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.     

Aspirin prevents apoptosis and NF-kappaB activation induced by H2O2 in hela cells

The classical pathway of nuclear factor-kappa B (NF-kappaB) activation by several inducers mainly involves the phosphorylation of IkappaBalpha by a signalsome complex composed of IkappaBalpha kinases (IKKalpha and IKKbeta). However, in some cell types hydrogen peroxide (H2O2) has been shown to activate an alternative pathway that does not involve the classical signalsome activation process. In this study, we demonstrate that H2O2 induced NF-kappaB activation in HeLa cells through phosphorylation and degradation of IkappaB proteins as shown by immunblot analysis. Our studies reveal that a commonly used non-steroid anti-inflammatory drug, acetylsalicylic acid (aspirin) prevents H2O2-induced NF-kappaB activation in a dose-dependent manner through inhibition of phosphorylation and degradation of IkappaBalpha and IkappaBbeta. Differential staining and DNA fragmentation analysis also show that aspirin preloading of HeLa cells also prevents H2O2-induced apoptosis in a dose-dependent manner with maximum efficiency at 10 mM concentration. Additionally, aspirin effectively prevents caspase-3 and caspase-9 (cysteinyl aspartate-specific proteases) activation by H2O2. These results suggest that NF-kappaB activation is involved in H2O2-induced apoptosis and aspirin may inhibit both processes simultaneously.     

Suppression of nuclear factor-kappa B activity by nitric oxide and hyperoxia in oxygen-resistant cells

Inhaled nitric oxide (iNO) is used clinically to treat pulmonary hypertension in newborns, often in conjunction with hyperoxia (NO/O2). Prolonged exposure to NO/O2 causes synergistic lung injury and death of lung epithelial cells. To explore the mechanisms involved, oxygen-resistant HeLa-80 cells were exposed to NO +/- O2. Exposure to NO and O2 induced a synergistic cytotoxicity, accompanied with apoptotic characteristics, including elevated caspase-3-like activity, Annexin V incorporation, and nuclear condensation. This apoptosis was associated with a synergistic suppression of NF-kappaB activity. Cells lacking functional NF-kappaB p65 subunit were more sensitive to NO/O2 than their wild type counterparts. This injury was partially rescued by transfection with a p65 expression construct, suggesting an inverse relationship between NF-kappaB and susceptibility to the cytotoxicity of NO/O2. Despite the reduced NF-kappaB activity in cells exposed to NO +/- O2, IkappaBalpha was degraded, suggesting that pathways regulating the steady-state levels of IkappaB were not involved. However, exposure to NO/O2 caused a marked reduction in nuclear localization and an increase in protein carbonyl formation of NF-kappaB p65 subunit. These results suggest that NO/O2-induced apoptosis occurs by suppressing NF-kappaB activity.     

Dysfunction of regulatory volume increase is a key component of apoptosis

Sustained cell shrinkage is a major hallmark of apoptotic cell death. In apoptotic cells, whole cell volume reduction, called apoptotic volume decrease (AVD), proceeds until fragmentation of cells. Under non-apoptotic conditions, human epithelial HeLa cells exhibited a slow regulatory volume increase (RVI) after osmotic shrinkage induced by exposure to hypertonic solution. When AVD was induced by treatment with a Fas ligand, TNF-alpha or staurosporine, however, it was found that HeLa cells failed to undergo RVI. When RVI was inhibited by combined application of Na+/H+ exchanger (NHE) and anion exchanger blockers, hypertonic stress induced prolonged shrinkage followed by caspase-3 activation in HeLa cells. Hypertonicity also induced apoptosis in NHE1-deficient PS120 fibroblasts, which lack the RVI response. When RVI was restored by transfection of these cells with NHE1, hypertonicity-induced apoptosis was completely prevented. Thus, it is concluded that RVI dysfunction is indispensable for the persistence of AVD and induction of apoptosis.     

TNF-alpha-induced sphingosine 1-phosphate inhibits apoptosis through a phosphatidylinositol 3-kinase/Akt pathway in human hepatocytes.

Human hepatocytes usually are resistant to TNF-alpha cytotoxicity. In mouse or rat hepatocytes, repression of NF-kappaB activation is sufficient to induce TNF-alpha-mediated apoptosis. However, in both Huh-7 human hepatoma cells and Hc human normal hepatocytes, when infected with an adenovirus expressing a mutated form of IkappaBalpha (Ad5IkappaB), which almost completely blocks NF-kappaB activation, >80% of the cells survived 24 h after TNF-alpha stimulation. Here, we report that TNF-alpha activates other antiapoptotic factors, such as sphingosine kinase (SphK), phosphatidylinositol 3-kinase (PI3K), and Akt kinase. Pretreatment of cells with N,N-dimethylsphingosine (DMS), an inhibitor of SphK, or LY 294002, an inhibitor of PI3K that acts upstream of Akt, increased the number of apoptotic cells induced by TNF-alpha in Ad5IkappaB-infected Huh-7 and Hc cells. TNF-alpha-induced activations of PI3K and Akt were inhibited by DMS. In contrast, exogenous sphingosine 1-phosphate, a product of SphK, was found to activate Akt and partially rescued the cells from TNF-alpha-induced apoptosis. Although Akt has been reported to activate NF-kappaB, DMS and LY 294002 failed to prevent TNF-alpha-induced NF-kappaB activation, suggesting that the antiapoptotic effects of SphK and Akt are independent of NF-kappaB. Furthermore, apoptosis mediated by Fas ligand (FasL) involving Akt activation also was potentiated by DMS pretreatment in Hc cells. Sphingosine 1-phosphate administration partially protected cells from FasL-mediated apoptosis. These results indicate that not only NF-kappaB but also SphK and PI3K/Akt are involved in the signaling pathway(s) for protection of human hepatocytes from the apoptotic action of TNF-alpha and probably FasL.     

A quantitative study of NF-kappaB activation by H2O2: relevance in inflammation and synergy with TNF-alpha

Although the germicide role of H(2)O(2) released during inflammation is well established, a hypothetical regulatory function, either promoting or inhibiting inflammation, is still controversial. In particular, after 15 years of highly contradictory results it remains uncertain whether H(2)O(2) by itself activates NF-kappaB or if it stimulates or inhibits the activation of NF-kappaB by proinflammatory mediators. We investigated the role of H(2)O(2) in NF-kappaB activation using, for the first time, a calibrated and controlled method of H(2)O(2) delivery--the steady-state titration--in which cells are exposed to constant, low, and known concentrations of H(2)O(2). This technique contrasts with previously applied techniques, which disrupt cellular redox homeostasis and/or introduce uncertainties in the actual H(2)O(2) concentration to which cells are exposed. In both MCF-7 and HeLa cells, H(2)O(2) at extracellular concentrations up to 25 microM did not induce significantly per se NF-kappaB translocation to the nucleus, but it stimulated the translocation induced by TNF-alpha. For higher H(2)O(2) doses this stimulatory role shifts to an inhibition, which may explain published contradictory results. The stimulatory role was confirmed by the observation that 12.5 microM H(2)O(2), a concentration found during inflammation, increased the expression of several proinflammatory NF-kappaB-dependent genes induced by TNF-alpha (e.g., IL-8, MCP-1, TLR2, and TNF-alpha). The same low H(2)O(2) concentration also induced the anti-inflammatory gene coding for heme oxygenase-1 (HO-1) and IL-6. We propose that H(2)O(2) has a fine-tuning regulatory role, comprising both a proinflammatory control loop that increases pathogen removal and an anti-inflammatory control loop, which avoids an exacerbated harmful inflammatory response.     

Caspase cleavage product lacking amino-terminus of IkappaBalpha sensitizes resistant cells to TNF-alpha and TRAIL-induced apoptosis

In response to a diverse array of signals, IkappaBalpha is targeted for phosphorylation-dependent degradation by the proteasome, thereby activating NF-kappaB. Here we demonstrate a role of the cleavage product of IkappaBalpha in various death signals. During apoptosis of NIH3T3, Jurkat, Rat-1, and L929 cells exposed to tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), Fas, serum deprivation, or TNF-alpha, respectively, IkappaBalpha was cleaved in a caspase-dependent manner. In vitro and in vivo cleavage assays and site-directed mutagenesis showed that caspase-3 cleaved IkappaBalpha between Asp31 and Ser32. Expression of the cleavage product lacking amino-terminus (1-31), DeltaIkappaBalpha, sensitized otherwise resistant NIH3T3 fibroblast cells to apoptosis induced by TNF-alpha or TRAIL, and HeLa tumor cells to TNF-alpha. DeltaIkappaBalpha was more pro-apoptotic compared to wild type or cleavage-resistant (D31E)IkappaBalpha mutant and the sensitization elicited by DeltaIkappaBalpha was as effective as that by the dominant negative mutant, (S32,36A)IkappaBalpha, in NIH3T3 cells. DeltaIkappaBalpha suppressed the transactivation of NF-kappaB induced by TNF-alpha or TRAIL, as reflected by luciferase-reporter activity. Conversely, expression of the p65 subunit of NF-kappaB suppressed TNF-alpha-, TRAIL-, and serum deprivation-induced cell death. On the contrary, DeltaIkappaBalpha was less effective at increasing the death rate of HeLa cells that were already sensitive to death signals including TRAIL, etoposide, or taxol. These results suggest that DeltaIkappaBalpha generated by various death signals sensitizes cells to apoptosis by suppressing NF-kappaB activity.     

Selective sensitization to tumor necrosis factor-alpha-induced apoptosis by blockade of NF-kappaB in primary glomerular mesangial cells.

Recent data have implicated nuclear factor kappaB (NF-kappaB) in the prevention of apoptosis in transformed cell lines exposed to tumor necrosis factor alpha (TNF-alpha). However, it is obscure whether NF-kappaB plays an anti-apoptotic role in nontransformed cells, and it is not clear whether NF-kappaB inhibits apoptosis triggered by other mediators. We investigated the effect of specific inhibition of NF-kappaB on cytokine-induced apoptosis of glomerular mesangial cells, which is important in determining the outcome of glomerulonephritis. Cultured rat mesangial cells were stably transfected with the dominant negative mutant inhibitor of NF-kappaB (IkappaBalphaM). IkappaBalphaM was resistant to stimulus-dependent degradation and suppressed NF-kappaB activation induced by TNF-alpha (10 ng/ml) or IL-1beta (10 ng/ml). IkappaBalphaM significantly sensitized mesangial cells to TNF-alpha-induced apoptosis in a dose- and time-dependent manner but had no significant effects on the level of apoptosis in the presence of proinflammatory or apoptosis-inducing stimuli including Fas ligand, IL-1alpha, IL-1beta, hydrogen peroxide, lipopolysaccharide, cycloheximide, or serum deprivation. Moreover, IkappaBalphaM-mediated sensitization to TNF-alpha overcame the protective effect of mesangial cell survival factors present in serum, which usually inhibit killing of mesangial cells by the proapoptotic stimuli used. These data show that inhibition of NF-kappaB selectively sensitizes primary adult glomerular mesangial cells to TNF-induced apoptosis but not to other mediators of cell death including the Fas ligand.     

Combination of tumor necrosis factor alpha and interferon alpha induces apoptotic cell death through a c-myc-dependent pathway in p53 mutant H226br non-small-cell lung cancer cell line.

We investigated the role of wild-type p53 and c-myc activity in apoptosis induced by a combination of natural human tumor necrosis factor alpha (TNF-alpha) and natural human interferon alpha (IFN-alpha). Studies were performed with two human non-small-cell lung cancer cell lines, H226b, which has wild-type p53, and H226br, which has a mutant p53. The combination of IFN-alpha and TNF-alpha significantly inhibited cell growth and induced apoptotic cell death of both H226b and H226br, compared with IFN-alpha or TNF-alpha alone. Treatment with one or both cytokines did not affect the expression level of p53 in both cell lines. These results suggest that the combination of IFN-alpha/TNF-alpha induces apoptotic cell death through a p53- independent pathway. The c-myc oncogene is known to be involved in apoptosis induced by TNF. Antisense c-myc oligonucleotides have been reported to modulate cell growth or apoptosis in several cell lines. Antisense oligodeoxynucleotides were added to the culture of H226br cells before the addition of IFN-alpha/TNF-alpha. Antisense c-myc inhibited IFN-alpha/TNF-alpha cytotoxicity and apoptotic cell death. In conclusion, this study provides support for the speculation that TNF-alpha/IFN-alpha induce apoptosis through a c-myc-dependent pathway rather than a p53-dependent pathway. (c)2001 Elsevier Science.     

Small interfering RNA-mediated silencing of mitochondrial NADP+-dependent isocitrate dehydrogenase enhances the sensitivity of HeLa cells toward tumor necrosis factor-alpha and anticancer drugs

Tumor necrosis factor-alpha (TNF-alpha) and several anticancer drugs induce the production of reactive oxygen species, which play an important causative role in apoptotic cell death. Recently, we demonstrated that the control of mitochondrial redox balance and the cellular defense against oxidative damage is one of the primary functions of mitochondrial NADP(+)-dependent isocitrate dehydrogenase (IDPm) by supplying NADPH for antioxidant systems. In the present report, we show that silencing of IDPm expression in HeLa cells greatly enhances apoptosis induced by TNF-alpha and anticancer drugs. Transfection of HeLa cells with an IDPm small interfering RNA (siRNA) markedly decreased activity of IDPm, enhancing the susceptibility of anticancer agent-induced apoptosis reflected by morphological evidence of apoptosis, DNA fragmentation, cellular redox status, mitochondria redox status and function, and the modulation of apoptotic marker proteins. These results indicate that IDPm may play an important role in regulating the apoptosis induced by TNF-alpha and anticancer drugs and the sensitizing effect of IDPm siRNA on the apoptotic cell death of HeLa cells offers the possibility of developing a modifier of cancer chemotherapy.     

Protein kinase PKR plays a stimulus- and virus-dependent role in apoptotic death and virus multiplication in human cells

The protein kinase regulated by double-stranded RNA (dsRNA), PKR, is implicated in a range of biologic processes, including apoptotic death and interferon antiviral responses, based in part on studies with mouse cells genetically deficient in Pkr. To test the role of the PKR protein in human cells, an RNA interference silencing strategy was used to generate stable HeLa cell lines with less than 2% of the PKR protein (PKR deficient) compared to either parental or control knockdown HeLa lines. Phosphorylation of the alpha subunit of eukaryotic initiation factor 2 on serine 51 was not detectably increased in response to dsRNA in PKR-deficient HeLa cells but was elevated severalfold in PKR-sufficient cells. PKR-deficient cells displayed reduced dsRNA-induced apoptosis compared to PKR-sufficient cell lines, whereas tumor necrosis factor alpha (TNF-alpha)-induced apoptosis was comparable between the HeLa lines. NF-kappaB was activated to a comparable extent in PKR-deficient and PKR-sufficient HeLa cells upon treatment with either dsRNA or TNF-alpha. The antiviral response against vesicular stomatitis virus was reduced in interferon-treated PKR-deficient compared to PKR-sufficient HeLa cells. However, the growth of two human viruses, adenovirus and reovirus, was unaffected by the PKR knockdown. Surprisingly, the yield of mutant adenovirus that fails to encode VAI RNA was not enhanced in PKR-deficient cells, indicating the importance of host factors in addition to PKR in conferring the VAI RNA phenotype.     

NF-kappaB activation and susceptibility to apoptosis after polyamine depletion in intestinal epithelial cells

The maintenance of intestinal mucosal integrity depends on a balance between cell renewal and cell death, including apoptosis. The natural polyamines, putrescine, spermidine, and spermine, are essential for mucosal growth, and decreasing polyamine levels cause G(1) phase growth arrest in intestinal epithelial (IEC-6) cells. The present study was done to determine changes in susceptibility of IEC-6 cells to apoptosis after depletion of cellular polyamines and to further elucidate the role of nuclear factor-kappaB (NF-kappaB) in this process. Although depletion of polyamines by alpha-difluoromethylornithine (DFMO) did not directly induce apoptosis, the susceptibility of polyamine-deficient cells to staurosporine (STS)-induced apoptosis increased significantly as measured by changes in morphological features and internucleosomal DNA fragmentation. In contrast, polyamine depletion by DFMO promoted resistance to apoptotic cell death induced by the combination of tumor necrosis factor-alpha (TNF-alpha) and cycloheximide. Depletion of cellular polyamines also increased the basal level of NF-kappaB proteins, induced NF-kappaB nuclear translocation, and activated the sequence-specific DNA binding activity. Inhibition of NF-kappaB binding activity by sulfasalazine or MG-132 not only prevented the increased susceptibility to STS-induced apoptosis but also blocked the resistance to cell death induced by TNF-alpha in combination with cycloheximide in polyamine-deficient cells. These results indicate that 1) polyamine depletion sensitizes intestinal epithelial cells to STS-induced apoptosis but promotes the resistance to TNF-alpha-induced cell death, 2) polyamine depletion induces NF-kappaB activation, and 3) disruption of NF-kappaB function is associated with altered susceptibility to apoptosis induced by STS or TNF-alpha. These findings suggest that increased NF-kappaB activity after polyamine depletion has a proapoptotic or antiapoptotic effect on intestinal epithelial cells determined by the nature of the death stimulus.     

Cytotoxicity and apoptosis induction of some selected marine bacteria metabolites

AIMS: To study the potential apoptosis effects of cytotoxic marine bacterial metabolites on human HeLa cell line. METHODS AND RESULTS: After HeLa cells were routinely cultured, tetrazolium-based colorimetric assay for cytotoxicity was performed to screen the marine bacteria extracts showing 12 strains active. To find the potential active strain with apoptosis mechanism, a battery of apoptosis assays, including AO/EB staining, TUNEL assay (terminal-deoxynucleotidyl transferase mediated nick end labelling), gel electrophoresis and flow cytometry, were used to determine whether apoptosis was involved in HeLa cell cytotoxicity of marine bacterial extracts. The results indicated that four strains could induce cell shrinkage, cell membrane blebbing, formation of apoptotic body and DNA fragmentation. CONCLUSIONS: Crude extracts of 12 of 153 strains of marine bacteria showed cytotoxic effects with ID50 ranged from 77.20 to 199.84 microg ml(-1), in which eight strains of bacteria were associated bacteria. The metabolites in the strains of QD1-2, NJ6-3-1, NJ1-1-1 and SS6-4 were able to induce HeLa cells apoptosis. Furthermore, the assessment by flow cytometry indicated that the hypodiploid apoptotic cells increased in a time-dependent manner, suggesting that induced apoptosis occurred from 24 h to 48 h after the extracts treatment. SIGNIFICANCE AND IMPACT OF THE STUDY: Our results suggested that the compounds from fermentation in these four marine bacterial strains could be candidates for developing apoptosis specific anti-tumour agents with lower toxicity. This study indicated that associated marine bacteria could be good source to find cytotoxic metabolites, and some cytotoxic marine bacterial metabolites could have apoptosis mechanisms.