Inhibition of farnesyltransferase reduces angiogenesis by interrupting endothelial cell migration

Inhibitors of farnesyltransferase (FTI) have been developed for cancer treatment for more than a decade. Aside from being a therapeutic target in tumor cells, little is known about the role of farnesyltransferase (FTase) in other physiological processes. In this study, we revealed the involvement of FTase in angiogenesis and showed that FTI inhibited angiogenesis by directly acting on endothelial cells. Inhibition of FTase interrupted cell migration in vitro and in vivo. In addition, we found that FTase was important for cell polarization, cell spreading and pseudopodia formation. We also found that FTase interacted with microtubule end binding protein 1 (EB1) and that this interaction was critical for the localization of EB1 to microtubule tips. Our findings thus offer novel insight into the functions of FTase in endothelial cells and provide valuable information for the use of FTI in cancer therapy.     

Inhibition of cytokine production by hymenialdisine derivatives

We describe herein the synthesis and biological activity of two indoloazepines that are structurally related to the marine sponge metabolite hymenialdisine. The natural product hymenialdisine was found to be a potent inhibitor of interleukin-2 (IC(50) = 2.4 microM) and tumor necrosis factor alpha (IC(50) = 1.4 microM) production. One of the hymenialdisine derived indoloazepines was found to also inhibit interleukin-2 (IC(50) = 3.5 microM) and tumor necrosis factor alpha (IC(50) = 8.2 microM) production.     

Inhibition of prostaglandin synthesis and leucocyte migration by flurbiprofen.

The carrageenan air pouch technique in the rat was used to investigate the effects of flurbiprofen, a non-steroidal anti-inflammatory agent, on prostaglandin production and leucocyte migration. tthere is a suggestion that flurbiprofen was capable of inhibiting prostaglandin synthesis at doses lower than those which were effective in reducing leucocyte migration. Therefore, these two effects may reflect independent actions of the drug. The possible clinical relevance of these experiments has been noted since the effects of flurbiprofen on prostaglandin synthesis and leucocyte migration are produced in the rat at peak plasma concentrations which are less than those found in man after a single dose of 50 mg.     

Inhibition of macrophage migration by C. botulinum exoenzyme C3

C3-like exoenzymes are produced by various microorganism including Clostridium botulinum (C3bot), Bacillus cereus and Staphylococcus aureus. C3bot is the prototype of C3-like exoenzymes that specifically ADP-ribosylates and thereby inactivates Rho(A/B/C). C3-like exoenzymes are not yet regarded as virulence factors, as the lack of cell entry domains results in a poor accessibility of the C3-like exoenzymes to cells. In this study, the sensitivity of various cell lines to C3bot has been reinvestigated. Primary monocytes as well as cultured macrophage-like cells including J774A.1 cells and RAW macrophages exhibit a tenfold higher sensitivity to C3bot than fibroblasts and epithelial cells. RhoA ADP-ribosylation by C3bot resulted in the formation of pronounced bipolar protrusions based on defective tail retraction. The formation of bipolar protrusion resulted in inhibited macrophage migration. These findings suggested that macrophages appear to be target cells of C3bot. Migration of macrophage is a prerequiste for their recruitment to the site of pathogen invasion or tissue damage. Inhibition of macrophage migration likely preserves the survival of C3-producing microorganisms. The observations of this study reinforce the paradigm of a role of C3-like exoenzymes as virulence factors.     

Inhibition of cell proliferation, invasion and migration by ursolic acid in human lung cancer cell lines

Ursolic acid (UA) is a pentacyclic triterpene naturally occurring in many plant foods. Apoptotic, anti-invasive and anti-migratory effects of UA at 2, 4, 8, or 16 μmol/L in human non-small cell lung cancer A549, H3255 and Calu-6 cell lines were examined. The impact of this compound upon associated biomarkers such as vascular endothelial growth factor (VEGF), intercellular adhesion molecule-1 (ICAM-1) and matrix metalloproteinase (MMP) was also evaluated. UA treatments concentration-dependently decreased cell viability, and lowered Na⁺-K⁺-ATPase activity (P < 0.05). This compound at 4–16 μmol/L concentration-dependently increased DNA fragmentation, and reduced VEGF and transforming growth factor beta1 levels in test cancer cells (P < 0.05). UA concentration-dependently suppressed ICAM-1 expression (P < 0.05). This compound significantly declined fibronectin expression (P < 0.05), but concentration-dependent effect was shown in H3255 cells only (P < 0.05). UA treatments significantly suppressed the expression of MMP-9 and MMP-2 (P < 0.05), and inhibited protein kinase C activity in test cell lines (P < 0.05). UA treatments also concentration-dependently reduced cell invasion (P < 0.05); however, this compound at 4–16 μmol/L significantly decreased cell migration (P < 0.05), and concentration-dependent effect was shown in A549 and Calu-6 cells (P < 0.05). These findings suggested that this triterpene was a potent anti-lung cancer agent, and it might be able to retard invasion and metastasis of lung cancer cells.     

Inhibition of cell colony formation by selenite: involvement of glutathione.

Selenium is an essential trace element that has been shown to have anticarcinogenic activity. One mechanism that has been proposed for this activity is a cytotoxic effect of selenium on tumor cells. As a means of assessing its cytotoxicity, we have examined the effect of selenite on tumor cell viability, using as an assay the ability of the cells to form colonies. We have found that brief exposure of HeLa cells to micromolar concentrations of selenite resulted in significant inhibition of colony formation, indicating that this is an assay for selenite cytotoxicity that is more sensitive than those that have been employed previously. In order to investigate the involvement of cellular glutathione in selenite cytotoxicity, we treated cells with buthionine sulfoximine (BSO) before selenite exposure. This treatment, which resulted in a 7-fold reduction in the level of intracellular glutathione, also caused a significant decrease in the inhibitory effect of selenite on colony formation. However, when cells were exposed to selenite that had previously been reacted with glutathione, the BSO-induced decrease in cytotoxicity was eliminated. In contrast, reaction of selenite with other sulfhydryl compounds, such as cysteine and mercaptoethylamine, did not restore its potency in BSO-treated cells. The simplest explanation for these results is that, for selenite to exert its inhibitory effect, it must react with intracellular glutathione to form the selenodiglutathione derivative.     

Inhibition of cytokine production by the herbicide atrazine. Search for nuclear receptor targets

The hematological toxicity of the commonly used triazine herbicides is a cause for concern. In a search for molecular targets of these compounds, as their effects paralleled those seen with dexamethasone (DEX), we first looked for interaction with the glucocorticoid receptor. In contrast to the effects on proliferation and cytokine production of DEX, those induced by atrazine were not prevented by the glucocorticoid antagonist RU486. Also, whereas DEX was able to inhibit the promoter activity of genes regulated by NF-kappaB, atrazine failed to do so. We next looked for interaction with members of the peroxisome proliferator-activated receptor (PPAR) family. No peroxisome proliferation was observed in the liver or kidneys of mice treated with atrazine. Moreover, no PPAR-mediated induction of promoter activity was seen on targets of PPARalpha, PPARgamma, or PPARdelta. Similarly, neither atrazine nor simazine were able to stimulate RORalpha-mediated promoter activity. Finally, no binding of atrazine to the AR was observed. In conclusion, the effects of atrazine-type herbicides most probably do not result from interaction with the above-mentioned nuclear receptors.     

Differential modulation of cytokine production in major depressive disorder by cortisol and dexamethasone

In major depressive disorder (MDD), there is increasing evidence of a relationship between neuroendocrine and immunological alterations. Therefore, we investigated the influence of cortisol and dexamethasone on the in vitro production of TNF-α and IL-6 in blood cells of depressed inpatients at admission, in the course of MDD and in healthy controls. Patients were psychopathologically classified as responders and non-responders after a 6-week antidepressant treatment. At admission in the responder subgroup, incubation with both steroids under basal conditions resulted in an increase of TNF-α levels, which decreased after treatment. After stimulation with phytohemagglutinin, an enhancement of TNF-α suppression by steroids was detectable after successful antidepressive treatment. A significant relationship was seen between the cortisol-induced modulation of TNF-α levels and the psychopathology in this subgroup. Under basal conditions, IL-6 levels were increased after treatment with both steroids. The data suggest a normalization of the altered effects of glucocorticoids on TNF-α production in the responder subgroup only.     

苦参碱对U251胶质瘤细胞的增殖抑制和原癌基因表达的影响

目的　探讨苦参碱对胶质瘤细胞株 (U2 5 1细胞株 )的抑制作用及其作用机制。方法　用MTT比色法检测不同浓度苦参碱对U2 5 1细胞的增殖抑制 ,流式细胞仪观察苦参碱对U2 5 1细胞周期的影响 ,RT PCR观察原癌基因C myc表达的变化。结果　苦参碱对U2 5 1细胞增殖抑制作用成剂量依赖性 ,当浓度为 0 10g·L-1时 ,对U2 5 1细胞增殖的抑制率达到 5 3 7%± 6 0 %。流式细胞仪分析显示 ,用 0 10g·L-1苦参碱培养 3d ,细胞周期中G0 /G1期所占比例增高 ,S期占细胞周期的比例减低。RT PCR结果显示 ,随着苦参碱作用剂量的增加 ,C myc基因的表达被明显抑制。结论　苦参碱对人胶质瘤细胞系U2 5 1的增殖具有明显的抑制作用 ,并对原癌基因C myc的表达具有抑制作用     

Inhibition of prostaglandin synthesis in brain of rat by dexamethasone: Lack of effect of dexamethasone phosphate ester and various hormonal steroids

The present study was designed in order to characterize the inhibitory effect of dexamethasone upon the synthesis of prostaglandins (PG) in the brain of the rat. Rats were treated with dexamethasone (20 mg/kg b.w.) and sacrificed 0–76 hr after administration of the drug. The rate of synthesis and release of PGE₂ was followed by 1 hr of incubation of slices of cortex taken from these rats, in Krebs-Ringer solution. A significant inhibition occurred at 8 hr and maximal inhibition (45%) was attained at 16 hr after injection. A gradual increase in the rate of synthesis up to control values occurred between 24 and 76 hr. A dose-response study, at the range of 2–40 mg/kg, showed that a significant decrease was noted at 6 mg/kg and it was maximal (45% inhibition) at 20 and 40 mg/kg. Administration of dexamethasone-sodium-phosphate, as well as other synthetic glucocorticoids and various steroidal hormones (20 μM), failed to inhibit the biosynthesis of prostaglandins under the same experimental conditions. The effect of dexamethasone and dexamethasone phosphate on synthesis of PGE₂ was also studied under in vitro conditions at 5 and 20μM. When slices of cortex from intact rats were incubated for 1 or 2hr in the presence of either dexamethasone or dexamethasone phosphate only dexamethasone was effective in inhibiting the synthesis of PGE₂.

The present results demonstrate that the inhibition of the synthesis of prostaglandins in brain by dexamethasone is both time- and dose-dependent. The lack of effect of closely related glucocorticoids demonstrate that the effect is highly specific to dexamethasone.     

Inhibition of gelatinase activity in human airway epithelial cells and fibroblasts by dexamethasone and beclomethasone.

The effects of dexamethasone and beclomethasone on gelatinase activity released from lung epithelial cells (A549, NCI-H292 and Calu-3 cell lines and NHBE primary cultures) and human lung fibroblasts (HLF) were investigated. All cells spontaneously released gelatin-degrading activity but the amounts were unaffected by treatment with glucocorticoids. Phorbol myristate acetate (PMA) increased the amount of gelatinase activity in conditioned media prepared from all cell types examined. In epithelial cells, PMA induced the expression of gelatinase B, whereas in HLF the increased gelatinase activity resulted from increased activation of gelatinase A. Dexamethasone and beclomethasone produced concentration-dependent inhibition of PMA-induced gelatinase activity in HLF and epithelial cell lines. In the epithelial cell lines, the inhibition of activity was associated with an attenuation of enzyme induction by PMA. In contrast, primary cultures of human bronchial epithelial cells were unresponsive to dexamethasone at concentrations that were maximally effective at inhibiting gelatinase activity induced in other cells.     

Inhibition of oxidant-induced barrier disruption and protein tyrosine phosphorylation in Caco-2 cell monolayers by epidermal growth factor

The effect of epidermal growth factor (EGF) on the H202-induced increase in paracellular permeability in Caco-2 and T-84 cell monolayers was evaluated to examine the role of EGF in intestinal mucosal protection from oxidative stress. Oxidative stress was induced by exposing cell monolayers to H2O2 or a mixture of xanthine oxidase + xanthine (XO + X). Paracellular permeability was assessed by measuring transepithelial electrical resistance (TER), sodium chloride dilution potential, and unidirectional flux of [3H]mannitol. H2O2 (0.1 to 5.0 mM) reduced TER and dilution potential and increased mannitol flux. Administration of EGF delayed H2O2 and XO + X-induced changes in TER, dilution potential, and [3H]mannitol flux. This protective effect of apically or basally administered EGF was concentration-related, with A50 (95% confidence limits) values of 2.1 (1.17 to 4.34) and 6.0 (4.37 to 8.34) nM, respectively. The EGF-mediated protection was prevented by treatment of cell monolayers with genistein (10 microM), a tyrosine kinase inhibitor. H2O2 and XO + X also induced tyrosine phosphorylation of a number of proteins in Caco-2 and T-84 cell monolayers. EGF treatment inhibited the oxidant-induced tyrosine phosphorylation of proteins, particularly those with a molecular mass of 110-220 kDa. Treatment of Caco-2 cells with anti-transforming growth factor-alpha antibodies potentiated the H2O2-induced changes in TER, dilution potential, and mannitol flux. These studies demonstrated that an EGF receptor-mediated mechanism delays oxidant-induced disruption of the epithelial barrier function, possibly by suppressing the oxidant-induced tyrosine phosphorylation of proteins.     

地塞米松抑制兔内毒素休克进程中血浆ET 1和NO的异常升高

目的　探讨地塞米松（Ｄｅｘ）抗毒作用原理。方法　兔ｉｖ 内毒素６００ μｇ·ｋｇ－ １ 诱发休克,３０ ｍｉｎ 时分别ｉｖ 生理盐水或Ｄｅｘ 、Ｌ ＮＭＡ、ｐｈｏｓｐｈｏｒａｍｉｄｏｎ （ｐｈｏｓ）, 测平均动脉压（ＭＡＰ） 、每搏心输出量（ＳＶ）、和血浆ＥＴ １ 与ＮＯ－３ 浓度。结果　休克兔血浆ＮＯ－３ 、ＥＴ １ 浓度升高, 血流动力学进行性紊乱直至死亡。Ｌ ＮＭＡ 和Ｐｈｏｓ 分别抑制血浆ＮＯ－３ 或ＥＴ １浓度异常升高及基础分泌,短暂改善血流动力学指标。Ｄｅｘ 不影响ＮＯ、ＥＴ １ 基础水平,但抑制休克时兔血浆ＮＯ－３和ＥＴ １ 异常升高、纠正血流动力学紊乱、提高动物生存率。结论　Ｄｅｘ 抑制休克兔血浆ＮＯ、ＥＴ １ 异常升高, 可能是其抗内毒素休克的重要机制之一     

Early changes in murine epidermal cell phenotype by contact sensitizers.

In order to develop an in vitro predictive assay for the detection of contact sensitizers, we investigated the possible modulation of the expression of cell-surface molecules in the early phases of treatment of murine epidermal cells (EC) with known contact sensitizers. After in vitro treatment of Balb/c EC with the strong contact sensitizer, TNBS, Langerhans cells (LCs) demonstrated a rapid up-regulation of CD45, CD40, CD32/16 (Fc gamma RII/III) and CD23 (Fc epsilon RII) molecules. CD45 and CD40 were also rapidly up-regulated on the dendritic epidermal T cells. Interestingly, after treatment with this severe sensitizer, a marked induction of CD40 expression was found on a CD45 negative population, most probably keratinocytes. In contrast to these cell-surface molecules, I-Ad/I-Ed and CD90.2 expression were unchanged. No change was observed on the expression of CD45 and CD40 after treatment with a mild or a weak contact sensitizer, citral and citronellal respectively. In contrast, like TNBS, they up-regulated the expression of CD32/16 and CD23 on LCs. The irritant sodium dodecyl sulfate had no effect on all these cell-surface molecules. Our results indicated that in vitro, chemicals with allergic potential induced early specific phenotype changes that may represent an early-activated state of the cells. This state may be responsible for initiating the afferent phase of contact sensitivity in vivo. Based on these findings, it might be possible to develop an in vitro assay to reduce the number of experimental animals for a fast screening of contact sensitizers and for discriminating between mild contact sensitizers and irritants.