Influence of natural killer cells and perforin­mediated cytolysis on the development of chemically induced lung cancer in A/J mice.

One alternative approach for the treatment of lung cancer might be the activation of the immune system using vaccination strategies. However, most of clinical vaccination trials for lung cancer did not reach their primary end points, suggesting that lung cancer is of low immunogenicity. To provide additional experimental information about this important issue, we investigated which type of immune cells contributes to the protection from lung cancer development. Therefore, A/J mice induced for lung adenomas/ adenocarcinomas by the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) were depleted of CD4+ or CD8+ T cells, CD11b+ macrophages, Gr-1+ neutrophils and asialo GM1+ natural killer (NK) cells. Subsequent analysis of tumour growth showed an increase in tumour number only in mice depleted of NK cells. Further asking by which mechanism NK cells suppressed tumour development, we neutralized several death ligands of the tumour necrosis factor (TNF) family known to be involved in NK cell-mediated cytotoxicity. However neither depletion of TNF-α, TNF-related apoptosis-inducing ligand, TNF-like weak inducer of apoptosis or FasL alone nor in combination induced an augmentation of tumour burden. To show whether an alternative cell death pathway is involved, we next generated A/J mice deficient for perforin. After challenging with NNK, mice deficient for perforin showed an increase in tumour number and volume compared to wild-type A/J mice. In summary, our data suggest that NK cells and perforin-mediated cytolysis are critically involved in the protection from lung cancer giving promise for further immunotherapeutic strategies for this disease.

Increased binding of anti-dsDNA antibodies to short oligonucleotides modified with topoisomerase I reveals a potential new enzyme function independent from DNA relaxation.

OBJECTIVE: Topoisomerase I (topo I) is a highly conserved enzyme which is known to reduce torsional stress at double-stranded (ds) DNA. Torsional stress induced by supercoiling of dsDNA requires either very long dsDNA existing in genomic DNA or circulation as presented in plasmid DNA. To enable DNA relaxation, topo I induce a transient single-strand break followed by stress-relieving rotation of the released DNA strand. Our group found by serendipity that the topo I inhibitor irinotecan is able to suppress murine systemic lupus erythematosus (SLE), an autoimmune disease which is characterized by the existence of pathogenic anti-dsDNA antibodies (abs). As a possible mechanism we demonstrated in the absence of immunosuppression an increased binding of anti-dsDNA abs to long genomic or circulated plasmid dsDNA modified with topo I. RESULTS: Here we show that this effect requires active site tyrosine of topo I which is known to facilitate DNA relaxation activity. Moreover, topo I enhanced anti-dsDNA abs binding to short linear oligonucleotides down to a size of 42 bp. Since oligonucleotides of such length are devoid of torsional stress and relaxation respectively, our results suggest a new and unknown function for the enzyme topo I.

Reversal of established lupus nephritis and prolonged survival of New Zealand black x New Zealand white mice treated with the topoisomerase I inhibitor irinotecan.

Systemic lupus erythematosus is a chronic autoimmune disorder that predominantly affects women of childbearing age. Lupus-associated glomerulonephritis is a major cause of mortality in these patients. Current treatment protocols for systemic lupus erythematosus include cyclophosphamide, prednisolone, azathioprine, and mycophenolate mofetil. However, in mice none of these agents alone or in combination were shown to reverse established proteinuria. Using New Zealand Black x New Zealand White F1 mice, we report that administration of the topoisomerase I inhibitor irinotecan from week 13 completely prevented the onset of proteinuria and prolonged survival up to at least 90 wk without detectable side effects. Furthermore, application of irinotecan to mice with established lupus nephritis, as indicated by grade 3+ (> or =300 mg/dl) and grade 4+ (> or =2000 mg/dl) proteinuria and, according to a median age of 35 wk, resulted in remission rates of 75% and 55%, respectively. Survival was significantly prolonged with 73 wk (grade 3+ and 4+ combined) versus 40 wk for control animals. Although total IgG and anti-dsDNA Abs in the serum and mesangial IgG deposits in the kidneys were not reduced in irinotecan-treated mice, subendothelial immune deposits were considerably diminished, suggesting a prevention of glomerular basement membrane disruption. This effect was accompanied by increased rates of ssDNA breaks and inhibition of renal cell apoptosis being different to what is known about irinotecan in anticancer therapy. In conclusion, our data provide evidence that irinotecan might represent an entirely new strategy for the treatment of systemic lupus erythematosus.

Cardiac glycosides initiate Apo2L/TRAIL-induced apoptosis in non-small cell lung cancer cells by up-regulation of death receptors 4 and 5.

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (Apo2L/TRAIL) belongs to the TNF family known to transduce their death signals via cell membrane receptors. Because it has been shown that Apo2L/TRAIL induces apoptosis in tumor cells without or little toxicity to normal cells, this cytokine became of special interest for cancer research. Unfortunately, cancer cells are often resistant to Apo2L/TRAIL-induced apoptosis; however, this can be at least partially negotiated by parallel treatment with other substances, such as chemotherapeutic agents. Here, we report that cardiac glycosides, which have been used for the treatment of cardiac failure for many years, sensitize lung cancer cells but not normal human peripheral blood mononuclear cells to Apo2L/TRAIL-induced apoptosis. Sensitization to Apo2L/TRAIL mediated by cardiac glycosides was accompanied by up-regulation of death receptors 4 (DR4) and 5 (DR5) on both RNA and protein levels. The use of small interfering RNA revealed that up-regulation of death receptors is essential for the demonstrated augmentation of apoptosis. Blocking of up-regulation of DR4 and DR5 alone significantly reduced cell death after combined treatment with cardiac glycosides and Apo2L/TRAIL. Combined silencing of DR4 and DR5 abrogated the ability of cardiac glycosides and Apo2L/TRAIL to induce apoptosis in an additive manner. To our knowledge, this is the first demonstration that glycosides up-regulate DR4 and DR5, thereby reverting the resistance of lung cancer cells to Apo2/TRAIL-induced apoptosis. Our data suggest that the combination of Apo2L/TRAIL and cardiac glycosides may be a new interesting anticancer treatment strategy.

Evidence that N-acetylcysteine inhibits TNF-alpha-induced cerebrovascular endothelin-1 upregulation via inhibition of mitogen- and stress-activated protein kinase.

N-acetylcysteine (NAC) is neuroprotective in animal models of acute brain injury such as caused by bacterial meningitis. However, the mechanism(s) by which NAC exerts neuroprotection is unclear. Gene expression of endothelin-1 (ET-1), which contributes to cerebral blood flow decline in acute brain injury, is partially regulated by reactive oxygen species, and thus a potential target of NAC. We therefore examined the effect of NAC on tumor necrosis factor (TNF)-alpha-induced ET-1 production in cerebrovascular endothelial cells. NAC dose dependently inhibited TNF-alpha-induced preproET-1 mRNA upregulation and ET-1 protein secretion, while upregulation of inducible nitric oxide synthase (iNOS) was unaffected. Intriguingly, NAC had no effect on the initial activation (i.e., IkappaB degradation, nuclear p65 translocation, and Ser536 phosphorylation) of NF-kappaB by TNF-alpha. However, transient inhibition of NF-kappaB DNA binding suggested that NAC may inhibit ET-1 upregulation by inhibiting (a) parallel pathway(s) necessary for full transcriptional activation of NF-kappaB-mediated ET-1 gene expression. Similar to NAC, the MEK1/2 inhibitor U0126, the p38 inhibitor SB203580, and the protein kinase inhibitor H-89 selectively inhibited ET-1 upregulation without affecting nuclear p65 translocation, suggesting that NAC inhibits ET-1 upregulation via inhibition of mitogen- and stress-activated protein kinase (MSK). Supporting this notion, cotreatment with NAC inhibited the TNF-alpha-induced rise in MSK1 and MSK2 kinase activity, while siRNA knock-down experiments showed that MSK2 is the predominant isoform involved in TNF-alpha-induced ET-1 upregulation.

Oxidative stress precedes peak systemic inflammatory response in pediatric patients undergoing cardiopulmonary bypass operation.

Oxidative stress seems to contribute to cardiopulmonary bypass (CPB)-related postoperative complications. Pediatric patients are particularly prone to these complications. With this in mind, we measured oxidative stress markers in blood plasma of 20 children undergoing elective heart surgery before, during, and up to 48 h after cessation of CPB, along with inflammatory parameters and full analysis of iron status. Ascorbate levels were decreased by approximately 50% (P < 0.001) at the time of aorta cross-clamp removal (or pump switch-off in 4 patients with partial CPB), and associated with corresponding increases in dehydroascorbate (P < 0.001, r = -0.80) and malondialdehyde (P < 0.01, r = -0.59). In contrast to the immediate oxidative response, peak levels of IL-6 and IL-8 were not observed until 3-12 h after CPB cessation. The early loss of ascorbate correlated with duration of CPB (P < 0.002, r = 0.72), plasma hemoglobin after cross-clamp removal (P < 0.001, r = 0.70), and IL-6 and IL-8 levels at 24 and 48 h after CPB (P < 0.01), but not with postoperative lactate levels, strongly suggesting that hemolysis, and not inflammation or ischemia, was the main cause of early oxidative stress. The correlation of ventilation time with early changes in ascorbate (P < 0.02, r = 0.55), plasma hemoglobin (P < 0.01, r = 0.60), and malondialdehyde (P < 0.02, r = 0.54) suggests that hemolysis-induced oxidative stress may be an underlying cause of CPB-associated pulmonary dysfunction. Optimization of surgical procedures or therapeutic intervention that minimize hemolysis (e.g., off-pump surgery) or the resultant oxidative stress (e.g., antioxidant treatment) should be considered as possible strategies to lower the rate of postoperative complications in pediatric CPB.

The Topoisomerase I Inhibitor Irinotecan and the Tyrosyl-DNA Phosphodiesterase 1 Inhibitor Furamidine Synergistically Suppress Murine Lupus Nephritis.

OBJECTIVE: The treatment of lupus nephritis is still an unmet medical need requiring new therapeutic approaches. Our group found recently that irinotecan, an inhibitor of topoisomerase I (topo I), reversed proteinuria and prolonged survival in mice with advanced lupus nephritis. While irinotecan is known to stabilize the complex of topo I and DNA, the enzyme tyrosyl-DNA phosphodiesterase 1 (TDP-1) functions in an opposing manner by releasing topo I from DNA. Therefore, we undertook this study to test whether the TDP-1 inhibitor furamidine has an additional effect on lupus nephritis when used in combination with irinotecan. METHODS: NZB/NZW mice were treated with low-dose irinotecan and furamidine either alone or in combination beginning at age 26 weeks. DNA relaxation was visualized using gel electrophoresis. Binding of anti-double-stranded DNA (anti-dsDNA) antibodies to DNA modified by topo I, TDP-1, and the topo I inhibitor camptothecin was determined by enzyme-linked immunosorbent assay. RESULTS: Compared to treatment with either agent alone, simultaneous treatment with low-dose irinotecan and furamidine significantly improved survival of NZB/NZW mice. Similar to what has been previously shown for irinotecan alone, the combination treatment did not change the levels of anti-dsDNA antibodies. In vitro, recombinant TDP-1 increased topo I-mediated DNA relaxation, resulting in enhanced binding of anti-dsDNA antibodies. In combination with topo I and camptothecin, TDP-1 reversed the inhibitory effects of camptothecin on DNA relaxation and anti-dsDNA binding. CONCLUSION: Affecting DNA relaxation by the enzymes topo I and TDP-1 and their inhibitors may be a promising approach for the development of new targeted therapies for systemic lupus erythematosus.

Cerebral vasculature is the major target of oxidative protein alterations in bacterial meningitis.

We have previously shown that antioxidants such as a-phenyl-tert-butyl nitrone or N-acetylcysteine attenuate cortical neuronal injury in infant rats with bacterial meningitis, suggesting that oxidative alterations play an important role in this disease. However, the precise mechanism(s) by which antioxidants inhibit this injury remain(s) unclear. We therefore studied the extent and location of protein oxidation in the brain using various biochemical and immunochemical methods. In cortical parenchyma, a trend for increased protein carbonyls was not evident until 21 hours after infection and the activity of glutamine synthetase (another index of protein oxidation) remained unchanged. Consistent with these results, there was no evidence for oxidative alterations in the cortex by various immunohistochemical methods even in cortical lesions. In contrast, there was a marked increase in carbonyls, 4-hydroxynonenal protein adducts and manganese superoxide dismutase in the cerebral vasculature. Elevated lipid peroxidation was also observed in cerebrospinal fluid and occasionally in the hippocampus. All of these oxidative alterations were inhibited by treatment of infected animals with N-acetylcysteine or alpha-phenyl-tert-butyl nitrone. Because N-acetylcysteine does not readily cross the blood-brain barrier and has no effect on the loss of endogenous brain antioxidants, its neuroprotective effect is likely based on extraparenchymal action such as inhibition of vascular oxidative alterations.

Cell death induced by down-regulation of heat shock protein 70 in lung cancer cell lines is p53-independent and does not require DNA cleavage.

OBJECTIVE: The inducible form of heat shock protein 70 is known to be overexpressed in tumors and seems to be necessary for the survival of tumor cells via an unknown mechanism. We therefore evaluated whether selective depletion of heat shock protein 70 induces cell death in lung cancer cells. METHODS: An adenovirus expressing antisense heat shock protein 70 and an adenovirus with beta-galactosidase were used for transduction of the lung cancer cell lines A549, NCI-H358, LXF-289, LOU-NH91, normal human bronchial epithelial cells, and normal lung fibroblasts IMR90. Cell death was determined by morphology, propidium iodide uptake, and trypan blue staining; DNA cleavage was assessed by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling. Expression of heat shock protein 70, heat shock cognate 70, and phosphorylated p53 was determined by Western blot analysis. RESULTS: Transduction of lung cancer cells with adenovirus expressing antisense heat shock protein 70 but not with adenovirus with beta-galactosidase resulted in extensive cell death after 96 hours (A549: 53.2 +/- 9.44% versus 12.9 +/- 6.6%; NCI-H358: 48.4 +/- 7.2% versus 25.2 +/- 1.4%; LXF-289: 58.8 +/- 6.5% versus 24.7 +/- 5.4%; LOU-NH91: 82.5 +/- 1.8% versus 38.5 +/- 2.6%). In contrast, adenovirus expressing antisense heat shock protein 70 showed much less cytotoxicity in normal human bronchial epithelial cells (16.0 +/- 0.5% versus 17.1 +/- 7.3%) and in normal lung fibroblasts IMR90 (17.2 +/- 3.6% versus 8.2 +/- 1.6%). After treatment with adenovirus expressing antisense heat shock protein 70, transactivation of p53 in A549 but not in NCI-H358, a cell line deleted for p53 has been observed. Furthermore, 22.0 +/- 3.0% of A549 cells treated for adenovirus expressing antisense heat shock protein 70 stained positive with terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling versus 10.2 +/- 4.0% treated with control virus. This effect but not cell death itself was blocked by treatment with 10 micromol/L zVAD-fmk, a broad caspase inhibitor. CONCLUSIONS: Selective down-regulation of heat shock protein 70 induces cell death in lung cancer but not in normal lung cells. The demonstrated effect is p53-independent and does not require DNA cleavage. The data suggest that gene transfer of antisense heat shock protein 70 might be useful in developing new strategies for the treatment of lung cancer.

Low-dose irinotecan improves advanced lupus nephritis in mice potentially by changing DNA relaxation and anti-double-stranded DNA binding.

OBJECTIVE: Despite clear advances in the treatment of systemic lupus erythematosus (SLE), many patients still present with refractory lupus nephritis, requiring new treatment strategies for this disease. This study was undertaken to determine whether reduced doses of the topoisomerase I (topo I) inhibitor irinotecan, which is known as a chemotherapeutic agent, suppress SLE in (NZB × NZW)F1 (NZB/NZW) mice, and to evaluate the potential mechanism by which irinotecan influences the course of SLE. METHODS: NZB/NZW mice were treated with low-dose irinotecan beginning at either 24 weeks of age or established glomerulonephritis, defined as proteinuria of grade ≥3+. Binding of anti-double-stranded DNA (anti-dsDNA) antibodies was measured by enzyme-linked immunosorbent assay (ELISA), and DNA relaxation was visualized by gel electrophoresis. RESULTS: Significantly reduced irinotecan doses improved lupus nephritis and prolonged survival in NZB/NZW mice. The lowest dose successfully used for the treatment of established murine lupus nephritis was >50 times lower than the dose usually used for chemotherapy in humans. As a mechanism, low-dose irinotecan reduced B cell activity. However, the levels of B cell activity in irinotecan-treated mice were similar to those in BALB/c mice of the same age, suggesting that irinotecan did not induce clear immunosuppression. In addition, incubation of dsDNA with topo I increased binding of murine and human anti-dsDNA antibodies, showing for the first time that relaxed DNA is more susceptible to anti-dsDNA antibody binding. This effect was reversed by addition of the topo I inhibitor camptothecin. CONCLUSION: Our findings indicate that topo I inhibition may be a novel and targeted therapy for SLE.

Inhibition of tissue transglutaminase sensitizes TRAIL-resistant lung cancer cells through upregulation of death receptor 5.

Tissue transglutaminase (TG2) is implicated in cellular processes such as apoptosis and cell migration. Its acyl transferase activity cross-links certain proteins, among them transcription factors were described. We show here that the TG2 inhibitor KCC009 reversed resistance to tumor necrosis factor-related apoptosis-inducing factor (TRAIL) in lung cancer cells. Sensitization required upregulation of death receptor 5 (DR5) but not of death receptor 4. Upregulation of DR5 involved the first intron of the DR5 gene albeit it was independent from p53 and nuclear factor kappa B. In conclusion, inhibition of tissue transglutaminase provides an interesting strategy for sensitization to TRAIL-induced apoptosis in p53-deficient lung cancer cells.

Cytotoxic effects of camptothecin and cisplatin combined with tumor necrosis factor-related apoptosis-inducing ligand (Apo2L/TRAIL) in a model of primary culture of non-small cell lung cancer.

BACKGROUND: The cytokine tumor-necrosis factor-related apoptosis-inducing ligand (Apo2L/TRAIL) has been shown to preferentially induce apoptosis in cancer cells. A previous study of our group demonstrated that non-small cell lung cancer cell lines can be sensitized to Apo2L/TRAIL-induced apoptosis by chemotherapeutic agents. The aim of the present study was the evaluation of these results in a model of primary culture of non-small cell lung cancer. MATERIALS AND METHODS: Lung cancer tissue and normal lung tissue obtained from 8 patients who underwent operation were cultured and treated with Apo2L/TRAIL alone and in combination with cisplatin and the topoisomerase I inhibitor camptothecin for different periods. Metabolic activity of the tissue was measured by alamar blue. Markers for apoptosis were determined by immunohistochemistry and Western blot. Expression of Apo2L/TRAIL receptors in primary lung cancer and normal lung tissue was evaluated by semi-quantitative RT-PCR. Statistics were performed using nonparametric repeated measures Anova with Dunnett's correction. RESULTS: Treatment with cisplatin, camptothecin and the combination of camptothecin and Apo2L/TRAIL demonstrated significant reduction of metabolic activity in tumor and normal lung tissue. In addition, the combination of camptothecin plus Apo2L/TRAIL revealed greater cytotoxic activity in cancer tissue compared with normal lung tissue. Importantly, no toxic activity of Apo2L/TRAIL alone in normal lung tissue was observed. CONCLUSION: Our results obtained in a model of primary culture of lung cancer suggest that the combination of Apo2L/TRAIL and camptothecin might present an effective strategy for the treatment of non-small cell lung cancer.

Differential effect of p47phox and gp91phox deficiency on the course of Pneumococcal Meningitis.

Bacterial meningitis is a severe inflammatory disease of the central nervous system and is characterized by massive infiltration of granulocytes into the cerebrospinal fluid (CSF). To assess the role of NADPH oxidase-derived reactive oxygen species (ROS) in pneumococcal meningitis, mice deficient in either the gp91 subunit (essential for functioning of the phagocyte enzyme) or the p47 subunit (essential for functioning of homologous enzymes in nonphagocytic cells) were intracisternally infected with live Streptococcus pneumoniae, and defined disease parameters were measured during the acute stage of infection. While none of the parameters measured (including CSF bacterial titers) were significantly different in gp91(-/-) and wild-type mice, the infection in p47(-/-) mice was associated with significantly increased inflammation of the subarachnoid and ventricular space, disruption of the blood-brain barrier, and the presence of interleukin-1 beta, tumor necrosis factor alpha, and matrix metalloproteinase 9 in the cortex. These changes were associated with approximately 10-fold-higher CSF bacterial titers in p47(-/-) mice than in wild-type mice (P < 0.001). In contrast to infection with live bacteria, the inflammatory response, including CSF leukocytosis, was significantly attenuated in p47(-/-) mice (but not gp91(-/-) mice) challenged with a fixed number of heat-inactivated pneumococci. Impairment of the host defense appeared to be responsible for the higher bacterial titers in p47(-/-) mice. Therefore, these results indicate that ROS generated by a gp91-independent NADPH oxidase(s) are important for establishing an adequate inflammatory response to pneumococcal CSF infection.