicate that APE1/Ref-1 inhibition by isoflavones impairs the radiation-induced transcription activity of NF-kappaB and HIF-1alpha. This mechanism could result in the inhibition of genes essential for tumor growth and angiogenesis, as demonstrated by inhibition of VEGF production and HUVECs tube formation. Our novel findings suggest that the increased responsiveness to radiation mediated by soy isoflavones could be due to pleiotropic effects of isoflavones blocking cell survival pathways induced by radiation including Src/STAT3/HIF-1alpha, APE1/Ref-1 and NF-kappaB.

A Small-Molecule Triptolide Suppresses Angiogenesis and Invasion of Human Anaplastic Thyroid Carcinoma Cells via Down-Regulation of the Nuclear Factor-κB Pathway

Anaplastic thyroid carcinoma (ATC) is among the most aggressive malignancies known and is characterized with rapid growth, early invasion, and complete refractoriness to current therapies. Here we report that triptolide, a small molecule from a Chinese herb, could potently inhibit proliferation in vitro, angiogenesis in vivo, and invasion in a Matrigel model in human ATC cell line TA-K cells at nanomolar concentrations. We further elucidate that triptolide inhibits the nuclear factor-kappaB (NF-kappaB) transcriptional activity via blocking the association of p65 subunit with CREB-binding protein (CBP)/p300 in the early stage and via decreasing the protein level of p65 in the late stage. Expression of the NF-kappaB targeting genes cyclin D1, vascular endothelial growth factor, and urokinase-type plasminogen activator is significantly reduced by triptolide in both TA-K and 8505C human ATC cell lines, which are well known to be critical for proliferation, angiogenesis, and invasion in solid tumors. Our findings suggest that triptolide may function as a small molecule inhibitor of tumor angiogenesis and invasion and may provide novel mechanistic insights into the potential therapy for human ATC.

Butanol fraction containing berberine or related compound from nexrutine® inhibits NFκB signaling and induces apoptosis in prostate cancer cells

Epidemiological and laboratory studies support the hypothesis that several plant components influence prostate carcinogenesis and holds promise for disease prevention. Previously we reported that Nexrutine (bark extract from Phellodendron amurense) inhibits proliferation of prostate cancer cells and prostate tumor development in the transgenic adenocarcinoma of mouse prostate (TRAMP) model through modulation of Akt signaling pathway. In the present investigation we conducted studies to further define the mechanism of action of Nexrutine and to identify the active component associated with its biological activity. Androgen-responsive, androgen-independent human prostate cancer cell lines and tissues from TRAMP mice fed Nexrutine(R) were used in these studies. Activity guided fractionation identified butanol fraction recapitulating the activities of Nexrutine assessed by proliferation assays, apoptotic assays (DAPI and TUNEL staining), transient transfections, gel shift assays and Western blotting. In addition ultra-performance liquid chromatography (UPLC) of butanol fraction was used to identify active component of Nexrutine. Butanol fraction recapitulated the activities of Nexrutine in (i) inhibiting proliferation; (ii) inducing apoptosis; and (iii) modulating transcriptional activity of NFkappaB in prostate cancer cells. Our data also indicates that both Nexrutine and butanol fraction modulates NFkappaB transcriptional activity by inhibiting IkappaBalpha phosphorylation. Expression of p65 and phosphorylated IkappaBalpha are high in tumors from TRAMP mice. In contrast dietary administration of Nexrutine reduced expression of p65 and phosphorylated IkappaBalpha in prostate from TRAMP mice. In addition using UPLC, we have identified berberine or closely related compound in the butanol fraction. The results suggest that berberine or closely related component of butanol fraction may be responsible for the observed biological activities and induce apoptosis in prostate cancer cells by targeting critical cell survival signaling pathways both in vitro and in vivo.

The DEAD‐box RNA helicase DDX1 interacts with RelA and enhances nuclear factor kappaB‐mediated transcription

DEAD-box RNA helicases constitute the largest family of RNA helicases and are involved in many aspects of RNA metabolism. In this study, we identified RelA (p65), a subunit of nuclear factor-kappaB (NF-kappaB), as a cellular co-factor of DEAD-box RNA helicase DDX1, through mammalian two hybrid system and co-immunoprecipitation assay. Additionally, confocal microscopy and chromatin immunoprecipitation assays confirmed this interaction. In NF-kappaB dependent reporter gene assay, DDX1 acted as a co-activator to enhance NF-kappaB-mediated transcription activation. The functional domains involved were mapped to the carboxy terminal transactivation domain of RelA and the amino terminal ATPase/helicase domain of DDX1. The DDX1 trans-dominant negative mutant lacking ATP-dependent RNA helicase activity lost it transcriptional inducer activity. Moreover, depletion of endogenous DDX1 by specific small interfering RNAs significantly reduced NF-kappaB-dependent transcription. Taken together, the results suggest that DDX1 may play an important role in NF-kappaB-mediated transactivation, and revelation of this regulatory pathway may help to explore the novel mechanisms for regulating NF-kappaB transcriptional activity.

6‐Shogaol suppressed lipopolysaccharide‐induced up‐expression of iNOS and COX‐2 in murine macrophages

Ginger, the rhizome of Zingiber officinale, is a traditional medicine with carminative effect, antinausea, anti-inflammatory, and anticarcinogenic properties. In this study, we investigated the inhibitory effects of 6-shogaol and a related compound, 6-gingerol, on the induction of nitric oxide synthase (NOS) and cyclooxygenase-2 (COX-2) in murine RAW 264.7 cells activated with LPS. Western blotting and reverse transcription-PCR analyses demonstrated that 6-shogaol significantly blocked protein and mRNA expression of inducible NOS (iNOS) and COX-2 in LPS-induced macrophages. The in vivo anti-inflammatory activity was evaluated by a topical 12-O-tetradecanoylphorbol 13-acetate (TPA) application to mouse skin. When applied topically onto the shaven backs of mice prior to TPA, 6-shogaol markedly inhibited the expression of iNOS and COX-2 proteins. Treatment with 6-shogaol resulted in the reduction of LPS-induced nuclear translocation of nuclear factor-kappaB (NF kappaB) subunit and the dependent transcriptional activity of NF kappaB by blocking phosphorylation of inhibitor kappaB (I kappaB)alpha and p65 and subsequent degradation of I kappaB alpha. Transient transfection experiments using NF kappaB reporter constructs indicated that 6-shogaol inhibits the transcriptional activity of NF kappaB in LPS-stimulated mouse macrophages. We found that 6-shogaol also inhibited LPS-induced activation of PI3K/Akt and extracellular signal-regulated kinase 1/2, but not p38 mitogen-activated protein kinase (MAPK). Taken together, these results show that 6-shogaol downregulates inflammatory iNOS and COX-2 gene expression in macrophages by inhibiting the activation of NF kappaB by interfering with the activation PI3K/Akt/I kappaB kinases IKK and MAPK.

HCMV IL-10 Suppresses Cytokine Expression in Monocytes Through Inhibition of Nuclear Factor-κB

Modulation of host immune responses is a common strategy for promoting virus persistence and avoiding clearance. Human cytomegalovirus (HCMV) is known to encode numerous immunomodulatory genes, including a homolog of the cytokine human interleukin-10 (hIL-10). While having limited sequence homology to hIL-10, cytomegalovirus IL-10 (cmvIL-10) shares many functional characteristics with the human cytokine and acts as a potent suppressor of the inflammatory immune response. The mechanism by which hIL-10 inhibits inflammatory cytokines involves a transcriptional block via inhibition of nuclear factor-kappaB (NF-kappaB) activity. To determine whether cmvIL-10 employs the same mechanism to inhibit cytokine production, the effect of cmvIL-10 on NF-kappaB signaling in monocytes was investigated. The results demonstrate that cmvIL-10 does inhibit NF-kappaB activation, as evidenced by reduced degradation of the NF-kappaB inhibitor IkappaB-alpha, and decreased transcription of the NF-kappaB-responsive genes tumor necrosis factor-alpha (TNF-alpha) and IL-1beta. These studies confirm that cmvIL-10 mediates cytokine suppression by blocking NF-kappaB transcriptional activity in human monocytes.

Insulin exhibits short-term anti-inflammatory but long-term proinflammatory effects in vitro

Although insulin is indispensable for maintaining glucose homeostasis, it is still controversial whether or not a high concentration of insulin is deleterious. We examined the effect of insulin on the transcriptional activity of NF-kappaB, which mediates the expression of a variety of inflammation/coagulation-related genes using hepatocyte cell lines in vitro. We found that insulin (1 nM) alone caused minimal increase in NF-kappaB-mediated transcription. On the other hand, when cells were simultaneously treated with proinflammatory cytokines such as TNFalpha, the following dual effect of insulin was observed: short-term (6h) suppressive, and long-term (36 h or later) stimulatory effects. The former effect was transient and appears to be mediated by the phosphatidylinositol 3 kinase (PI(3)K) signaling pathway. The latter effect, in contrast, was more pronounced, enhancing the TNFalpha-stimulated NF-kappaB-dependent transcription by more than sevenfold. This positive effect was NF-kappaB-specific, and was eliminated by mitogen-activated protein kinase (MAPK) inhibitors. Altogether, our data suggest that insulin has short-term anti-inflammatory but long-term proinflammatory effects. From a clinical standpoint, this implies that low basal and periodically high plasma insulin is beneficial, whereas a sustained rise in plasma insulin, as often seen in patients with obesity, may induce atherothrombotic disorders, because of the NF-kappaB-mediated overexpression of proinflammatory/procoagulant/antifibrinolytic proteins in the liver.

Maintenance of constitutive IkappaB kinase activity by glycogen synthase kinase-3alpha/beta in pancreatic cancer.

Constitutive nuclear factor kappaB (NF-kappaB) activation is among the many deregulated signaling pathways that are proposed to drive pancreatic cancer cell growth and survival. Recent reports suggest that glycogen synthase kinase-3beta (GSK-3beta) plays a key role in maintaining basal NF-kappaB target gene expression and cell survival in pancreatic cancer cell lines. However, the mechanism by which GSK-3beta facilitates constitutive NF-kappaB signaling in pancreatic cancer remains unclear. In this report, we analyze the contributions of both GSK-3 isoforms (GSK-3alpha and GSK-3beta) in regulating NF-kappaB activation and cell proliferation in pancreatic cancer cell lines (Panc-1 and MiaPaCa-2). We show that GSK-3 isoforms are differentially required to maintain basal NF-kappaB DNA binding activity, transcriptional activity, and cell proliferation in Panc-1 and MiaPaCa-2 cells. Our data also indicate that IkappaB kinase (IKK) subunits are not equally required to regulate pancreatic cancer-associated NF-kappaB activity and cell growth. Importantly, we provide the first evidence that GSK-3 maintains constitutive NF-kappaB signaling in pancreatic cancer by regulating IKK activity. These data provide new insight into GSK-3-dependent NF-kappaB regulation and further establish GSK-3 and IKK as potential therapeutic targets for pancreatic cancer.

Caspase‐3 activation as a key factor for HBx‐transformed cell death

Nuclear factor-kappa B (NF-kappaB) activation has been associated with the tumorigenic growth of hepatitis B virus X protein (HBx)-transformed cells. This study was aimed to find a key target for treatment of HBx-mediated cancers. NF-kappaB activation, endoplasmic reticulum-stress (ER-stress), caspase-3 activation, and cell proliferation were evaluated after Chang/HBx cells permanently expressing HBx viral protein were treated with inhibitors of NF-kappaB, proteasome and DNA topoisomerase. Inhibition of NF-kappaB transcriptional activity by transient transfection with mutant plasmids encoding Akt1 and glycogen synthase kinase-3beta (GSK-3beta), or by treatment with chemical inhibitors, wortmannin and LY294002, showed little effect on the survival of Chang/HBx cells. Furthermore, IkappaBalpha (S32/36A) mutant plasmid or other NF-kappaB inhibitors, 1-pyrrolidinecarbonidithioic acid and sulphasalazine, were also shown to have little effect on the cell proliferation. By contrast, proteasome inhibitor-1 (Pro1) and MG132 enhanced the HBx-induced ER-stress response and the subsequent activation of caspase-12, -9 and -3 and reduced cell proliferation. Camptothecin (CPT), however, triggered activation of caspase-3 without induction of caspase-12, and reduced cell proliferation. In addition, CPT-induced cell death was reversed by pre-treatment with z-DEVD, a caspase-3-specific inhibitor. Detailed exploitation of the regulators of caspase-3 activation could open the gate for finding an efficient target for development of anticancer therapeutics against HBx-transformed hepatocellular carcinoma.

Delphinidin, an anthocyanidin in pigmented fruits and vegetables, induces apoptosis and cell cycle arrest in human colon cancer HCT116 cells

Because of unsatisfactory treatment options for colon cancer, there is a need to develop novel preventive approaches for this malignancy. One such strategy is through chemoprevention by the use of non-toxic dietary substances and botanical products. Delphinidin, an anthocyanidin in pigmented fruits and vegetables, possesses strong anti-oxidant and anti-inflammatory properties. In the present study, we investigated the antiproliferative and proapoptotic properties of delphinidin in human colon cancer HCT116 cells. We found that treatment of cells with delphinidin (30-240 microM; 48 h) resulted in (i) decrease in cell viability (ii) induction of apoptosis, (iii) cleavage of PARP, (iv) activation of caspases-3, -8, and -9, (v) increase in Bax with a concomitant decrease in Bcl-2 protein, and (vi) G2/M phase cell cycle arrest. NF-kappaB provides a mechanistic link between inflammation and cancer, and is a major factor controlling the ability of both pre-neoplastic and malignant cells to resist apoptosis-based tumor surveillance mechanisms. We therefore, determined the effect of delphinidin on NF-kappaB signaling pathway. The immunoblot, ELISA and EMSA analysis demonstrated that the treatment of HCT116 cells with delphinidin resulted in the inhibition of (i) IKKalpha, (ii) phosphorylation and degradation of IkappaBalpha, (iii) phosphorylation of NF-kappaB/p65 at Ser(536), (iv) nuclear translocation of NF-kappaB/p65, (v) NF-kappaB/p65 DNA binding activity, and (vi) transcriptional activation of NF-kappaB. Our results suggest that delphinidin treatment of HCT116 cells suppressed NF-kappaB pathway, resulting in G2/M phase arrest and apoptosis. We suggest that delphinidin could have potential in inhibiting colon cancer growth.

Clotrimazole Ameliorates Intestinal Inflammation and Abnormal Angiogenesis by Inhibiting Interleukin-8 Expression through a Nuclear Factor-κB-Dependent Manner

Increased interleukin (IL)-8 plays an important role not only in activation and recruitment of neutrophils but also in inducing exaggerated angiogenesis at the inflamed site. In the present study, we investigated the fact that clotrimazole (CLT) inhibits intestinal inflammation, and the inhibitory action is mediated through suppression of IL-8 expression. In the trinitrobenzene sulfonic acid (TNBS)-induced rat colitis model, CLT dose-dependently protected from the TNBS-induced weight loss, colon ulceration, and myeloperoxidase activity increase. In the lesion site, CLT also suppressed the TNBS-induced angiogenesis, IL-8 expression, and nuclear factor (NF)-kappaB activation. In a cellular model of colitis using tumor necrosis factor (TNF)-alpha-stimulated HT29 colon epithelial cells, treatment with CLT significantly suppressed TNF-alpha-mediated IL-8 induction and NF-kappaB transcriptional activity revealed by a luciferase reporter gene assay. Furthermore, cotreatment with CLT and pyrrolidine dithiocarbamate, a NF-kappaB inhibitor, synergistically reduced the NF-kappaB transcriptional activity as well as IL-8 expression. In an in vitro angiogenesis assay, CLT suppressed IL-8-induced proliferation, tube formation, and invasion of human umbilical vein endothelial cells. The in vivo angiogenesis assay using chick chorioallantoic membrane also showed that CLT significantly inhibited the IL-8-induced formation of new blood vessels. Taken together, these results suggest that CLT may prevent the progression of intestinal inflammation by not only down-regulating IL-8 expression but also inhibiting the action of IL-8 in both colon epithelial and vascular endothelial cells during pathogenesis of intestinal inflammation.

Fermented guava leaf extract inhibits LPS‐induced COX‐2 and iNOS expression in Mouse macrophage cells by inhibition of transcription factor NF‐κB

The goal of this study was to elucidate the antiinflammatory activities of Psidium guajava L. (guava) leaf. To improve the functionality of guava leaf, it was fermented with Phellinus linteus mycelia, Lactobacillus plantarum and Saccharomyces cerevisiae. The ethanol extract from fermented guava leaf inhibited lipopolysaccharide (LPS)-induced nitric oxide (NO) and prostaglandin E(2) (PGE(2)) production. Western blot analysis showed that fermented guava leaf extract decreased LPS-induced inducible nitric oxide synthase (iNOS) and the cyclooxygenase-2 (COX-2) protein level in RAW 264.7 cells. To investigate the mechanism involved, the study examined the effect of fermented guava leaf extract on LPS-induced nuclear factor-kappaB (NF-kappaB) activation. Fermented guava leaf extract significantly inhibited LPS-induced NF-kappaB transcriptional activity. Immunochemical analysis revealed that fermented guava leaf extract suppressed LPS-induced degradation of I-kappaBalpha. Taken together, the data indicate that fermented guava leaf extract is involved in the inhibition of iNOS and COX-2 via the down-regulation of NF-kappaB pathway, revealing a partial molecular basis for the antiinflammatory properties of fermented guava leaf extract.

Inhibition of NF-κB-mediated transcription and induction of apoptosis in human breast cancer cells by epoxypseudoisoeugenol-2-methyl butyrate

Breast cancer is one of the most prevalent woman cancers. Genomic instability, accumulative mutations, and subsequent changes in intracellular signaling cascades play key roles in the development of human breast cancers. Activation of nuclear factor-kappaB (NF-kappaB) has been implicated in oncogenesis of breast cancers and is known to be associated with resistance to anticancer agents and apoptosis. Blocking NF-kappaB signaling may represent a therapeutic strategy in breast cancer therapy. The objective of this study is to investigate the in vitro effects of epoxypseudoisoeugenol-2-methyl butyrate (EPB), a phenylpropranoid isolated from Pimpinella corymbosa, on the activation of NF-kappaB, cell growth, cell cycle progression and apoptosis in MCF-7 (estrogen-dependent) and BT-549 (estrogen-independent) breast cancer cells. Transcriptional activity of NF-kappaB was measured by cell based reporter gene assay. Cell proliferation was determined by MTT assay. Cell cycle analysis was carried out by flow cytometry and apoptosis was observed by DAPI staining assy. EPB inhibited the NF-kappaB-mediated transcription activity induced by tumor necrosis factor-alpha (TNF-alpha) and phorbol myristate acetate (PMA) in MCF-7 cells. EPB also inhibited constitutive NF-kappaB transcriptional activity in BT-549 cells. EPB inhibited the proliferation of both MCF-7 and BT-549 cells in a concentration- and time-dependent manner. EPB induced cell cycle arrest in G(1)/G(0) phase and apoptosis in both MCF-7 and BT 549 cells. These in vitro results indicated that EPB has a potential for use against both hormone-dependent and hormone-independent breast cancers and its effects seem to be mediated by inhibiting the NF-kappaB activity.

The PIM1 Kinase Is a Critical Component of a Survival Pathway Activated by Docetaxel and Promotes Survival of Docetaxel-treated Prostate Cancer Cells

A defining characteristic of solid tumors is the capacity to divide aggressively and disseminate under conditions of nutrient deprivation, limited oxygen availability, and exposure to cytotoxic drugs or radiation. Survival pathways are activated within tumor cells to cope with these ambient stresses. We here describe a survival pathway activated by the anti-cancer drug docetaxel in prostate cancer cells. Docetaxel activates STAT3 phosphorylation and transcriptional activity, which in turns induces expression of the PIM1 gene, encoding a serine-threonine kinase activated by many cellular stresses. Expression of PIM1 improves survival of docetaxel-treated prostate cancer cells, and PIM1 knockdown or expression of a dominant-negative PIM1 protein sensitize cells to the cytotoxic effects of docetaxel. PIM1 in turn mediates docetaxel-induced activation of NFkappaB transcriptional activity, and PIM1 depends in part on RELA/p65 proteins for its prosurvival effects. The PIM1 kinase plays a critical role in this STAT3 --> PIM1 --> NFkappaB stress response pathway and serves as a target for intervention to enhance the therapeutic effects of cytotoxic drugs such as docetaxel.