Analysis of In Vivo Nuclear Factor-B Activation during Liver Inflammation in Mice: Prevention by Catalase Delivery

ABSTRACT Nuclear factor-B (NF-B) is a transcription factor that plays crucial roles in inflammation, immunity, cell proliferation, and apoptosis. Until now, there have been few studies of NF-B activation in whole animals because of experimental difficulties. Here, we show that mice receiving a simple injection of plasmid vectors can be used to examine NF-B activation in the liver. Two plasmid vectors, pNF-B-Luc (firefly luciferase gene) and pRL-SV40 (Renilla reniformis luciferase gene), were injected into the tail vein of mice by the hydrodynamics-based procedure, an established method of gene transfer to mouse liver. Then, the ratio of the firefly and R. reniformis luciferase activities (F/R) was used as an indicator of the NF-B activity in the liver. Injection of thioacetamide or lipopolysaccharide plus D-galactosamine increased the F/R ratio in the liver, and this was significantly (P Ͻ 0.001) inhibited by an intravenous injection of catalase derivatives targeting liver nonparenchymal cells. Imaging the firefly luciferase expression in live mice clearly demonstrated that the catalase derivatives efficiently prevented the NF-B-mediated expression of the firefly luciferase gene. Plasma transaminases and the survival rate of mice supported the findings obtained by the luminescence-based analyses. Thus, this method, which requires no genetic recombination techniques, is highly sensitive to the activation of NF-B and allows us to continuously examine the activation in live animals. In conclusion, this novel, simple, and sensitive method can be used not only for analyzing the NF-B activation in the organ under different inflammatory conditions but also for screening drug candidates for the prevention of liver inflammation

Mitochondrial targeted catalase suppresses invasive breast cancer in mice

<p>Abstract</p> <p>Background</p> <p>Treatment of invasive breast cancer has an alarmingly high rate of failure because effective targets have not been identified. One potential target is mitochondrial generated reactive oxygen species (ROS) because ROS production has been associated with changes in substrate metabolism and lower concentration of anti-oxidant enzymes in tumor and stromal cells and increased metastatic potential.</p> <p>Methods</p> <p>Transgenic mice expressing a human catalase gene (mCAT) were crossed with MMTV-PyMT transgenic mice that develop metastatic breast cancer. All mice (33 mCAT positive and 23 mCAT negative) were terminated at 110 days of age, when tumors were well advanced. Tumors were histologically assessed for invasiveness, proliferation and metastatic foci in the lungs. ROS levels and activation status of p38 MAPK were determined.</p> <p>Results</p> <p>PyMT mice expressing mCAT had a 12.5 per cent incidence of high histological grade primary tumor invasiveness compared to a 62.5 per cent incidence in PyMT mice without mCAT. The histological grade correlated with incidence of metastasis with 56 per cent of PyMT mice positive for mCAT showing evidence of pulmonary metastasis compared to 85.4 per cent of PyMT mice negative for mCAT with pulmonary metastasis (p ≤ 0.05). PyMT tumor cells expressing mCAT had lower ROS levels and were more resistant to hydrogen peroxide-induced oxidative stress than wild type tumor cells, suggesting that mCAT has the potential of quenching intracellular ROS and subsequent invasive behavior. The metastatic tumor burden in PyMT mice expressing mCAT was 0.1 mm²/cm²of lung tissue compared with 1.3 mm²/cm²of lung tissue in PyMT mice expressing the wild type allele (p ≤ 0.01), indicating that mCAT could play a role in mitigating metastatic tumor progression at a distant organ site. Expression of mCAT in the lungs increased resistance to hydrogen peroxide-induced oxidative stress that was associated with decreased activation of p38MAPK suggesting ROS signaling is dependent on p38MAPK for at least some of its downstream effects.</p> <p>Conclusion</p> <p>Targeting catalase within mitochondria of tumor cells and tumor stromal cells suppresses ROS-driven tumor progression and metastasis. Therefore, increasing the antioxidant capacity of the mitochondrial compartment could be a rational therapeutic approach for invasive breast cancer.</p> <p>Please see related commentary article: <url>http://www.biomedcentral.com/1741-7015/9/62</url></p

Combining cisplatin with cationized catalase decreases nephrotoxicity while improving antitumor activity

Cisplatin is frequently used to treat solid tumors; however, nephrotoxicity due to its reactive oxygen species-mediated effect limits its use. We tested the ability of cationized catalase, a catalase derivative, to inhibit nephrotoxicity in cisplatin-treated mice. Immunohistochemical analysis showed that the catalase derivative concentrated in the kidney more efficiently than native catalase. Repeated intravenous doses of cationized catalase significantly decreased cisplatin-induced changes in serum creatinine, blood urea nitrogen, nitrite/nitrate levels, lactic dehydrogenase activity, and renal total glutathione and malondialdehyde contents. In addition, cationized catalase effectively blunted cisplatin-induced proximal tubule necrosis but had no significant effect on the cisplatin-induced inhibition of subcutaneous tumor growth. Repeated doses of catalase, especially cationized catalase, significantly increased the survival of cisplatin-treated tumor-bearing mice preventing cisplatin-induced acute death. Our studies suggest that catalase and its derivatives inhibit cisplatin-induced nephrotoxicity, thus improving the efficiency of cisplatin to treat solid tumors

Molecular profiling of a lethal tumor microenvironment, as defined by stromal caveolin-1 status in breast cancers

Breast cancer progression and metastasis are driven by complex and reciprocal interactions, between epithelial cancer cells and their surrounding stromal microenvironment. We have previously shown that a loss of stromal Cav-1 expression is associated with an increased risk of early tumor recurrence, metastasis and decreased overall survival. To identify and characterize the signaling pathways that are activated in Cav-1 negative tumor stroma, we performed gene expression profiling using laser microdissected breast cancer-associated stroma. Tumor stroma was laser capture microdissected from 4 cases showing high stromal Cav-1 expression and 7 cases with loss of stromal Cav-1. Briefly, we identified 238 gene transcripts that were upregulated and 232 gene transcripts that were downregulated in the stroma of tumors showing a loss of Cav-1 expression (p ≤ 0.01 and fold-change ≥1.5). Gene set enrichment analysis (GSEA) revealed “stemness,” inflammation, DNA damage, aging, oxidative stress, hypoxia, autophagy and mitochondrial dysfunction in the tumor stroma of patients lacking stromal Cav-1. Our findings are consistent with the recently proposed “Reverse Warburg Effect” and the “Autophagic Tumor Stroma Model of Cancer Metabolism.” In these two complementary models, cancer cells induce oxidative stress in adjacent stromal cells, which then forces these stromal fibroblasts to undergo autophagy/mitophagy and aerobic glycolysis. This, in turn, produces recycled nutrients (lactate, ketones and glutamine) to feed anabolic cancer cells, which are undergoing oxidative mitochondrial metabolism. Our results are also consistent with previous biomarker studies showing that the increased expression of known autophagy markers (such as ATG16L and the cathepsins) in the tumor stroma is specifically associated with metastatic tumor progression and/or poor clinical outcome