Cannabinoid signalling in TNF-alpha induced IL-8 release

The molecular events mediating the immunomodulatory properties of cannabinoids have remained largely unresolved. We have therefore investigated the molecular mechanism(s) through which R-(+)-[2,3-dihydro-5-methyl-3-[(morpholinyl)methyl] pyrrolo[1,2,3-de]-1,4-benzoxazinyl]-(1-napthanlenyl) methanone (WIN55212-2) modulate production of interleukin-8 (IL-8) in HT-29 cells. Release of IL-8 induced by tumor necrosis factor-alpha (TNF-alpha) was determined by enzyme-linked immunosorbent assay (ELISA). Changes in expression of inhibitory kappa B (IkappaB) were monitored by Western blotting and activation of nuclear factor-kappa B (NF-kappaB) was determined in electrophoretic mobility shift assay (EMSAs). TNF-alpha induced release of IL-8 was inhibited by WIN55212-2 which also blocked the degradation of IkappaB-alpha and activation of NF-kappaB induced by TNF-alpha. These data provide strong evidence that WIN55212-2 may modulate IL-8 release by negatively regulating the signaling cascade leading to the activation of NF-kappaB. These findings highlight a potential mechanism for the immunomodulatory properties of cannabinoids and contribute towards acquiring a clear understanding of the role of cannabinoids in inflammation.     

Role of cyclooxygenases in angiogenesis

Angiogenesis is the process by which new blood vessels are formed. This process supports normal physiology as well as contributes to progression of disease. Progressive rheumatoid arthritis and growth of tumors are two pathologies to which angiogenesis contributes. In arthritis, we know that prostaglandins (PGs) and the enzyme cyclooxygenase-2, which catalyses prostaglandin production, are inflammatory mediators. These mediators are involved in rheumatoid arthritis and cancer-induced angiogenic processes. We discuss, herein, recent findings on the expression of cyclooxygenases in both rheumatoid arthritis and human cancer, and the links between COX-2, PGs, and angiogenesis. We also propose a model for the possible mechanistic interaction of the various cell types involved in angiogenesis.     

Role of aminopeptidase in angiogenesis

Microvessels are composed of endothelial cells and surrounding pericytes. Angiogenesis, a neo-vessel formation from pre-existing microvessels, is a complex phenomenon, which requires following sequential steps: detachment of pre-existing pericytes for vascular destabilization, extracellular matrix turnover, migration, proliferation, tube formation by endothelial cells (ECs), and reattachment of pericytes for vascular stabilization. Aminopeptidases regulate the N-terminal modification of proteins and peptides for maturation, activation or degradation, and thereby relate to a variety of biological processes. Recently, three aminopeptidases have been reported to be involved in angiogenesis. They include type 2 methionine aminopeptidase, aminopeptidase N, and adipocyte-derived leucine aminopeptidase/puromycin insensitive leucyl-specific aminopeptidase. This review will focus on the possible role of these aminopeptidases in angiogenesis.     

Role of p38 MAPK in the selective release of IL-8 induced by chemical allergen in naive THp-1 cells.

At present, the assessment of the allergenic potential of chemicals is carried out using animal models. Over the last decade, several in vitro methods mainly using primary dendritic cells have been proposed to identify the potential of chemicals to induce skin sensitization to meet current animal welfare and public opinions. The major limitations of such tests are the donor-to-donor variability, the low levels in the source, and a possible shortage of human sources.The aim of the present investigation was to establish an in vitro test to identify chemical allergens using the human promyelocytic cell line THP-1 in order to avoid some of these difficulties. We investigated whether the chemokine interleukin-8 or CXCL8 (IL-8) production could provide a methodology for the detection of both respiratory and contact allergens. THP-1 cells were exposed to contact allergens (cinnamaldehyde, dinitrochlorobenzene, nickel sulfate, penicillin G, p-phenylenediamine, tetramethylthiuram disulfide), to respiratory allergens (ammonium hexachloroplatinate, diphenylmethane diisocyanate, trimellitic anhydride) and to irritants (salicylic acid, phenol, sodium lauryl sulphate). Following 48 h of incubation, the release of IL-8 was evaluated by sandwich ELISA. IL-8 production was significantly increased after stimulation with all allergens tested, with the exception of trimellitic anhydride, whereas irritants exposure failed to induce IL-8 release. The lack of IL-8 production by trimellitic anhydride can be explained by the rapid hydrolysis of this chemical in water to trimellitic acid, which is not an allergen. In contrast to IL-8 release, CD54 and CD86 expression did not provide a sensitive method failing to correctly identify approximately 30% of the tested compounds. Although CD86 appears to be a more sensitive marker than CD54 when discriminating allergens from irritants neither of these markers provided robust methodology. We also investigated if a common activation pathway in allergen-induced IL-8 production involving p38 mitogen-activated protein kinase could be identified. By Western blot analysis we could indeed demonstrate p38 activation by all chemical allergens tested and, using the selective p38 MAPK inhibitor SB203580, a significant modulation of allergen-induced IL-8 release could be achieved in all cases.Our data suggests that production of IL-8 by na?ve THP-1 cells may represent a promising in vitro model for the screening of potential chemical allergens and activation of p38 MAPK represents a common pathway triggered by allergens.     

Role of c-jun N-terminal kinase in the induced release of GM-CSF,RANTES and IL-8 from human airway smooth muscle cells

1. Human airway smooth muscle cells (HASMC) contribute to airway inflammation in asthma by virtue of their capacity to produce several inflammatory mediators including IL-8, GM-CSF and RANTES. The intracellular signal pathway underlying the production of these cytokines in HASMC is not entirely elucidated. 2. We examined the role of the mitogen-activated protein kinase (MAPK) c-jun N-terminal kinase (JNK) in TNFalpha- and IL-1beta-induced GM-CSF, RANTES and IL-8 production in HASMC by using a novel specific inhibitor for JNK (SP600125). 3. Confluent HASMC were treated with TNFalpha or IL-1beta (10 ng ml(-1)) for 24 h in the presence or absence of SP600125 (1-100 micro M). JNK activity was determined by a kinase assay. Phosphorylation of JNK, p38 MAPK and ERK was examined by Western blotting. Culture supernatants were assayed for GM-CSF, RANTES and IL-8 content by ELISA. 4. Maximum TNFalpha- or IL-1beta-induced phosphorylation of JNK in HASMC occurred after 15 min and returned to baseline levels after 4 h. SP600125 inhibited TNFalpha- and IL-1beta-induced JNK activity in HASMC as shown by the reduced phosphorylation of its substrate c-jun. Furthermore, GM-CSF, RANTES and to a lesser extent IL-8 release from HASMC treated with TNFalpha and IL-1beta was inhibited dosedependently by SP600125. 5. JNK activation is involved in TNFalpha- and IL-1beta-induced GM-CSF, RANTES and IL-8 production from HASMC. JNK may therefore represent a critical pathway for cytokine production in HASMC.     

Role of inflammatory mediators in angiogenesis

The angiogenic process involves several cell types and mediators, which interact to establish a specific microenvironment suitable for the formation of new capillaries from pre-existing vessels. Angiogenesis occurs in several physiological and pathological conditions, such as embryo development and wound healing, diabetic retinopathy and tumours. Inflammatory cells, namely monocytes/macrophages, T lymphocytes and neutrophils, fully participate in the angiogenic process by secreting cytokines that may affect endothelial cell (EC) functions, including EC proliferation, migration and activation. Angiogenesis is the result of a net balance between the activities exerted by positive and negative regulators. With regards to inflammatory cells and endothelium cross-talk, such balance is conceptually very similar to that of pro-inflammatory and anti-inflammatory mediators that modulate an appropriate inflammatory response. In this review we will mainly discuss the relevance of both physiological and pathological inflammatory processes in angiogenesis, with particular regards to microenvironmental contribution. We will also describe some of the most relevant pro-inflammatory cytokines in the modulation of the angiogenic process. Furthermore, we will concentrate on what has been recently reported about the mechanism by which some of these cytokines are induced during inflammation to promote a suitable microenvironment for angiogenesis and tumour progression. Pro-angiogenic cytokines, such as IL-1 and TNF, and anti-angiogenic cytokines such as IFN-gamma and IL-12, will be briefly described. We will try to provide a rationale for the use of both cytokines and cytokine blockades as novel potential pharmaceutical targets to modulate angiogenesis in chronic inflammation as well as in cancer.     

葡萄膜黑色素瘤高转移相关基因的筛选和分析

目的探讨葡萄膜黑色素原位瘤与转移瘤差异表达基因及其作用;探索乳腺癌1号基因相关蛋白1(BAP1)缺失的葡萄膜黑色素瘤(UM)差异表达基因以及预测BAP1靶标,探讨BAP1缺失对UM转移作用及其机制。方法生信分析筛选UM转移差异表达基因及其功能,对公共数据库GEO中的数据集GSE27-831,GSE48863以及GSE39171中芯片数据以2为底数进行对数转换以及标准化(P<0.05,|logFC|>1)等预处理。为了进一步捕捉术语之间的关系,选择丰富的术语的子集,并将其作为网络图绘制,其中相似性>0.3的项由边缘连接。我们从20个簇中的每个簇中选择具有最佳P值的项,约束每个集群不超过15个术语,总共不超过250个术语。该网络用CytoSCAPE可视化插件,其中每个节点表示1个丰富的术语,并用其簇的名称着色。对于每个给定的基因列表,利用以下数据库进行蛋白质-蛋白质相互作用富集分析:BioGrid、InWeb_IM、OmniPath。所得到的网络至少包含与另一个列表成员形成交互的蛋白质子集。如果网络中包含3～500个蛋白质,则进一步应用分子复合检测(MCODE)算法来识别紧密连接的网络组件。路径和过程富集分析被独立地应用于每个MCODE组件,并且保留3个最佳评分(按P值,P<0.05,FDR<0.001生物途径视为有价值)术语作为对应组件的功能描述。结果我们运用生物信息学技术对转移性和非转移性UM组织样本和BAP1沉默和正常表达UM细胞样本基于微阵列对基因组数据进行基因本体富集(GO)分析和京都基因和基因组百科全书(KEGG)分析,结果显示差异基因主要富集在补体凝血级联途径。结论 BAP1在转移性UM的异常低表达,UM中BAP1突变可能通过影响肿瘤血管生成促进肿瘤浸润转移。     

Fisetin induces apoptosis through mitochondrial apoptosis pathway in human uveal melanoma cells

Fisetin, a diatery flavonoid, been reported that possess anticancer effects in various cancers. The purpose of the study was to investigate the antitumor effects of fisetin in cultured uveal melanoma cell lines and compared with normal retinal pigment epithelial (RPE) cells. MTT assay was used for evaluating cytotoxic effects of fisetin. Flow cytometry study was used for the determination of apoptosis. JC‐1 fluorescent reader was used to determine mitochondrial transmembrane potential changes. The results shown that fisetin dose‐dependently decreased the cell viability of uveal melanoma cells but not influenced the cell viability of RPE cells. Apoptosis of uveal melanoma cells was induced by fisetin efficiently. Fisetin inhibited antiapoptotic Bcl‐2 family proteins and damaged the mitochondrial transmembrane potential. The levels of proapoptotic Bcl‐2 proteins, cytochrome c, and various caspase activities were increased by fisetin. In conclusion, fisetin induces apoptosis of uveal melanoma cells selectively and may be a promising agent to be explored for the treatment of uveal melanoma.     

The role of CD18 in IL-8 induced dermal and synovial inflammation.

1. The intradermal administration of endothelial IL-8 (IL-8(1-77) or monocyte derived IL-8 (IL-8(1-72) to rabbits produced a concentration-dependent increase in plasma extravasation and an accumulation of polymorphonuclear leukocytes (PMNs) when measured over a 3 h time period. When plasma extravasation and PMN accumulation were measured over a 30 min time period no significant increases in PMN accumulation or plasma extravasation were observed in response to IL-8 alone. However, under these conditions, the addition of prostaglandin E2 (100 pmol) produced a significant potentiation of IL-8-induced plasma extravasation. There was no significant difference between the biological activities of IL-8(1-77) and IL-8(1-72). 2. Plasma extravasation and PMN accumulation induced by IL-8 were inhibited in rabbits pretreated with the monoclonal antibody designated IB4 (1 mg kg-1, i.v.) directed against the common beta chain (CD18) of the leukocyte integrins. 3. The intra-articular administration to rabbits of IL-8(1-77) (1 nmol) resulted 24 h later in the appearance of a mixed population of leukocytes (PMNs and mononuclear cells) in synovial lavage fluid. Biochemical analyses revealed the presence of an increased level of sulphated proteoglycans (sPG) and of the metalloproteinase stromelysin. Pretreatment of rabbits with IB4 (3 mg kg-1, i.v.) inhibited the accumulation of PMNs but had no effect on the mononuclear infiltrate nor on the levels of sPG or stromelysin. 4. The intradermal or intra-articular injection of E. coli-derived endotoxin induced similar inflammatory changes to those observed with IL-8.(ABSTRACT TRUNCATED AT 250 WORDS)     

The role of angiogenesis inhibitors in the management of melanoma

Metastatic melanoma has a very poor prognosis and systemic therapies - both cytotoxic and biological - have not improved outcome in this disease so far. For this reason, novel therapeutic strategies are urgently required. Angiogenesis represents a relevant process to modulate in melanoma, as pro-angiogenic ligands and their receptors are overexpressed and have been found to correlate with disease progression and prognosis. The angiogenic axis may be targeted at many different levels, which are still being defined. This article presents an overview of the importance of angiogenesis in melanoma and draws attention to some of the key molecules that are currently being targeted rationally within clinical studies. We discuss a number of anti-angiogenic and anti-vascular agents and their mechanisms of action. An overview of the efficacy and toxicity of these treatments in clinical trials performed so far in melanoma is presented and future directions for anti-angiogenic strategies in melanoma are considered.     

Berberine hydrochloride counteracts enhanced IL-8 expression induced by SN 38 in AGS cells

Abstract

IL-8 over-expression could enhance cancer metastasis. In present study, berberine hydrochloride (BER) triggered proliferative inhibition and G2/M arrest in AGS cells, down-regulated protein expression of cyclin B1, Bcl-2, up-regulated expression of p21, p53 and cleaved caspase 3, but showed no effect on protein expression of CHOP, Bip, and caspase 4. BER could down-regulate the enhanced IL-8 expression through down-regulating ERK1/2 and p38 MAPK over-activation induced by SN 38. The increased IL-8 mediated adhesive ability of AGS cells to HUVECs induced by SN 38, could be reduced by BER. Thus, BER could reduce the side-effect of SN 38 in clinic.     

Role of matrikins in melanoma progression

Expression of melanoma invasiveness, ultimately leading to the formation of metastases, requires that cancer cells break through the successive skin barriers (dermo-epidermal junction, dermis) constituted of various extracellular matrix constituents. In order to facilitate their progression, melanoma cells express, in concert with stromal cells, a group of proteolytic systems which degrade this extracellular structures. However, proteolysis of basement membrane, collagen or elastic fibers can uncover cryptic sites or/and liberate matrix fragments whose properties appeared distinct from their intact macromolecule counterparts. Those fragments, called matrikines, are able to empede or to accelerate melanoma progression ex vivo and in vivo. Non-collagenous domains of basement membrane collagens, which behave like potent "matstatins", are seen as potential pharmacological agents in melanoma.     

广州城区大气细颗粒物对PC-12细胞IL-8表达的影响

目的 研究广州城区大气细颗粒物(PM2.5)对PC-12细胞IL-8表达的影响,并从p38 MAPK途径探索其作用机制,促进对PM2.5毒性的认识.方法 采集广州城区大气中的PM2.5,对PC-12细胞染毒,设对照组、不同浓度PM2.5组和SB203580+PM2.5组(用20 μ mol/L的SB230580预处理1h后再给予100μg/ml的PM2.5),Trizol法提取RNA用定量PCR法检测细胞IL-8表达情况,RIRP法提取细胞总蛋白,用Western blot法检测p38 MAPK的磷酸化改变.结果 定量PCR检测显示用25、50和100μg/ml的PM2.5染毒后PC-12细胞IL-8表达明显增高;Western blot结果显示PM2.5可磷酸化激活p38 MAPK信号分子,加入p38 MAPK抑制剂SB230580可以抑制PM2.5诱导的IL-8表达.结论 PM2.5可通过p38 MAPK途径诱导PC-12细胞表达细胞因子IL-8,这可能是其导致神经细胞毒性的机制之一.     

caspase-8在卡铂诱导人食管鳞癌细胞凋亡中的作用研究

目的：探讨caspase-8在卡铂诱导人食管鳞癌细胞凋亡中的作用。方法：流式细胞术分别检测对照组及经卡铂处理的实验组人食管鳞癌细胞株Eca-109、TE-1、TE-10的凋亡率,同时采用RT-PCR及westernblotting检测各组细胞表达caspase-8mRNA及蛋白的变化情况。结果：用终浓度40μg/ml的卡铂可诱导人食管鳞癌细胞株Eca-109、TE-1、TE-10凋亡,同时发现经卡铂处理组Eca-109、TE-1、TE-10细胞表达caspase-8mRNA及蛋白水平均呈显著升高趋势。结论：卡铂可增加caspase-8在食管鳞癌细胞中的表达,从而诱导人食管鳞癌细胞凋亡,这可能是卡铂治疗食管鳞癌的分子机制之一。     

Targeting IL-8 in colorectal cancer

Introduction: Colorectal cancer is the leading cause of death from gastrointestinal malignancy in the US. Chemokines and their receptors are being recognized as key regulators of cancers and increasingly as therapeutic targets for metastatic cancers, including colorectal cancer. Several studies have demonstrated that IL-8 and its receptor CXCR2 are two of the most significantly upregulated chemokines in colorectal cancer. IL-8 through binding to its receptors can act not only on inflammatory responses and infectious diseases, but also on cancer cells via their receptors to promote migration, invasion and proliferation, and in vivo angiogenesis. Therefore, IL-8 and CXCR2 may be important therapeutic targets against colorectal cancer.

Areas covered: This review provides an update on the roles of IL-8 and its receptors in colorectal cancer preclinical models and translational relevance: i) Increased expression of IL-8 and/or its receptors has been characterized in colon cancer cells; ii) IL-8 signaling pathway in colorectal cancer cells; iii) targeting IL-8 expression, or receptor-targeted strategies in colorectal cancer, eliminates the redundant function of IL-8 signaling and determines the effects of suppressing IL-8 signaling on tumor progression and development.

Expert opinion: IL-8 and its receptor CXCR2 may function as significant regulatory factors within the tumor microenvironment and be important therapeutic targets in colorectal cancers. Not only may they lead to antitumor properties, but also they may chemosensitize the tumor toward the current chemotherapy.     

Treosulfan and gemcitabine in metastatic uveal melanoma patients: results of a multicenter feasibility study

No effective treatment currently exists for metastatic uveal melanoma. However, recent results obtained by an ATP-based tumor chemosensitivity assay have shown consistent activity of treosulfan+gemcitabine in up to 80% of tumor specimens tested. In this study we describe the first clinical results observed with this drug combination at different European centers in patients with metastatic uveal melanoma. Clinical case series of patients with metastatic uveal melanoma were treated with treosulfan+gemcitabine at seven different centers. Fourteen patients, 13 previously untreated and one pretreated with chemoimmunotherapy, were included in the study. Patients received treosulfan+gemcitabine in four different dose regimens. The response rates, progression-free and overall survival, and toxicity were evaluated. The analysis of 14 patients revealed one complete response, three partial responses and a stable disease in eight cases. The objective response rate was 28.6%, the median overall survival was 61 weeks [95% confidence interval (CI) 54-133 weeks], the progression-free survival was 28.5 weeks (95% CI 13-62 weeks) and the 1-year survival rate was 80%. The drugs were well tolerated. The most common side-effects were leuko- and thrombocytopenia. These preliminary results suggest potential therapeutic benefit of treosulfan+gemcitabine treatment in metastatic uveal melanoma and warrant further controlled studies.     

吞噬细胞运动蛋白1在IL-8诱导的乳腺癌细胞的侵袭和转移中发挥重要作用

目的探讨吞噬细胞运动蛋白1(engulfment and cell motility 1,ELMO1)在IL-8诱导的乳腺癌细胞侵袭和转移过程中的作用。方法采用趋化运动实验检测不同浓度IL-8刺激下乳腺癌细胞的趋化运动能力;采用Western blot检测乳腺癌细胞中ELMO1的表达情况;利用小RNA干扰技术,瞬时转染乳腺癌细胞MDA-MB-231,用过表达质粒上调乳腺癌细胞MCF-7中ELMO1的表达;应用趋化运动实验和Transwell侵袭实验检测各组转染细胞的趋化和侵袭能力。结果趋化运动实验结果显示,在IL-8刺激下,乳腺癌细胞MDA-MB-231和MCF-7的运动能力明显增强,具有剂量依赖性;Western blot结果显示ELMO1在si ELMO1/MDA-MB-231细胞中的表达明显降低,而在MCF-7/ELMO1细胞中的表达明显增高;趋化运动实验结果显示在IL-8刺激下,Si ELMO1/MDA-MB-231细胞组的趋化运动能力明显降低,MCF-7/ELMO1细胞组的趋化运动能力明显增强;Transwell侵袭实验结果显示在IL-8刺激下,敲除ELMO1明显降低MDA-MB-231细胞的侵袭能力,过表达ELMO1明显增强MCF-7细胞的侵袭能力。结论 IL-8能促进MDA-MB-231和MCF-7细胞的趋化运动和侵袭能力,而ELMO1在IL-8诱导的乳腺癌细胞趋化和侵袭作用中发挥重要作用。     

CCK-8对LPS诱导小鼠骨髓来源树突状细胞分泌IL-12的影响

目的探讨八肽胆囊收缩素(cholecystokinin-octopep-tide,CCK-8)对脂多糖(lipopolysaccharide,LPS)诱导小鼠骨髓来源的树突状细胞(bone marrow-derived dendritic cell,BM-DC)分泌IL-12的影响。方法应用免疫荧光技术观察BM-DC表面CCK受体表达情况,酶联免疫吸附(ELISA)方法检测CCK-8对LPS诱导BM-DC分泌IL-12的影响,Western blot技术检测CCK-8对LPS诱导BM-DC细胞p38MAPK磷酸化的影响。结果BM-DC表面存在CCK-1R和CCK-2R;CCK-8促进LPS对BM-DC表达IL-12的诱导作用呈剂量依赖性(10-10、10-8、10-6mol·L-1);并能增加其p38磷酸化水平;CCK的1、2受体拮抗剂CR1409或CR2945均可减弱CCK-8的此种效应。结论CCK-8剂量依赖性促进了LPS诱导BM-DC分泌的IL-12,该作用由CCK-1R和CCK-2R介导,可能是通过促进p38MAPK的磷酸化作用而实现的。