A trip through the signaling pathways of melanoma

Many cellular signaling pathways are involved in the development of cancer. Depending on the tumor entity, the nature as well as the mode of activation can differ. Some signaling pathways frequently show changes as all tumor cells have to fulfill some basic requirements such as independence from growth factors or insensitivity against apoptosis. In this review, the possibilities of a tumor to manipulate signaling pathways to reach these goals are exemplified based on an archetypical melanoma cell. In addition, new therapeutic options based on the knowledge of signaling pathways will be discussed.     

核因子kB在大鼠胃缺血再灌注损伤中的表达及意义

目的研究核因子kB（NF-kB）在大鼠胃缺血再灌注损伤中的表达及意义。方法采用大鼠胃缺血再灌注（gustric ischemia/reperfusion, CI／R）模型（夹闭腹腔动脉30min后再灌注），分别于再灌注0．5、1、3、24h取胃，计算胃黏膜损伤指数。应用免疫组化、Western blot方法检测胃黏膜NF-kB p65。结果大鼠GUR后引起胃黏膜损伤，再灌注1h时损伤最明显，随后降低，24h接近正常。GI／R后胃黏膜NF—kB阳性细胞数和蛋白表达量增多，与胃黏膜损伤变化规律一致。结论NF-kB在大鼠CI／R损伤过程中发挥着重要作用。     

低分子肝素对缺血再灌注大鼠肾组织核因子-κB表达的影响

探讨低分子肝素对缺血再灌注大鼠肾组织核因子-κB（NF-κB）表达的影响。建立大鼠IRI模型，健康WistaI大鼠80只随机分为正常对照组、假手术组、模型未治疗组、LMWH治疗组，后两组又分别分为术后1、3、6、24h组。检测各组血清肌酐（Scr）水平及中性粒细胞（PMNs）细胞间黏附分子-1（ICAM-1）表达；通过光镜和免疫组织化学方法观察各组大鼠肾组织形态学及趋化因子NF-κB表达变化。结果表明：（1）肾缺血再灌注未治疗组造模后1h，Scr水平虽然没有明显变化，但ICAM-1、NF-κB表达增多，肾小管坏死积分值亦较假手术组明显增加（P〈0．01）；（2）缺血再灌注6h以后，两组Scr浓度明显增高（P〈0．01），但LMWH治疗组SCr、ICAM-1、NF-κB表达水平及肾小管坏死积分值均明显低于模型未治疗组（P〈0．05）；（3）肾组织中NF-κB表达与肾小管损伤积分值呈现良好的相关性（r=0．71，P〈0、01）；而NF-κB与ICAM-1间则呈现显著正相关（r=0．62，P〈0．05）。由此说明：（1）ICAM-1、NF-κB在肾缺血再灌注早期的瞬时表达，可能参与了炎症早期的白细胞迁移与浸润，与肾损伤的发生密切相关；（2）LMWH可通过减少ICAM-1及NF-κB的表达，阻抑炎症反应过程，减轻肾组织损伤。     

小白菊内酯抑制紫杉醇诱导的肿瘤细胞凋亡

目的　探讨NF κB在紫杉醇诱导肿瘤细胞凋亡中的作用及小白菊内酯对紫杉醇诱导凋亡的影响。方法　以人乳癌BCap37细胞和人表皮KB细胞为研究对象 ,用 5,1 0和 2 0 μmol·L-1 小白菊内酯预处理细胞 ,以DNA凋亡梯状条带、DNA含量、MTT、细胞甩片及电泳迁移率变动分析 (EMSA)法检测它对紫杉醇诱导细胞凋亡的影响并探索其作用靶点。结果　通过检测DNA凋亡梯状条带、DNA含量和细胞生存率 ,2 0μmol·L-1 小白菊内酯能显著抑制紫杉醇所诱导的BCap37和KB细胞凋亡 ,但不影响紫杉醇诱导肿瘤细胞G2 /M期阻滞。EMSA实验表明小白菊内酯能抑制紫杉醇诱导激活NF κB。结论　小白菊内酯通过抑制NF κB的激活来抑制紫杉醇所诱导的肿瘤细胞凋亡 ,而紫杉醇诱导肿瘤细胞凋亡的过程可能与G2 /M期阻滞无关     

Effects of Simvastatin on NF-kB-DNA Binding Activity and Monocyte Chemoattractant Protein-1 Expression in a Rabbit Model of Atherosclerosis

To observe the effects of simvastatin on nuclear factor kappaB （NF-kB）-DNA binding activity and on the expression of monocyte chemoattractant protein-1 （MCP-1） in atherosclerotic plaque in rabbits and to explore the anti-atherosclerotic properties beyond its lipid-lowering effects. Thirty-six New Zealand male rabbits were randomly divided into low-cholesterol group （LC）, high- cholesterol group （HC）, high-cholesterol＋ simvastatin group （HC＋S） and then were fed for 12 weeks. At the end of the experiment, standard enzymatic assays, electrophoretic mobility shift as- say （EMSA）, immunohistochemical staining, and morphometry were performed to observe serum lipids, NF-kB-DNA binding activity, MCP-1 protein expression, intirna thickness and plaque area of aorta respectively in all three groups. Our results showed that the serum lipids, NF-kB-DNA binding activity, expression of MCP-1 protein, intima thickness, and plaque area of aorta in the LC and HC＋S groups were significantly lower than those in the HC group （P〈0.05）. There was no significant difference in the serum lipids between the LC and HC＋S groups （P〉0.05）, but the NF-kB-DNA binding activity, the expression of MCP-1 protein and the intirna thickness and plaque area of aorta in the HC＋S group were significantly decreased as compared to the LC group （P〈0. 05）. This study demonstrated that simvastatin could decrease atherosclerosis by inhibiting the NF-kB-DNA binding activity and by reducing the expression of MCP-1 protein.     

Overexpression of the nuclear factor kappaB inhibitor A20 is neurotoxic after an excitotoxic injury to the immature rat brain

Abstract

Background: The zinc finger protein A20 is an ubiquitinating/deubiquitinating enzyme essential for the termination of inflammatory reactions through the inhibition of nuclear factor kappaB (NF-kappaB) signaling. Moreover, it also shows anti-apoptotic activities in some cell types and proapoptotic/pronecrotic effects in others. Although it is known that the regulation of inflammatory and cell death processes are critical in proper brain functioning and that A20 mRNA is expressed in the CNS, its role in the brain under physiological and pathological conditions is still unknown.

Methods: In the present study, we have evaluated the effects of A20 overexpression in mixed cortical cultures in basal conditions: the in vivo pattern of endogenous A20 expression in the control and N-methyl-d-aspartate (NMDA) excitotoxically damaged postnatal day 9 immature rat brain, and the post-injury effects of A20 overexpression in the same lesion model.

Results: Our results show that overexpression of A20 in mixed cortical cultures induced significant neuronal death by decreasing neuronal cell counts by 45±9%. In vivo analysis of endogenous A20 expression showed widespread expression in gray matter, mainly in neuronal cells. However, after NMDA-induced excitotoxicity, neuronal A20 was downregulated in the neurodegenerating cortex and striatum at 10–24 hours post-lesion, and it was re-expressed at longer survival times in reactive astrocytes located mainly in the lesion border. When A20 was overexpressed in vivo 2 hours after the excitotoxic damage, the lesion volume at 3 days post-lesion showed a significant increase (20·8±7·0%). No A20-induced changes were observed in the astroglial response to injury.

Conclusions: A20 is found in neuronal cells in normal conditions and is also expressed in astrocytes after brain damage, and its overexpression is neurotoxic for cortical neurons in basal mixed neuron–glia culture conditions and exacerbates postnatal brain excitotoxic damage.     

丙型肝炎病毒核心蛋白细胞内信号传导途径的探讨

目的：研究丙型肝炎病毒核心蛋白致病作用的细胞内信号传导途径（STPs)。方法：在前期工作已证实HCV核心蛋白激活NF－κB介导的细胞内STPs这一结论的基础上，对核心蛋白活化NF－κB的机制和作用位点进行了测定。用0．4μg pCXN2-core与pNF-κB共转染Hela细胞3h后分别加入5mmol IKKβ的抑制剂乙酰水杨酸和25μmol消炎痛（作为对照）各0．5ml，继续转染33h，测定荧光素酶比活性，以此检测乙酰水杨酸对NF－κB介导的STPs的抑制作用。以pCXN2-core转染Cos-7细胞36h，收获细胞溶解液，用Western印迹分析检测IκBα表达，确定IκBα的降解情况。用pCXN2-core、pMEKKΔ（表达非活性MEKK的质粒）、pNF-κB共转Hela细胞36h，测定荧光素酶比活性，确定HCV核心蛋白是否通过MEKK活化NF－κB介导的STPs。结果：（1）HCV核心蛋白活化NF－κB介导的STPs被乙酰水杨酸抑制；（2）pCXN2-core转染后的细胞溶 解液中IκBα的量较pCXN2转染后的少；（3）HCV核心蛋白活化NF－κB介导的STPs的作用不被MEKKΔ阻断。结论：（1）乙酰水杨酸通过抑制KK的IκBα磷酸化作用，抑制IκB降解，阻断NF－κB的活化，表明核心蛋白作用位点可能在IKK或其上游某个部位；（2）在表达HCV核心蛋白的细胞存在IκBα降解的增加，造成NF－κB的活化；（3）核心蛋白的作用位点处于MEKK作用点下游。（4）HCV核心蛋白可能的细胞内信号传导途径是：HCV核心蛋白直接作用于IKK→IKK磷酸化IκBα→IκBα降解→NF－κB脱离抑制从胞浆转入胞核，与DNA结合发挥作用。     

实验性结肠炎大鼠内毒素-Toll样受体4-核因子κB信号通路的变化及益生菌的作用

目的探讨和分析实验性结肠炎大鼠血浆内毒素水平的变化及结肠Toll样受体4（TLR4）和核因子κB（NF-κB）表达及意义,并分析益生菌的作用。方法30只雄性W istar大鼠均分为正常对照组（NC组）、模型对照组（UC组）和益生菌治疗组（PC组）;UC和PC组建立2,4,6-三硝基苯磺酸（TNBS）实验性结肠炎大鼠模型,PC组大鼠给予双歧三联活菌悬液（2.2×10^9CFU/只）治疗,1次/d,共4周。光镜下观察肠黏膜炎性反应并评分;鲎试验检测各组大鼠血浆内毒素水平;W estern印迹法和实时荧光定量PCR法检测大鼠结肠TLR4和NF-κB p65蛋白及mRNA的表达,分析其变化及益生菌的作用。结果PC组炎性反应评分较UC组明显降低（P〈0.05）,但高于NC组（P〈0.01）;PC组血浆内毒素明显低于UC组（P〈0.05）,但高于NC组（P〈0.01）;UC组TLR4和NF-κB p65的蛋白及mRNA表达明显高于PC组和NC组（P〈0.05、0.01）,PC组高于NC组（P〈0.01）。结论内毒素-TLR4-NF-κB信号通路参与了大鼠实验性结肠炎的发生和发展,减少内毒素的产生、降低大鼠结肠TLR4和NF-κB表达可能是益生菌减轻大鼠结肠炎症的机制之一。     

膀胱移行细胞癌组织中NF-κB p65、uPA、Bcl-2的表达变化及其意义

目的观察膀胱移行细胞癌（下称膀胱癌）组织中NF—κBp65和尿激酶型纤溶酶原激活物（uPA）及Bcl-2的表达变化,并探讨其相关性。方法采用免疫组化SP法对40例膀胱癌组织和10例正常膀胱组织中NF—κBp65、uPA、Bcl-2进行测定,分析三者与膀胱癌分级、分期、侵袭转移及复发的关系及NF-κBp65表达与uPA、Bcl-2的相关性。结果NF-κBp65、uPA、Bcl-2在膀胱癌组织中的表达均明显高于正常膀胱组织（P〈0．05）;NF—κBp65和uPA与膀胱癌病理分级、分期、淋巴结转移有关（P均〈0．05）,Bcl-2与膀胱癌病理分级、分期有关（P〈0．05）,与淋巴结转移无关（P〉0．05）;膀胱癌组织中,NF—κBp65表达与uPA、Bcl-2表达呈正相关（r分别为0．388、0．462,P均〈0．05）。结论在膀胱癌组织中NF-κBp65、uPA、Bcl-2均呈高表达,并与膀胱癌的临床病理学特征有关;NF—κBp65可能通过调控uPA、Bcl-2的表达促进膀胱癌的进展;NF—κBp65、uPA、Bcl-2可以作为判断膀胱癌侵袭性、转移及预后的生物学标志物。