Effect of Zn treatment on wild type and MT-null cell lines in relation to apoptotic and/or necrotic processes and on MT isoform gene expression

It has been shown in various systems that zinc is able to antagonize the catalytic properties of the redox-active transition metals iron and copper, although the process is still unclear. Probably, the protective effect of Zn against oxidative stress is mainly due to the induction of a scavenger metal binding protein such as metallothionein (MT), rather than a direct action. To support this hypothesis, in this study, the effects of Zn, Cu, Fe, Zn + Cu and Zn + Fe treatments were investigated in a fibroblast cell line corresponding to an SV40-transformed MT-1/-2 mutant (MT-/-), and in wild type (MT+/+), by valuing metal concentrations and apoptotic and/or necrotic processes. We also investigated the synthesis of MT and the levels of both MT-1 and MT-2 mRNAs. In MT+/+ cells, co-treatment with Zn + Fe caused a decrease in Fe content compared to treatment with Fe alone. After Zn and Zn + Cu exposure the expression of MT-1 and MT-2 isoforms increased with a concomitant increase in MT synthesis. Annexin V-FITC and propidium iodide staining revealed necrotic or apoptotic cells in terminal stages, especially after Fe treatments. Immunofluorescent staining with an anti-ssDNA Mab and annexin detected a lower signal in co-treated cells compared to the single treatments in both cell lines. The intensity and quantity of fluorescence resulting from anti-ssDNA and Annexin V staining of MT null cells was higher compared to wild type cells. These results suggest that Zn alone does not completely exert an anti-oxidant effect against Cu and Fe toxicity, but that induction of MT is necessary.     

Melanoma differentiation-associated gene-7/IL-24 gene enhances NF-kappa B activation and suppresses apoptosis induced by TNF

Melanoma differentiation-associated gene-7 (mda-7), also referred to as IL-24, is a novel growth regulatory cytokine that has been shown to regulate the immune system by inducing the expression of inflammatory cytokines, such as TNF, IL-1, and IL-6. Whether the induction of these cytokines by MDA-7 is mediated through activation of NF-kappaB or whether it regulates cytokine signaling is not known. In the present report we investigated the effect of MDA-7 on NF-kappaB activation and on TNF-induced NF-kappaB activation and apoptosis in human embryonic kidney 293 cells. Stable or transient transfection with mda-7 into 293 cells failed to activate NF-kappaB. However, TNF-induced NF-kappaB activation was significantly enhanced in mda-7-transfected cells, as indicated by DNA binding, p65 translocation, and NF-kappaB-dependent reporter gene expression. Mda-7 transfection also potentiated NF-kappaB reporter activation induced by TNF receptor-associated death domain and TNF receptor-associated factor-2. Cytoplasmic MDA-7 with deleted signal sequence was as effective as full-length MDA-7 in potentiating TNF-induced NF-kappaB reporter activity. Secretion of MDA-7 was not required for the potentiation of TNF-induced NF-kappaB activation. TNF-induced expression of the NF-kappaB-regulated gene products cyclin D1 and cyclooxygenase-2, were significantly up-regulated by stable expression of MDA-7. Furthermore, MDA-7 expression abolished TNF-induced apoptosis, and suppression of NF-kappaB by IkappaBalpha kinase inhibitors enhanced apoptosis. Overall, our results indicate that stable or transient MDA-7 expression alone does not substantially activate NF-kappaB, but potentiates TNF-induced NF-kappaB activation and NF-kappaB-regulated gene expression. Potentiation of NF-kappaB survival signaling by MDA-7 inhibits TNF-mediated apoptosis.     

Indirubin enhances tumor necrosis factor-induced apoptosis through modulation of nuclear factor-kappa B signaling pathway

Although indirubin is known to exhibit anti-cancer and anti-inflammatory activities, very little is known about its mechanism of action. In this study, we investigated whether indirubin mediates its effects through interference with the NF-kappaB pathway. As examined by the DNA binding of NF-kappaB, we found that indirubin suppressed tumor necrosis factor (TNF)-induced NF-kappaB activation in a dose- and time-dependent manner. Indirubin also suppressed the NF-kappaB activation induced by various inflammatory agents and carcinogens. Further studies showed that indirubin blocked the phosphorylation and degradation of IkappaB alpha through the inhibition of activation of IkappaB alpha kinase and phosphorylation and nuclear translocation of p65. NF-kappaB reporter activity induced by TNFR1, TNF receptor-associated death domain, TRAF2, TAK1, NF-kappaB-inducing kinase, and IKKbeta was inhibited by indirubin but not that induced by p65 transfection. We also found that indirubin inhibited the expression of NF-kappaB-regulated gene products involved in antiapoptosis (IAP1, IAP2, Bcl-2, Bcl-xL, and TRAF1), proliferation (cyclin D1 and c-Myc), and invasion (COX-2 and MMP-9). This correlated with enhancement of the apoptosis induced by TNF and the chemotherapeutic agent taxol in human leukemic KBM-5 cells. Indirubin also suppressed cytokine-induced cellular invasion. Overall, our results indicate that anti-cancer and anti-inflammatory activities previously assigned to indirubin may be mediated in part through the suppression of the NF-kappaB activation pathway.     

Transfection of human endothelial cells with HIV-1 tat gene activates NF-kappa B and enhances monocyte adhesion

Human immunodeficiency virus (HIV)-1 Tat released from HIV-1-infected monocytes is believed to enter other cells via an integrin-facilitated pathway, resulting in altered gene expression. Indeed, exogenous Tat protein can increase cell adhesion molecule gene expression in human endothelial cells. Signaling pathways initiated by Tat in endothelial cells are not known. We evaluated the ability of endogenous tat to stimulate monocyte adhesion via activation of nuclear factor-kappaB (NF-kappaB) within human umbilical vein endothelial cells. Transfection with pcTat, but not control vector DNA, increased NF-kappaB binding activity, NF-kappaB luciferase reporter activity, and monocyte adhesion. pcTat also increased kappaB-dependent HIV-1-LTR-CAT reporter activity 28-fold compared with a 3-fold increase produced by transfection with an equivalent amount of pcTax (from human leukemia virus). The pcTat-induced increase in pNF-kappaB-Luc activity and monocyte adhesion to endothelial cells was blocked by cotransfection with dominant-negative mutant IkappaBalpha and by incubation with 10 mM aspirin. We conclude that monocyte adhesion to human endothelial cells stimulated by pcTat is mediated via an NF-kappaB-dependent mechanism. Furthermore, inhibition studies using aspirin suggest that pcTat-stimulated NF-kappaB activation and monocyte adhesion occur via a redox-sensitive mechanism.     

Molecular mechanism of tumor necrosis factor-alpha production in 1-->3-beta-glucan (zymosan)-activated macrophages

The molecular details of 1-->3-beta-glucans, a fungal cell wall component, induced inflammatory responses are not well understood. In the present study, we conducted a systematic analysis of the molecular events leading to tumor necrosis factor (TNF)-alpha production after glucan stimulation of macrophages. We demonstrated that activation of nuclear factor kappaB (NF-kappaB) is essential in zymosan A (a source of 1-->3-beta-glucans)-induced TNF-alpha production in macrophages (RAW264.7 cells). Zymosan A-induced TNF-alpha protein production was associated with an increase in the TNF-alpha gene promoter activity. Activation of the TNF-alpha gene promoter was dependent on activation of NF-kappaB. Time course studies indicated that DNA binding activity of NF-kappaB preceded TNF-alpha promoter activity. Inhibition of NF-kappaB activation led to a dramatic reduction in both TNF-alpha promoter activity and TNF-alpha protein production in the response to zymosan A. Mutation of a major NF-kappaB binding site (kappa3) in the gene promoter resulted in a significant decrease in the induction of the gene promoter by zymosan A, while mutation of Egr or CRE sites failed to inhibit the response to zymosan. Together, these results strongly suggest that NF-kappaB is involved in signal transduction of 1-->3-beta-glucans-induced TNF-alpha expression.