Helicobacter pylori infection activates NF-kappaB signaling pathway to induce iNOS and protect human gastric epithelial cells from apoptosis

Helicobacter pylori infection induces apoptosis and inducible nitric oxide synthase (iNOS) expression in gastric epithelial cells. In this study, we investigated the effects of NF-kappaB activation and iNOS expression on apoptosis in H. pylori-infected gastric epithelial cells. The suppression of NF-kappaB significantly increased caspase-3 activity and apoptosis in H. pylori-infected MKN-45 and Hs746T gastric epithelial cell lines as well as primary gastric epithelial cells. An NF-kappaB signaling pathway via NF-kappaB-inducing kinase and IkappaB kinase-beta activation was found to be involved in the inhibition of apoptosis in H. pylori-infected gastric epithelial cells. In gastric epithelial cells transfected with retrovirus containing IkappaBalpha superrepressor, iNOS mRNA and protein levels were reduced, indicating that H. pylori infection induced the expression of iNOS by activating NF-kappaB. Moreover, a NO donor, S-nitroso-N-acetylpenicillamine (100 microM), decreased caspase-3 activity and apoptosis in NF-kappaB-suppressed cells infected with H. pylori. These results suggest that NF-kappaB activation may play a role in protecting gastric epithelial cells from H. pylori-induced apoptosis by upregulating endogenous iNOS.     

5-Fluorouracil enhances apoptosis sensitivity of T lymphocytes mediated by CD3 epsilon

Previous studies by our laboratory have reported that the T cell receptor (TCR) TCR/CD3 complex could mediate activation as well as apoptosis of T lymphocytes. Two tyrosine residues in the ITAM (immuno-receptor tyrosine-based activation motifs) of CD3 epsilon were required for apoptosis signalling of Jurkat T lymphocytes. Stable cell lines TJK and T3JK produced from CD8(-) Jurkat T lymphocytes by transfection with wild-type and mutant CD8 epsilon (fusion of the extracellular and transmembrane domains of human CD8 alpha to the intracellular domain of mouse CD3 epsilon), were used with CD8(-) Jurkat T lymphocytes for studying the role of single intact CD3 epsilon. 5-Fluorouracil (5-FU), a chemotherapeutic drug can induce cell death of many tumour cell lines. In the present experiments, we examined the expression of caspase-3, p53 and Bid in the three cell lines induced by 5-FU and/or anti-CD8 antibody. We found high expression of p53 during activation-induced cell death of TJK cells mediated by anti-CD8 antibody and apoptosis of TJK and T3JK induced by 5-FU, implicating p53 involvement in apoptosis of leukemia cells induced by anti-CD8 antibody and 5-FU. We also detected the active form of caspase-3 and Bid in apoptotic leukemia cells after treatment with 5-FU and/or anti-CD8 antibody, indicating that the drug and antibody induced cell death through caspase-3 and the signal pathway may involve the Bcl-2 protein family. Our results showed that combined treatment with 5-FU and anti-CD8 antibody could enhance the rate of apoptosis induced by 5-FU or anti-CD8 antibody through increased expression of p53 and by promoting activation of caspase-3 and Bid. This suggests that the combination of 5-FU and anti-CD8 antibody may play an important role in inducing apoptosis of leukemia cells.     

Ethanol induced NF-{kappa}B activation protects against cell injury in cultured rat gastric mucosal epithelium

Ethanol is a well-established irritant inducing inflammation in gastric mucosa, but the effects at the cellular level remain unclear. This study investigates NF-kappaB activation in gastric mucosal cells by ethanol and assesses the effects of heat shock pretreatment in this ulcerogenic situation. Rat gastric mucosal epithelia were exposed to ethanol for different time periods. Heat shock was induced by incubating the cells at 42 degrees C for 1 h prior to the experiments. For evaluation of NF-kappaB activation, the nuclear fraction of the cell lysates was analyzed with an EMSA or an ELISA-based assay. Caspase-3 (a promoter of apoptosis) activity was measured with a time-resolved fluorescence based assay, cell viability with a tetrazolium assay, and cell membrane integrity with a LDH assay. Ethanol (1-5%) induced NF-kappaB activation, reaching a maximum after 3 h, and also led to moderately increased COX-2 expression. Heat shock pretreatment and the intracellular calcium chelator BAPTA were able to inhibit ethanol-induced NF-kappaB activation. Heat shock pretreatment decreased ethanol-induced caspase-3 activation, decreased cell membrane damage, and retained cellular viability. Inhibition of NF-kappaB activation by NEMO-binding peptide, by decreasing RelA expression, or by inhibiting COX-2 activity by CAY-14040 promoted the effects of ethanol, such as increased caspase-3 activity and decreased cell viability. In conclusion, ethanol induces NF-kappaB activation via a calcium-dependent pathway and induces COX-2 expression. Inhibition of the NF-kappaB activation or COX-2 activity potentiates apoptosis and cell damage induced by ethanol, suggesting a protective role for NF-kappaB activation and COX-2 expression.     

Treatment of human colon carcinoma cell lines with anti-neoplastic agents enhances their lytic sensitivity to antigen-specific CD8+ cytotoxic T lymphocytes

Certain anti-neoplastic agents at subtoxic doses may exert immunomodulatory effects, which alter the expression of specific tumor cell surface molecules. We reasoned that potential increases in tumor cell surface markers, such as those important for facilitating effector-target contact, as well as triggering cell death pathways, might then improve antigen (Ag)-specific T-cell-mediated tumor cytolysis. Here, in a human colon carcinoma cell model in vitro, we examined whether the anti-neoplastic agents 5-fluorouracil (5-FU), CPT-11 or cisplatin (CDDP) could upregulate the expression of specific tumor cell surface markers, which may then enhance productive lytic interactions between CD8+ CTL and Ag-bearing tumor cells. Based on our earlier studies, IFN-gamma treatment was included as a control for sensitization to CTL-mediated lysis. Pretreatment of the SW480 primary colon carcinoma cell line with IFN-gamma, 5-FU, CPT-11 or CDDP enhanced ICAM-1 and Fas expression, resulting in Ag-specific CTL-mediated lysis involving Fas-dependent and -independent mechanisms. In contrast, pretreatment of the SW620 metastatic isolate, derived from the same patient, with IFN-gamma, CPT-11 or CDDP, but not 5-FU, enhanced ICAM-1 expression, resulting in Ag-specific CTL-mediated lysis via Fas-independent mechanisms only. Flow cytometric-based assays were then developed to measure the effects of drug treatment on caspase signaling and apoptosis incurred by tumor targets after interaction with CTL. We found that the lytic enhancement caused by drug treatment of SW480 or SW620 targets was accompanied by an increase in caspase-3-like protease activity. A peptide-based caspase inhibitor abrogated CTL-mediated apoptosis, suggesting that "chemomodulation" involved regulation of the caspase pathway. These results revealed for the first time an important role for components of the caspase pathway, such as caspase-3-like proteases, in the sensitization of human colon carcinoma cells by anti-neoplastic agents to Ag-specific CTL. Thus, certain anti-neoplastic agents may display unique immunoregulatory properties that facilitate human colon carcinoma death by engaging the lytic capacity of Ag-specific CTL, which may have implications for chemoimmunotherapy strategies.     

A20 inhibits oxidized low-density lipoprotein-induced apoptosis through negative Fas/Fas ligand-dependent activation of caspase-8 and mitochondrial pathways in murine RAW264.7 macrophages

A20 was originally characterized as a TNF-inducible gene in human umbilical vein endothelial cells. As an NF-kappaB target gene, A20 is also induced in many other cell types by a wide range of stimuli. Expression of A20 has been shown to protect from TNF-induced apoptosis and also functions via a negative-feedback loop to block NF-kappaB activation induced by TNF and other stimuli. To date, there are no reports on whether A20 can protect OxLDL-induced apoptosis in macrophages. For the first time we report that A20 expression blocks OxLDL-mediated cell toxicity and apoptosis. OxLDL induced the expression of Fas and FasL, and the subsequent caspase-8 cleavage and treatment with a neutralizing ZB4 anti-Fas antibody blocked apoptosis induced by OxLDL. Expression of dominant negative FADD efficiently prevented OxLDL-induced apoptosis and caspase-8 activation. A20 expression significantly attenuated the increased expression of Fas and FasL, and Fas-mediated apoptosis. These findings suggest that A20-mediated protection from OxLDL may occur at the level of Fas/FADD-caspase-8 and be FasL dependent. Treatment of RAW264.7 cells with OxLDL induces a series of time-dependent events, including the release of cytochrome c, Smac and Omi from the mitochondria to the cytosol, activation of caspase-9, -6, -2, and -3, which are blocked by A20 expression. No cleaved form of Bid was detected, even treatment with OxLDL for 48 h. Expression of dominant negative FADD also efficiently prevented OxLDL-induced the above apoptotic events. The release of cyto c, Smac and Omi from mitochondria to cytosol, activated by OxLDL treatment, and the activation of caspase-9 may not be a downstream event of caspase-8-mediated Bid cleavage. Therefore, the protective effect of A20 on mitochondrial apoptotic pathway activated by OxLDL may be dependent on FADD. A20 expression reversed OxLDL-mediated G(0)/G(1) stage arrest by maintaining the expression of cyclin B1, cyclin D1, and cyclin E, and p21 and p73. Thus, A20 expression blocks OxLDL-mediated apoptosis in murine RAW264.7 macrophages through disrupting Fas/FasL-dependent activation of caspase-8 and the mitochondria pathway.     

Suppression of tumor necrosis factor-mediated apoptosis by nuclear factor kappaB-independent bone morphogenetic protein/Smad signaling

The activation of nuclear factor kappaB (NF-kappa B) plays a pivotal role in the regulation of tumor necrosis factor (TNF)-mediated apoptosis. However, little is known about the regulation of TNF-mediated apoptosis by other signaling pathways or growth factors. Here, unexpectedly, we found that bone morphogenetic protein (BMP)-2 and BMP-4 inhibited TNF-mediated apoptosis by inhibition of caspase-8 activation in C2C12 cells, a pluripotent mesenchymal cell line that has the potential to differentiate into osteoblasts depending on BMP stimulation. Utilizing both a trans-dominant IkappaBalpha inhibitor of NF-kappaB expressed in C2C12 cells and IkappaB kinase beta-deficient embryonic mouse fibroblast, we show that BMP-mediated survival was independent of NF-kappaB activation. Rather, the antiapoptotic activity of BMPs functioned through the Smad signaling pathway. Thus, these findings provide the first report of a BMP/Smad signaling pathway that can inhibit TNF-mediated apoptosis, independent of the prosurvival activity of NF-kappaB. Our results suggest that BMPs not only stimulate osteoblast differentiation but can also promote cell survival during the induction of bone formation, offering new insight into the biological functions of BMPs.     

The novel triterpenoid 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO) potently enhances apoptosis induced by tumor necrosis factor in human leukemia cells

Tumor necrosis factor (TNF) is a potent activator of the nuclear factor-kappaB (NF-kappaB) pathway that leads to up-regulation of anti-apoptotic proteins. Hence, TNF induces apoptosis in the presence of inhibitors of protein or RNA synthesis. We report that a novel triterpenoid, 2-cyano-3,12-dioxooleana-1,9,-dien-28-oic acid (CDDO) inhibits NF-kappaB-mediated gene expression at a step after translocation of activated NF-kappaB to the nucleus. This effect appears specific for the NF-kappaB pathway as CDDO does not inhibit gene expression induced by the phorbol ester 12-0-tetradecanoylphorbol-13-acetate (TPA). CDDO in combination with TNF caused a dramatic increase in apoptosis in ML-1 leukemia cells that was associated with activation of caspase-8, cleavage of Bid, translocation of Bax, cytochrome c release, and caspase-3 activation. Experiments with caspase inhibitors demonstrated that caspase-8 was an initiator of this pathway. TNF also induced a transient activation of c-Jun N-terminal kinase (JNK), which upon addition of CDDO was converted to a sustained activation. The activation of JNK was also dependent on caspase-8. Sustained activation of JNK is frequently pro-apoptotic, yet inhibition of JNK did not prevent Bax translocation or cytochrome c release, demonstrating its lack of involvement in CDDO/TNF-induced apoptosis. Apoptosis was acutely induced by CDDO/TNF in every leukemia cell line tested including those that overexpress Bcl-x(L), suggesting that the mitochondrial pathway is not required for apoptosis by this combination. These results suggest that the apoptotic potency of the CDDO/TNF combination occurs through selective inhibition of NF-kappaB-dependent anti-apoptotic proteins, bypassing potential mitochondrial resistance mechanisms, and thus may provide a basis for the development of novel approaches to the treatment of leukemia.     

Analysis of Ras-dependent signals that prevent caspase-3 activation and apoptosis induced by cytokine deprivation in hematopoietic cells

In hematopoietic cells, Ras has been implicated in signaling pathways that prevent apoptosis triggered by deprivation of cytokines, such as interleukin-3 (IL-3). However, the mechanism whereby Ras suppresses cell death remains incompletely understood. We have investigated the role of Ras in IL-3 signal transduction by using the cytokine-dependent BaF3 cell line. Herein, we show that the activation of the pro-apoptotic protease caspase-3 upon IL-3 removal is suppressed by expression of activated Ras, which eventually prevents cell death. For caspase-3 suppression, the Raf/extracellular signal-regulated kinase (ERK)- or phosphatidylinositol 3-kinase (PI3-K)/Akt-mediated signaling pathway downstream of Ras was required. However, inhibition of both pathways did not block activated Ras-dependent suppression of cell death-associated phenotypes, such as nuclear DNA fragmentation. Thus, a pathway that is independent of both Raf/ERK and PI3-K/Akt pathways may function downstream of Ras, preventing activated caspase-3-initiated apoptotic processes. Conditional activation of c-Raf-1 also suppressed caspase-3 activation and subsequent cell death without affecting Akt activity, providing further evidence for a PI3-K/Akt-independent mechanism.     

X-linked inhibitor of apoptosis (XIAP) inhibits c-Jun N-terminal kinase 1 (JNK1) activation by transforming growth factor beta1 (TGF-beta1) through ubiquitin-mediated proteosomal degradation of the TGF-beta1-activated kinase 1 (TAK1)

Active NF-kappaB renders malignant hepatocytes refractory to the growth inhibitory and pro-apoptotic properties of transforming growth factorbeta1 (TGF-beta1). NF-kappaB counteracts TGF-beta1-induced apoptosis through up-regulation of downstream target genes, such as XIAP and Bcl-X(L), which in turn inhibit the intrinsic pathway of apoptosis. In addition, induction of NF-kappaB by TGF-beta1 inhibits JNK signaling, thereby attenuating TGF-beta1-induced cell death of normal hepatocytes. However, the mechanism involved in the negative cross-talk between the NF-kappaB and JNK pathways during TGF-beta1 signaling has not been determined. In this study, we have identified the XIAP gene as one of the critical mediators of NF-kappaB-mediated suppression of JNK signaling. We show that NF-kappaB plays a role in the up-regulation of XIAP gene expression in response to TGF-beta1 treatment and forms a TGF-beta1-inducible complex with TAK1. Furthermore, we show that the RING domain of XIAP mediates TAK1 polyubiquitination, which then targets this molecule for proteosomal degradation. Down-regulation of TAK1 protein expression inhibits TGF-beta1-mediated activation of JNK and apoptosis. Conversely, silencing of XIAP promotes persistent JNK activation and potentiates TGF-beta1-induced apoptosis. Collectively, our findings identify a novel mechanism for the regulation of JNK activity by NF-kappaB during TGF-beta1 signaling and raise the possibility that pharmacologic inhibition of the NF-kappaB/XIAP signaling pathway might selectively abolish the pro-oncogenic activity of TGF-beta1 in advanced hepatocellular carcinomas (HCCs) without affecting the pro-apoptotic effects of TGF-beta1 involved in normal liver homeostasis.     

c-Myc sensitizes cells to tumor necrosis factor-mediated apoptosis by inhibiting nuclear factor kappa B transactivation

Nuclear factor kappaB (NF-kappaB) plays a key role in suppression of tumor necrosis factor (TNF)-mediated apoptosis by inducing a variety of anti-apoptotic genes. Expression of c-Myc has been shown to sensitize cells to TNF-mediated apoptosis by inhibiting NF-kappaB activation. However, the precise step in the NF-kappaB signaling pathway and apoptosis modified by c-Myc has not been identified. Using the inducible c-MycER system and c-Myc null fibroblasts, we found that expression of c-Myc inhibited NF-kappaB activation by interfering with RelA/p65 transactivation but not nuclear translocation of NF-kappaB. Activation of c-Myc promoted TNF-induced release of cytochrome c from mitochondria to the cytosol because of the inhibition of NF-kappaB. Furthermore, we found that NF-kappaB-inducible gene A1 was attenuated by expression of c-Myc and that the restoration of A1 expression suppressed c-Myc-induced TNF sensitization. Our results elucidate the molecular mechanisms by which c-Myc increases cell susceptibility to TNF-mediated apoptosis, indicating that c-Myc may exhibit its pro-apoptotic activities by repression of cell survival genes.