ABSTRACT
Chronic inflammation increases the risk of development of several types of malignancies including colon cancer. It also represents a paradigm for the connection between inflammation and cancer in terms of epidemiology and mechanistic studies in preclinical models. A key component of inflammation promoting cancer is the transcription factor NF-κB, which is known to play a critical role in the regulation of the inducible nitric oxide synthase (iNOS) gene. iNOS is an enzyme dominantly expressed during inflammatory reactions. Although synthesis of high amounts of nitric oxide (NO) by iNOS has been demonstrated in pathophysiological processes, such as acute or chronic inflammation and tumorigenesis, the role of iNOS activity in these diseases is still not well understood. Analysis of human biopsies of colitis and colon cancer using immunohistochemistry revealed elevated iNOS protein expression levels, which were strongly paralleled by increased expression of nitrotyrosine suggesting that iNOS has been highly activated in these tissues. These results were corroborated in an in vitro study showing the presence of high iNOS levels in a colon cancer cell line (HT-29) following inflammatory stimuli (TNF-α, peroxynitrite). In addition, the involvement of metastatic processes in the colon biopsies was assessed by means of in situ zymography of MMP activation. MMP 2 (gelatinase A) activation was higher in histopathological sections of colitis and cancer compared to controls. Overall, these data strengthen the findings that in inflammation and colon cancer in humans, iNOS expression and tyrosine nitration may be an indicator of cancer development and progression.
Subject(s)
Colitis/genetics , Colitis/metabolism , Colonic Neoplasms/chemistry , Colonic Neoplasms/genetics , Nitric Oxide Synthase Type II/analysis , Nitric Oxide Synthase Type II/genetics , Tyrosine/analogs & derivatives , HT29 Cells , Humans , Immunohistochemistry , NF-kappa B/metabolism , Nitric Oxide Synthase Type II/metabolism , Peroxynitrous Acid/pharmacology , Real-Time Polymerase Chain Reaction , Tumor Cells, Cultured , Tyrosine/analysisABSTRACT
BACKGROUND: Peroxynitrite has been proposed to activate Nuclear Factor κappa beta (NF-κB) in a non-canonical or aberrant pathway and to contribute to pathology of many diseases. METHODS: The activation of NF-κB by peroxynitrite was assessed by Western blot, immunoprecipitation, RT-PCR, and image stream analysis. RESULTS: Our work showed that in HT-29 cancer cells, peroxynitrite can cause nitration of Inhibitory protein kappa B alpha (IκBα) at the expense of its phosphorylation. This led to a decrease in the degradation and re-synthesis of IκBα. Similar findings were observed for mRNA levels assessed by RT-PCR. Exposure of HT-29 cells to p38 inhibitor SB202190, prior to stimulation, resulted in a dramatic decrease of IκBα kinase and IκBα phosphorylation and caused an increase of peroxynitrite-mediated nitration of IκBα, indicating that peroxynitrite may activate NF-κB via dual mechanism of IκBα phosphorylation which is p38 dependent, as well as IκBα nitration. CONCLUSION: Our findings demonstrate a possible interaction of the p38 pathway with the NF-κB pathway under peroxynitrite stimulation.
Subject(s)
Colonic Neoplasms/metabolism , I-kappa B Kinase/metabolism , I-kappa B Proteins/metabolism , NF-kappa B/metabolism , Peroxynitrous Acid/pharmacology , Reactive Nitrogen Species/metabolism , Blotting, Western , Cell Line, Tumor , Cell Nucleus/metabolism , Colonic Neoplasms/genetics , Colonic Neoplasms/pathology , Enzyme Inhibitors/pharmacology , Humans , I-kappa B Kinase/genetics , I-kappa B Proteins/genetics , Image Processing, Computer-Assisted , Imidazoles/pharmacology , Immunoprecipitation , NF-KappaB Inhibitor alpha , NF-kappa B/genetics , Phosphorylation , Protein Transport , Pyridines/pharmacology , RNA, Messenger/genetics , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction , p38 Mitogen-Activated Protein Kinases/metabolismABSTRACT
BACKGROUND: Exhaustive exercise is associated with increased metabolic rate and accelerated generation of reactive oxygen species. Cigarette smoke also contains oxidants that may participate in the development of atherosclerosis. However, data on the association between exercise and smoking are sparse. METHODS: A homogenous group of 30 young men (15 smokers and 15 nonsmokers; mean age, 23.7 +/- 2.6 years), healthy, trained subjects, were assessed before and after a standard maximal exercise test. RESULTS: Exercise led to increased protein oxidation (carbonyl assay) in both smokers (+17.7%, P < 0.001) and nonsmokers (+19.1%, P < 0.05), elevation in plasma conjugated dienes (+ 157%, P < 0.04), and plasma lipid peroxides (+14%, P < 0.059) in smokers versus nonsmokers after exercise. Plasma antioxidants levels were significantly lower in the smoking group, with reduction in total carotenoids (-36.5%, P < 0.001), vitamin A (-80%, P < 0.001), and vitamin E (-64%, P < 0.002), compared with nonsmokers. A significant rise in leakage of muscle enzymes (CPK, LDH) and urine proteins (microalbumin and myoglobin) occurred in all subjects after exercise. CRP levels were higher in smokers compared with nonsmokers before and after exercise. CONCLUSIONS: Our results suggest that unnoticed interaction exists between smoking and intense exercise, which indicates that smokers are more susceptible to oxidative insults probably due to lower antioxidant capacity.
