Sunil K. Manna

Interleukin-8 Induces Nuclear Transcription Factor-κB through a TRAF6-dependent Pathway

Considering the potential role of interleukin-8 (IL-8) in inflammation, angiogenesis, tumorigenesis, and metastasis, we investigated the molecular mechanism involved in IL-8-mediated signaling. In this report we provide evidence that like TNF, an inducer of NF-κB and also a NF-κB-dependent gene product, IL-8 induces NF-κB in a unique pathway. IL-8 induces NF-κB activation in a dose-dependent manner in different cell types as detected by a DNA-protein binding assay. IL-8 induces NF-κB-dependent reporter gene expression as well as ICAM-1, VCAM-1, and Cox-2 expression. IL-8 also induces IκBα phosphorylation followed by degradation and p65 translocation. IL-8 induces c-Jun N-terminal kinase (JNK) and mitogen-activated protein kinase (MAPK) in a dose- and time-dependent manner. IL-8-induced NF-κB activation is for the most part unaltered when cells are transfected with dominant-negative TRADD, FADD, or TRAF2, but is inhibited with dominant-negative TRAF6-, NIK-, IKK-, or IκBα-transfected cells. The data suggest that IL-8-induced NF-κB activation proceeds through a TRAF2-independent but TRAF6-dependent pathway, followed by recruitment of IRAK and activation of IKK. IL-8-induced NF-κB activation is not observed in a cell-permeable peptide that has TRAF6 binding motif-treated cells or IRAK-deficient cells. IL-8-induced NF-κB activation proceeds mostly through interaction with TRAF6 and partially through the Rho-GTPase pathways. This is the first report that IL-8 induces NF-κB in a distinct pathway, and activation of NF-κB and its dependent genes may be one of the pathways of IL-8-induced inflammation and angiogenesis.

Biochanin-A, an isoflavon, showed anti-proliferative and anti-inflammatory activities through the inhibition of iNOS expression, p38-MAPK and ATF-2 phosphorylation and blocking NFκB nuclear translocation

Biochanin-A, an isoflavone, existing in red clover, cabbage and alfalfa, has an inhibitory and apoptogenic effect on certain cancer cells. However, the actual mechanism by which this compound inhibits proliferation and induces apoptosis in cancer cells and the mechanism of its anti-inflammatory activities have not been well characterized. In this study, we have investigated the anti-inflammatory and anti-proliferative activity of Biochanin-A. The effects of Biochanin-A on RAW 264.7, HT-29 cell lines and mouse peritoneal macrophages have been investigated in vitro. Cell proliferation and anti-inflammatory effects were analyzed by 3-(4-5-dimethylthiozol-2-yl)2-5-diphenyl-tetrazolium bromide (MTT) assay, (3)H-thymidine incorporation assay, Western blot, cytokines estimation, Luciferase assay, Electrophoretic mobility shift assay (EMSA) and Kinase assay. Present investigation demonstrated that, Biochanin-A inhibited lipopolysacharide (LPS)-induced nitric oxide(NO) production in macrophage and showed dose dependent inhibition of inducible nitric oxide synthase (iNOS) expression. The induction of NF-κB binding activity by LPS was inhibited markedly by co-incubation with different doses of Biochanin-A. Biochanin-A inhibited the LPS-induced IkB kinase (IKK) activity and nuclear factor kappa beta (NF-κB) activation associated with the inhibition of iNOS expression. LPS-induced phosphorylation of IκBα and p38 MAPK was blocked by Biochanin-A and it inhibited IL-6, IL-1β and TNF-α production in RAW264.7 cells indicating its anti-inflammatory activity in association with anti-proliferation. Biochanin-A is important for the prevention of phosphorylation and degradation of IκBα, thereby blocking NF-κB activation, which in turn leads to decreased expression of the iNOS, thus preventing proliferation and inflammation.

α-melanocyte-stimulating hormone down-regulates CXC receptors through activation of neutrophil elastase

Considering the role of interleukin‐8 (IL‐8) in a large number of acute and chronic inflammatory diseases, the regulation of IL‐8‐mediated biological responses is important. Alpha‐melanocyte‐stimulating hormone (α‐MSH), a tridecapeptide, inhibits most forms of inflammation by an unknown mechanism. In the present study, we have found that α‐MSH interacts predominantly with melanocortin‐1 receptors and inhibits several IL‐8‐induced biological responses in macrophages and neutrophils. It down‐regulated receptors for IL‐8 but not for TNF, IL‐4, IL‐13 or TNF‐related apoptosis‐inducing ligand (TRAIL) in neutrophils. It down‐regulated CXCR type 1 and 2 but not mRNA levels. α‐MSH did not inhibit IL‐8 binding in purified cell membrane or affinity‐purified CXCR. IL‐8 or anti‐CXCR Ab protected against α‐MSH‐mediated inhibition of IL‐8 binding. The level of neutrophil elastase, a specific serine protease, but not cathepsin G or proteinase 3 increased in α‐MSH‐treated cells, and restoration of CXCR by specific neutrophil elastase or serine protease inhibitors indicates the involvement of elastase in α‐MSH‐induced down‐regulation of CXCR. These studies suggest that α‐MSH inhibits IL‐8‐mediated biological responses by down‐regulating CXCR through induction of serine protease and that α‐MSH acts as a potent immunomodulator in neutrophil‐driven inflammatory distress.

A Study of Nuclear Transcription Factor-Kappa B in Childhood Autism

Background Several children with autism show regression in language and social development while maintaining normal motor milestones. A clear period of normal development followed by regression and subsequent improvement with treatment, suggests a multifactorial etiology. The role of inflammation in autism is now a major area of study. Viral and bacterial infections, hypoxia, or medication could affect both foetus and infant. These stressors could upregulate transcription factors like nuclear factor kappa B (NF-κB), a master switch for many genes including some implicated in autism like tumor necrosis factor (TNF). On this hypothesis, it was proposed to determine NF-κB in children with autism. Methods Peripheral blood samples of 67 children with autism and 29 control children were evaluated for NF-κB using electrophoretic mobility shift assay (EMSA). A phosphor imaging technique was used to quantify values. The fold increase over the control sample was calculated and statistical analysis was carried out using SPSS 15. Results We have noted significant increase in NF-κB DNA binding activity in peripheral blood samples of children with autism. When the fold increase of NF-κB in cases (nu200a=u200a67) was compared with that of controls (nu200a=u200a29), there was a significant difference (3.14 vs. 1.40, respectively; p<0.02). Conclusion This finding has immense value in understanding many of the known biochemical changes reported in autism. As NF-κB is a response to stressors of several kinds and a master switch for many genes, autism may then arise at least in part from an NF-κB pathway gone awry.

Cardiac glycoside induces cell death via FasL by activating calcineurin and NF-AT, but apoptosis initially proceeds through activation of caspases.

Decrease in caspase activity is a common phenomenon in drug resistance. For effective therapeutic intervention, detection of such agents, which affects other pathway independent of caspases to promote cell death, might be important. Oleandrin, a polyphenolic glycoside induced cell death through activation of caspases in a variety of human tumour cells. In this report we provide evidence that besides caspases activation, oleandrin interacts with plasma membrane, changes fluidity of the membrane, disrupts Na+/K+-ATPase pump, enhances intracellular free Ca2+ and thereby activates calcineurin. Calcineurin, in turns, activates nuclear transcription factor NF-AT and its dependent genes such as FasL, which induces cell death as a late response of oleandrin. Cell death at early stages is mediated by caspases where inhibitors partially protected oleandrin-mediated cell death in vector-transfected cells, but almost completely in Bcl-xL-overexpressed cells. Overall, our data suggest that oleandrin might be important therapeutic molecule in case of tumors where cell death pathway occurs due to deregulation of caspase-mediated pathway

α-Melanocyte-stimulating hormone inhibits lipopolysaccharide-induced biological responses by downregulating CD14 from macrophages

Monocytes/macrophages are the first cells involved in inflammation. α‐Melanocyte‐stimulating hormone (α‐MSH) is known to possess an anti‐inflammatory role induced by a variety of stimuli; however, the molecular mechanisms underlying these effects are not clearly defined. In this report we provide evidence that α‐MSH inhibited serum‐activated lipopolysaccharide (SA‐LPS)‐induced proteolytic enzyme release, oxidative burst response, reactive oxygen intermediate generation, nitric oxide production, and adhesion molecule expression in monocyte‐derived macrophages. α‐MSH also inhibited SA‐LPS‐induced nuclear transcription factor κB activation not only in macrophages, but also in a T‐cell line and human neutrophils isolated from fresh blood. α‐MSH downregulated CD14, but not interleukin‐1 receptor, tumor necrosis factor receptor 1 or 2 from the surface of macrophages. Anti‐CD14 antibody was unable to protect α‐MSH‐mediated downregulation of CD14. Overall, our results suggest that α‐MSH exerts its anti‐inflammatory effect by a novel mechanism in macrophages through downregulating of the endotoxin receptor CD14.

α‐Melanocyte‐stimulating hormone induces cell death in mast cells: involvement of NF‐κB

Mast cells play a major role in the initiation of inflammation and allergic reactions. As cell numbers are tightly controlled by the interplay of factors affecting cell proliferation, development, and death the regulation of mast cell number may be important. Melanocyte‐stimulating hormone inhibits most forms of inflammation by an unknown mechanism. In the present study, we have found that the α‐melanocyte‐stimulating hormone (α‐MSH) inhibited endotoxin‐mediated nuclear transcription factor κB (NF‐κB) activation in different cells correlated with the expression of α‐MSH receptors. We have also found for the first time that it induces cell death alone or in endotoxin‐stimulated mast cells. α‐MSH‐mediated apoptosis was not observed in NF‐κB overexpressed cells. The inhibitory effect of α‐MSH was mediated through generation of cAMP, as inhibitors of adenylate cyclase and of protein kinase A reversed its inhibitory effect. Overall, our results suggest that NF‐κB is the key molecule involved in α‐MSH‐mediated cell death and this may help to regulate mast cell‐mediated inflammation.

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

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

Azadirachtin interacts with retinoic acid receptors and inhibits retinoic acid-mediated biological responses

Considering the role of retinoids in regulation of more than 500 genes involved in cell cycle and growth arrest, a detailed understanding of the mechanism and its regulation is useful for therapy. The extract of the medicinal plant Neem (Azadirachta indica) is used against several ailments especially for anti-inflammatory, anti-itching, spermicidal, anticancer, and insecticidal activities. In this report we prove the detailed mechanism on the regulation of retinoic acid-mediated cell signaling by azadirachtin, active components of neem extract. Azadirachtin repressed all trans-retinoic acid (ATRA)-mediated nuclear transcription factor κB (NF-κB) activation, not the DNA binding but the NF-κB-dependent gene expression. It did not inhibit IκBα degradation, IκBα kinase activity, or p65 phosphorylation and its nuclear translocation but inhibited NF-κB-dependent reporter gene expression. Azadirachtin inhibited TRAF6-mediated, but not TRAF2-mediated NF-κB activation. It inhibited ATRA-induced Sp1 and CREB (cAMP-response element-binding protein) DNA binding. Azadirachtin inhibited ATRA binding with retinoid receptors, which is supported by biochemical and in silico evidences. Azadirachtin showed strong interaction with retinoid receptors. It suppressed ATRA-mediated removal of retinoid receptors, bound with DNA by inhibiting ATRA binding to its receptors. Overall, our data suggest that azadirachtin interacts with retinoic acid receptors and suppresses ATRA binding, inhibits falling off the receptors, and activates transcription factors like CREB, Sp1, NF-κB, etc. Thus, azadirachtin exerts anti-inflammatory and anti-metastatic responses by a novel pathway that would be beneficial for further anti-inflammatory and anti-cancer therapies.

Late phase activation of nuclear transcription factor kappaB by doxorubicin is mediated by interleukin-8 and induction of apoptosis via FasL

Doxorubicin is one of the most effective molecules used in the treatment of various tumors. Contradictory reports often open windows to understand the doxorubicin-mediated signaling to exert its apoptosis effect. In this report, we provide evidences that doxorubicin induced biphasic induction of nuclear factor kappaB (NF-κB) of immediate activation followed by decrease in the amount of RelA (p65) subunit possibly by inducing the activity of proteasome, but not proteases. Further induction of NF-κB was observed through interleukin 8 (IL-8), expressed by doxorubicin treatment. Increased amount of IL-8 induced apoptosis via increase in the releases of intracellular Ca2+, activation of calcineurin, nuclear translocation of nuclear factor activated T cell (NF-AT), and NF-AT-dependent FasL expression. Anti-IL-8 or -FasL antibody, dominant negative TRAF6 (TRAF6-DN), or TRAF6 binding peptide (TRAF6-BP) inhibited doxorubicin-mediated late phase induction of NF-κB and diminished cell death. Thus, our study clearly demonstrated that doxorubicin-mediated cell death is obtained through expression of IL-8. IL-8-mediated calcification is required for enhancement of doxorubicin-mediated cell death. Overall, this study will help to understand the much studied chemotherapeutic drug, doxorubicin-mediated cell signaling cascade to exert its effect during chemotherapy.