Sensitization of TNF-induced cytotoxicity in lung cancer cells by concurrent suppression of the NF-kappaB and Akt pathways

Blockage of either nuclear factor-kappaB (NF-kappaB) or Akt sensitizes cancer cells to TNF-induced apoptosis. In this study, we investigated the undetermined effect of concurrent blockage of these two survival pathways on TNF-induced cytotoxicity in lung cancer cells. The results show that Akt contributes to TNF-induced NF-kappaB activation in lung cancer cells through regulating phosphorylation of the p65/RelA subunit of NF-kappaB. Although individually blocking IKK or Akt partially suppressed TNF-induced NF-kappaB activation, concurrent suppression of these pathways completely inhibited TNF-induced NF-kappaB activation and downstream anti-apoptotic gene expression, and synergistically potentiated TNF-induced cytotoxicity. Moreover, suppression of Akt inhibited the Akt-mediated anti-apoptotic pathway through dephosphorylation of BAD. These results indicate that concurrent suppression of NF-kappaB and Akt synergistically sensitizes TNF-induced cytotoxicity through blockage of distinct survival pathways downstream of NF-kappaB and Akt, which may be applied in lung cancer therapy.     

Nitric oxide disrupts H2O2-dependent activation of nuclear factor kappa B. Role in sensitization of human tumor cells to tumor necrosis factor-alpha -induced cytotoxicity

Tumor necrosis factor alpha (TNF-alpha) exerts its effect by two distinct signaling pathways. It can trigger cytotoxicity in sensitive target cells. TNF-alpha can also promote nuclear factor kappaB (NF-kappaB) activity and regulate the expression of genes that interfere with apoptosis and thus conferring resistance to several apoptotic stimuli. We have observed that interferon-gamma (IFN-gamma) sensitizes human ovarian carcinoma cell lines to TNF-alpha-mediated apoptosis and further, IFN-gamma induces the expression of the inducible nitric-oxide synthase (iNOS) and the generation of nitric oxide (NO). This study examines the role of NO in the sensitization of the ovarian carcinoma cell line AD10 to TNF-alpha-mediated cytotoxicity. Treatment of AD10 cells with the NOS inhibitor l-NMA blocked the IFN-gamma-dependent sensitization whereas NO donors (S-nitroso-N-acetylpenicillamine) sensitized these cells to TNF-alpha cytotoxicity. Analysis of the activation status of NF-kappaB upon treatment with NO donors confirmed the inhibitory role of NO on both the NF-kappaB DNA-binding property and its activation. Moreover, the inhibition of NF-kappaB nuclear translocation by NO donors directly correlated with the intracellular concentration of H(2)O(2) and was reversed by the addition of exogenous H(2)O(2). These findings show that NO might interfere with TNF-alpha-dependent NF-kappaB activation by interacting with O(2) and reducing the generation of H(2)O(2), a potent NF-kappaB activator. Therefore, NO-mediated disruption of NF-kappaB activation results in the removal of anti-apoptotic/resistance signals and sensitizes tumor cells to cytotoxic cytokines like TNF-alpha.     

Tumor necrosis factor and reactive oxygen species cooperative cytotoxicity is mediated via inhibition of NF-kappaB

BACKGROUND: Tumor necrosis factor alpha (TNFalpha) plays a key role in pathogenesis of brain injury. However, TNFalpha exhibits no cytotoxicity in primary cultures of brain cells. This discrepancy suggests that other pathogenic stimuli that exist in the setting of brain injury precipitate TNFalpha cytotoxicity. The hypothesis was tested that reactive oxygen species (ROS), that are released early after brain injury, act synergistically with TNFalpha in causing cell death. MATERIALS AND METHODS: Cultured human and rat brain capillary endothelial cells (RBEC), and cortical astrocytes were treated with TNFalpha alone or together with different doses of H2O2, and apoptotic cell death and DNA fragmentation were measured by means of 3'-OH-terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) and Hoechst fluorescence assay, respectively. The effect of H2O2 on TNFalpha-induced activation of nuclear factor kappa B (NF-kappaB) was measured by Western blots of cytoplasmic and nuclear extracts of RBEC using anti-inhibitor of NF-kappaB (IkappaB) and anti-p65 subunit of NF-kappaB antibodies. Nuclear translocation of NF-kappaB was investigated by immunofluorescent staining of RBEC with anti-p65 antibodies. RESULTS: TNFalpha alone had no cytotoxic effect in brain endothelial cells and astrocytes at concentrations up to 100 ng/ml. Co-treatment with 5-10 microM of H2O2 caused a two-fold increase in the number of apoptotic cells 24 hr later. Similar doses (1-3 microM) of H2O2 initiated early DNA fragmentation. H2O2 inhibited TNFalpha-induced accumulation of p65 in the nucleus, although it had no effect on degradation of the IkappaB in cytoplasm. Immunostaining confirmed that H2O2 inhibited p65 transport to the nucleus. CONCLUSIONS: Reactive oxygen species could act synergistically with TNFalpha in causing cytotoxicity via inhibition of a cytoprotective branch of TNFalpha signaling pathways, which starts with NF-kappaB activation.     

Modulatory effects of ozone on THP-1 cells in response to SP-A stimulation

Ozone (O(3)), a major component of air pollution and a strong oxidizing agent, can lead to lung injury associated with edema, inflammation, and epithelial cell damage. The effects of O(3) on pulmonary immune cells have been studied in various in vivo and in vitro systems. We have shown previously that O(3) exposure of surfactant protein (SP)-A decreases its ability to modulate proinflammatory cytokine production by cells of monocyte/macrophage lineage (THP-1 cells). In this report, we exposed THP-1 cells and/or native SP-A obtained from bronchoalveolar lavage of patients with alveolar proteinosis to O(3) and studied cytokine production and NF-kappaB signaling. The results showed 1) exposure of THP-1 cells to O(3) significantly decreased their ability to express TNF-alpha in response to SP-A; TNF-alpha production, under these conditions, was still significantly higher than basal (unstimulated) levels in filtered air-exposed THP-1 cells; 2) exposure of both THP-1 cells and SP-A to O(3) did not result in any significant differences in TNF-alpha expression compared with basal levels; 3) O(3) exposure of SP-A resulted in a decreased ability of SP-A to activate the NF-kappaB pathway, as assessed by the lack of significant increase and decrease of the nuclear p65 subunit of NF-kappaB and cytoplasmic IkappaBalpha, respectively; and 4) O(3) exposure of THP-1 cells resulted in a decrease in SP-A-mediated THP-1 cell responsiveness, which did not seem to be mediated via the classic NF-kappaB pathway. These findings indicate that O(3) exposure may mediate its effect on macrophage function both directly and indirectly (via SP-A oxidation) and by involving different mechanisms.     

Nickel compounds render anti-apoptotic effect to human bronchial epithelial Beas-2B cells by induction of cyclooxygenase-2 through an IKKbeta/p65-dependent and IKKalpha- and p50-independent pathway

The carcinogenicity of nickel compounds has been well documented both in vitro and in vivo; however, the molecular mechanisms by which nickel compounds cause cancers are far from understood. Because suppression of apoptosis is thought to contribute to carcinogenesis, we investigated the mechanisms implicated in nickel-induced anti-apoptotic effect in human bronchial epithelial (Beas-2B) cells. We found that exposure of Beas-2B cells to nickel compounds resulted in increased cyclooxygenase-2 (COX-2) expression and that small interfering RNA (siCOX-2) knockdown of COX-2 expression resulted in increased cell sensitivity to nickel-triggered cell apoptosis, demonstrating that COX-2 induction has an anti-apoptotic effect on Beas-2B cells. Overexpression of IKKbeta-KM, a kinase inactive mutant of IKKbeta, blocked NF-kappaB activation and COX-2 induction by nickel compounds, indicating that activated NF-kappaB may be a mediator for COX-2 induction. To further explore the contribution of the NF-kappaB pathway in COX-2 induction and in protection from nickel exposure, mouse embryonic fibroblasts deficient in IKKbeta, IKKalpha, p65, and p50 were analyzed. Loss of IKKbeta impaired COX-2 induction by nickel exposure, whereas knockout of IKKalpha had a marginal effect. Moreover, the NF-kappaB p65, and not the p50 subunit, was critical for nickel-induced COX-2 expression. In addition, a deficiency of IKKbeta or p65 rendered cells more sensitive to nickel-induced apoptosis as compared with those in wild type cells. Finally, it was shown that reactive oxygen species H(2)O(2) were involved in both NF-kappaB activation and COX-2 expression. Collectively, our results demonstrate that COX-2 induction by nickel compounds occurs via an IKKbeta/p65 NF-kappaB-dependent but IKKalpha- and p50-independent pathway and plays a crucial role in antagonizing nickel-induced cell apoptosis in Beas-2B cells.     

Induction of apoptosis and NF-kappaB by quercetin in growing murine L1210 lymphocytic leukaemic cells potentiated by TNF-alpha

Polyphenol quercetin induced apoptosis in proliferating murine L1210 lymphocytic cells. DNA damage, as well as apoptosis and withdrawal from the cell cycle were transient. The above mentioned death promoting activity of quercetin was enhanced by physiological concentrations of TNF-alpha. At the same time, indices of cell viability dropped. However, the extent and tendency of the initially enhanced cell mortality steadily diminished throughout the experiment. After 12 h the G2/M phase reappeared. After 24 h all indices almost returned to control levels indicating either the selection of subpopulation of unaffected leukaemic cells or cells developing resistance to the treatment. A DNA ladder of oligonucleosomes was observed for apoptogenic treatments. We conclude that quercetin unmasked cell death, promoting the activity of TNF-alpha. However, after 12 and 24 h of exposure, surviving cells could complete the cell cycle and finally recover. At the same time, increased NF-kappaB activation was demonstrated by immunoblotting of the immunoreactive RelA/p65 subunit in nuclear extracts. Exposure to TNF-alpha or quercetin was crucial for increased activity of NF-kappaB, which may implicate an increasing resistance to their cytotoxicity.     

Bcl-2 overexpression protects photooxidative stress-induced apoptosis of photoreceptor cells via NF-kappaB preservation

We recently showed that photooxidative stress on cultured photoreceptor cells results in down-modulation of NF-kappaB activity which then leads to apoptosis of cultured 661W photoreceptor cells. In an effort to further delineate the mechanism of photoreceptor cell death, we sought to determine the effects of Bcl-2 overexpression on cell survivability. Wild-type 661W cells were transfected with the plasmid construct pSFFV-neo-Bcl-2 and several clones were isolated. All clones demonstrated increased Bcl-2 mRNA and protein levels, with the B4 clone exhibiting the greatest enhancement. On exposure to visible light the B4 cells were protected from undergoing apoptosis when compared with the mock transfected cells, as ascertained by TUNEL apoptosis assay and formazan based estimation of cell viability. The Bcl-2 overexpressing cells also maintained a higher Bcl-2/Bax ratio, suggesting that this ratio is important in protection from photooxidative stress. Electrophoretic mobility shift assays for NF-kappaB demonstrated higher activity in both nuclear and cytosolic fractions of the B4 photoreceptors compared with the 661W wild-type cells at all light exposure time points. Furthermore, the findings of the gel shift assays were further supported by immunocytochemistry for NF-kappaB which revealed that protein levels of the RelA subunit of NF-kappaB were protected in the nucleus as well as in the cytoplasm of Bcl-2 overexpressing B4 cells exposed to light compared to the 661W cells. These results suggest that Bcl-2 overexpression protects NF-kappaB protein levels and activity in the nucleus, indicating that preservation of NF-kappaB binding activity in the nucleus may be essential for photoreceptor cells to survive photooxidative damage induced apoptosis.     

Arsenic trioxide represses constitutive activation of NF-kappaB and COX-2 expression in human acute myeloid leukemia, HL-60

It has been proposed that eukaryotic nuclear factor nuclear factor kappa-B (NF-kappaB) and cyclooxygenase-2 (COX-2) are implicated in the pathogenesis of many human diseases including cancer. Arsenic has been widely used in medicine in Oriental countries. Recent studies have shown that arsenic trioxide (As(2)O(3)) could induce in vitro growth inhibition and apoptosis of malignant lymphocytes, and myeloma cells. However, the molecular mechanisms by which As(2)O(3) initiates cellular signaling toward cell death are still unclear. In the present study, the effects of As(2)O(3) on NF-kappaB and COX-2 expression in HL-60 cells were investigated. As(2)O(3) suppressed DNA-binding activity of NF-kappaB composed of p65/p50 heterodimer through preventing the degradation of IkappaB-alpha and the nuclear translocation of p65 subsequently as well as interrupting the binding of NF-kappaB with their consensus sequences. Inhibitory effect of As(2)O(3) on NF-kappaB DNA activity was dependent upon intracellular glutathione (GSH) and H(2)O(2) level, but not superoxide anion. Futhermore, we found that As(2)O(3) also downregulated the expression of COX-2, which has NF-kappaB binding site on its promoter through repressing the NF-kappaB DNA-binding activity.