Modulation of AP-1 by Natural Chemopreventive Compounds in Human Colon HT-29 Cancer Cell Line

PURPOSE: Activator protein-1 (AP-1) has been implicated as playing important roles in apoptosis and cancer development. In this work, we studied several natural chemopreventive compounds for their potential chemopreventive properties in the modulation of AP-1 signaling pathway in HT-29 colon cancer cells. METHODS: The HT-29 cells were transfected with AP-1-luciferase reporter gene, and one of the stable clones (C-4) was used for subsequent experiments. The HT-29 C-4 cells were treated for 1 h with various natural chemopreventive agents and challenged with AP-1 stimulators such as 12-O-tetradecanoylphorbol-13-acetate (TPA) or hydrogen peroxide (H2O2) for 6 h. The c-Jun N-terminal kinase (JNK) was examined to understand the effect of these compounds on the upstream signaling activator of AP-1. The protein expression level of endogenous cyclin D1, a gene that is under the control of AP-1, was also analyzed after treatments with the agents. In addition, cell death induced by these compounds was evaluated by MTS assay [3-(4.5-dimethylthiazol-2-yl)-5-(3-arboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt]. RESULTS: TPA and H2O2 treatments strongly induced AP-1-luciferase activity as expected. Phenethyl isothiocyanate, sulforaphane, curcumin, and resveratrol increased AP-1-luciferase activity dose-dependently and then decreased at higher doses in the presence or absence of TPA. Allyl isothiocyanate and (-)-epigallocatechin-3-gallate (EGCG) increased AP-1-luciferase activity dose-dependently up to 50 and 100 microM. Other tea catechins and procyanidin dimers, however, had little or no effect on AP-1-luciferase activity. The JNK activity was induced by the isothiocyanates and EGCG. Most of the chemopreventive compounds induced cell death in a dose-dependent manner, with the exception of epicatechin (EC) and the procyanidins, which had little effect. The expression of endogenous cyclin D1 protein was well correlated with those of AP-1-luciferase assay. CONCLUSION: Taken together, these results suggest that natural chemopreventive compounds may have differential biological functions on the signal transduction pathways such as AP-1 in the intervention of colon cancer progression and carcinogenesis.     

Biologic therapy of inflammatory bowel disease

Advancing knowledge regarding the biology of chronic inflammation has led to the development of specific biologic therapies that mechanistically target individual inflammatory pathways. Many biologic therapies are being evaluated for the treatment of the chronic inflammatory bowel diseases, Crohn's disease and ulcerative colitis. Biologic compounds proven to be effective for Crohn's disease include monoclonal antibodies to tumor necrosis factor (infliximab and CDP571) and to the leukocyte adhesion molecule alpha4 integrin (natalizumab). Other biologic compounds for which there is insufficient evidence to judge efficacy for inflammatory bowel disease include: p55 tumor necrosis factor binding protein (onercept); interferon alpha; interferon beta-1a; anti-interferon gamma antibody; anti-interleukin 12 antibody; p65 anti-sense oligonucleotide (blocks NF-kappaB); granulocyte colony stimulating factor, and granulocyte macrophage colony stimulating factor; anti-interleukin 2 receptor antibody; epidermal growth factor; keratinocyte growth factor 2 (repifermin); human growth hormone; anti-CD4 antibody; and anti-alpha4beta7 antibody. Biologic therapies that have been proven ineffective for inflammatory bowel disease include: interleukin 10; interleukin 11; anti-sense intercellular adhesion molecule-1; and the tumor necrosis factor receptor fusion protein etanercept. Based on the early successes of infliximab, CDP571 and natalizumab, it seems certain that biologic therapy will play an important role in the future treatment of inflammatory bowel disease.     

Nicotine affects the signaling of the death pathway, reducing the response of head and neck cancer cell lines to DNA damaging agents

BACKGROUND: Growing evidence suggests that tobacco can affect the responsiveness of cancer cells to treatment, particularly those of head and neck cancer. This article describes the effects of nicotine on the signaling of the death pathway, resulting in a decreased cytotoxicity of various anticancer agents such as cisplatin and gamma-radiation. METHODS: Colony-forming assays (CFA), using the head and neck cancer cell lines UMSCC10b and UMSCC5 and DNA fragmentation assays, were used to determine the effect of nicotine on cytotoxicity of various anticancer agents, whereas PCR and a JNK activity test were used to study the effect of nicotine on message expression levels and activity of the JNK signaling pathway. RESULTS: Nicotine consistently reduced the cytotoxic effect of DNA-damaging agents, such as cisplatin, UV, and gamma radiation, in UMSCC10b cells, increasing their IC(50) values by twofold, 1.7-fold, and 1.8-fold, respectively. These results were confirmed in a second squamous cell carcinoma cell line (UMSCC5), demonstrating an increase in IC(50) values for cDDP by twofold and 1.9-fold in the UMSCC10b andUMSCC5, respectively. In addition, nicotine reduced the DNA fragmentation 48 h after cDDP exposure in UMSCC10b and UMSCC5 cell lines by 30% and 33%, respectively. The latter, however, was not the result of an effect of nicotine on either the uptake of cDDP or repair of the cDDP-DNA-adducts. To further substantiate the adverse effect of nicotine, the JNK and gadd153 signaling pathways were studied. JNK activity was decreased by 1.8-fold, as well as the expression of its downstream target c-jun (1.9-fold), when tumor cells were exposed to cisplatin in the presence of nicotine. In addition, the gadd153 message was affected and reduced by 1.8-fold. CONCLUSIONS: Nicotine adversely affects the cytotoxicity of DNA-damaging agents. Nicotine does not interfere with the repair of the damage but directly affects the signaling of the death pathway, reducing the signaling of the JNK1 pathway. The latter results in a decrease in efficacy of the anticancer treatment in tumors exposed to nicotine.     

Oxidative stress, ER stress, and the JNK pathway in type 2 diabetes

Pancreatic -cell dysfunction and insulin resistance are observed in type 2 diabetes. Under diabetic conditions, oxidative stress and ER stress are induced in various tissues, leading to activation of the JNK pathway. This JNK activation suppresses insulin biosynthesis and interferes with insulin action. Indeed, suppression of the JNK pathway in diabetic mice improves insulin resistance and ameliorates glucose tolerance. Thus, the JNK pathway plays a central role in pathogenesis of type 2 diabetes and may be a potential target for diabetes therapy.     

Eating ourselves to death (and despair): The contribution of adiposity and inflammation to depression

Obesity and related metabolic conditions are of epidemic proportions in most of the world, affecting both adults and children. The accumulation of lipids in the body in the form of white adipose tissue in the abdomen is now known to activate innate immune mechanisms. Lipid accumulation causes adipocytes to directly secrete the cytokines interleukin (IL) 6 and tumor necrosis factor α (TNFα), but also monocyte chemoattractant protein 1 (MCP-1), which results in the accumulation of leukocytes in fat tissue. This sets up a chronic inflammatory state which is known to mediate the association between obesity and conditions such as cardiovascular disease, type 2 diabetes, and cancer. There is also a substantial literature linking inflammation with risk for depression. This includes the observations that: (1) people with inflammatory diseases such as multiple sclerosis, cardiovascular disease, and psoriasis have elevated rates of depression; (2) many people administered inflammatory cytokines such as interferon α develop depression that is indistinguishable from depression in non-medically ill populations; (3) a significant proportion of depressed persons show upregulation of inflammatory factors such as IL-6, C-reactive protein, and TNFα; (4) inflammatory cytokines can interact with virtually every pathophysiologic domain relevant to depression, including neurotransmitter metabolism, neuroendocrine function, and synaptic plasticity. While many factors may contribute to the association between inflammatory mediators and depression, we hypothesize that increased adiposity may be one causal pathway. Mediational analysis suggests a bi-directional association between adiposity and depression, with inflammation possibly playing an intermediary role.     

永久性大脑中动脉闭塞大鼠纹状体P—SAPK/JNK表达与神经元凋亡的相关性

目的探讨永久性大脑中动脉闭塞（permanent middle cerebral artery occlusion,pMCAO）大鼠纹状体P-SAPK／JNK表达与神经元凋亡的相关性。方法54只Sprague—Dawley雄性大鼠随机分为假手术组以及pMCAO1h、3h、6h、12h和24h组,每组9只。应用TUNEL法检测纹状体凋亡神经元,免疫组织化学染色法检测纹状体P—SAPK／JNK核转位,Western印迹法检测P-SAPK／JNK蛋白表达。结果pMCAO 1h纹状体TUNEL和P—SAPK／JNK阳性细胞数量显著增多（P=0．0001）,6h达高峰,12h时TUNEL阳性细胞减少,但仍高于假手术组（P=0．0002）。Western印迹分析显示,pMCAO后纹状体P—SAPK／JNK蛋白表达水平显著增高,且时程变化与免疫组化染色结果一致。纹状体神经元凋亡与P—SAPK／JNK蛋白表达呈显著正相关（r=0．984,P=0．0004）。结论脑缺血可能通过激活P-SAPK／JNK诱导纹状体神经元凋亡。     

支架蛋白JLP基因敲除小鼠的构建及其肾脏表型的初步观察

目的 繁殖和鉴定支架蛋白JLP基因敲除小鼠,对其肾脏表型进行初步观察。方法 将JLP基因敲除杂合小鼠进行配种繁殖,提取子代小鼠的组织DNA,用PCR技术检测小鼠的基因型,并用Western blot法验证基因敲除效果。通过PAS病理染色等观察肾脏微观结构。结果 成功繁殖并获得子代小鼠,子代小鼠有3种基因型,包括纯合子（JLP+/+、JLP-/-）和杂合子（JLP+/-）。PCR检测基因型的方法方便且结果准确。结论 笔者成功建立了JLP基因敲除小鼠模型,能够进一步探究发现JLP基因对生物体的作用及机制。