Upregulation of vascular endothelial growth factor by cobalt chloride-simulated hypoxia is mediated by persistent induction of cyclooxygenase-2 in a metastatic human prostate cancer cell line

Upregulation of vascular endothelial growth factor (VEGF) expression induced by hypoxia is crucial event leading to neovascularization. Cyclooxygenase-2, an inducible enzyme that catalyzes the formation of prostaglandins (PGs) from arachidonic acid, has been demonstrated to be induced by hypoxia and play role in angiogenesis and metastasis. To investigate the potential effect of COX-2 on hypoxia-induced VEGF expression in prostate cancer. We examined the relationship between COX-2 expression and VEGF induction in response to cobalt chloride (CoCl₂)-simulated hypoxia in three human prostate cancer cell lines with differing biological phenotypes. Northern blotting and ELISA revealed that all three tested cell lines constitutively expressed VEGF mRNA, and secreted VEGF protein to different degrees (LNCaP > PC-3 > PC3ML). However, these cell lines differed in the ability to produce VEGF in the presence of CoCl₂-simulated hypoxia. CoCl₂ treatment resulted in 40% and 75% increases in VEGF mRNA, and 50% and 95% in protein secretion by LNCaP and PC-3 cell lines, respectively. In contrast, PC-3ML cell line, a PC-3 subline with highly invasive, metastatic phenotype, exhibits a dramatic upregulation of VEGF, 5.6-fold in mRNA and 6.3-fold in protein secretion after treatment with CoCl₂. The upregulation of VEGF in PC-3ML cells is accompanied by a persistent induction of COX-2 mRNA (6.5-fold) and protein (5-fold). Whereas COX-2 expression is only transiently induced in PC-3 cells and not affected by CoCl₂ in LNCaP cells. Moreover, the increases in VEGF mRNA and protein secretion induced by CoCl₂ in PC-3ML cells were significantly suppressed following exposure to NS398, a selective COX-2 inhibitor. Finally, the effect of COX-2 inhibition on CoCl₂-induced VEGF production was reversed by the treatment with exogenous PGE₂. Our data demonstrate that VEGF induction by cobalt chloride-simulated hypoxia is maintained by a concomitant, persistent induction of COX-2 expression and sustained elevation of PGE₂ synthesis in a human metastatic prostate cancer cell line, and suggest that COX-2 activity, reflected by PGE₂ production, is involved in hypoxia-induced VEGF expression, and thus, modulates prostatic tumor angiogenesis. This revised version was published online in July 2006 with corrections to the Cover Date.     

Selective prostaglandin G/H synthase (PGHS)-2 inhibitors show greater inhibitory activities on human PGHS-2 than on murine PGHS-2 in intact cells

Excess eicosanoid formation during inflammation has been attributed to the expression of the gene coding for the inducible isoform of prostaglandin G/H synthase (PGHS-2). Human and murine PGHS-2 proteins differ in 73 out of the 604 amino acids. When comparing the inhibitory effects of a panel of PGHS-inhibitors in a whole cell human and murine PGHS-2 assay carried out under identical conditions, classical NSAIDs with the exception of aspirin and tenoxicam showed similar inhibitory effects on both human and murine PGHS-2 enzymes. However, the PGHS-2 selective inhibitors nimesulide, flosulide and NS398 showed a much greater inhibition of human PGHS-2. We suggest that these differences could be due to the genetic differences of human and murine PGHS-2. Formerly R&D Division of Hafslund Nycomed Pharma AG, Austria.     

NS-398诱导肝癌细胞凋亡的实验研究

目的 :研究选择性环氧化酶 2 (COX 2 )抑制剂NS 398对人肝癌细胞HepG2 凋亡的影响 ,及其相关蛋白Bcl 2在细胞凋亡中的作用。方法 :通过体外细胞培养 ,应用荧光显微镜、透射电镜、流式细胞仪观察NS 398对HepG2 的凋亡诱导作用 ,应用免疫细胞化学法观察Bcl 2在人肝癌细胞HepG2 凋亡中的表达。结果 :NS 398处理HepG2 细胞后 ,电镜下可见到细胞核固缩、染色质凝集成新月型紧靠核膜周边 ,核碎裂、染色质片断化等典型的细胞凋亡形态变化。荧光显微镜及流式细胞检测则未见凋亡征象。免疫细胞化学分析显示 ,NS 398处理后的HepG2 细胞 ,其Bcl 2表达较对照组明显下降。结论 :COX 2选择性抑制剂NS 398对人肝癌HepG2 细胞有显著的凋亡诱导作用 ;抗凋亡基因Bcl 2在NS 398诱导的HepG2 细胞凋亡中有重要的调控作用。     

选择性COX-2抑制剂NS-398的制备

目的制备选择性COX-2抑制剂NS-398。方法以2-氟硝基苯为起始原料,经4步反应化合成化合物NS-398。结果与结论用该方法可以在温和条件下合成出该药,最终产品纯度大于99%。     

Overactive bladder treatments in early phase clinical trials

‘Overactive bladder’ (OAB) is a syndrome that is characterised by symptoms of urgency, with or without urge urinary incontinence, usually with frequency and nocturia . It is a highly prevalent condition affecting 17% of the general population, with a significant negative effect on quality of life, impairing several areas with physical, social, emotional and sexual limitations. The prevalence of OAB increases with age in both men and women . The pathophysiology is multifactorial and not yet fully understood. Non-surgical treatment is the mainstay of therapy for OAB. The available options include biofeedback, electrical stimulation, bladder training, pharmacotherapy or a combination of these options. Nevertheless pharmacotherapy is still the treatment of choice for OAB symptoms . The pharmacological treatment of OAB is generally directed towards the central or the peripheral neural control pathways or the detrusor muscle . The antimuscarinic drugs are the most commonly used. In the US, approved antimuscarinics include oxybutynin, tolterodine, trospium chloride, solifenacin and darifenacin. Although this class of drugs has been shown to be more effective than placebo in specific meta-analyses , it has been reported that ≤ 80% of the patients discontinue the treatment within 6 months, mainly for the low drug compliance due to the high incidence of side effects . Therefore, there is a strong need to identify drugs with novel mechanisms of action, which could provide equal or even better efficacy and overall greater acceptability than antimuscarinic drugs. At present, several other specific molecular targets identified within detrusor muscle and/or neural systems are under investigation for the development of more specific treatments of OAB. This article provides an up-to date review of drugs that are in investigational preclinical and early stage (Phase I and II) clinical trials for the treatment of OAB.     

食管肿瘤干细胞抗辐射特性的实验研究

目的 :探讨食管肿瘤干细胞的抗辐射特性。方法 :采用无血清培养基培养人食管癌细胞ECA109,分离食管肿瘤干细胞,MTT法检测不同浓度环氧化酶-2选择性抑制剂NS398(2.5、5.0、10.0、20.0、40.0、80.0μmol/L)对细胞增殖的抑制作用,克隆形成实验检测亲本细胞和细胞球的增敏效应,成球实验分析NS398联合X线照射对细胞成球能力的影响,Western blot检测细胞β-catenin蛋白的表达水平。结果:NS398对亲本细胞和细胞球的增殖抑制作用均具有时间、浓度依赖性。20μmol/L NS398作用后,亲本细胞Do、Dq和SF2值均减小(P<0.05),放射增敏比(sensitization enhancement ratio,SER)为1.17;20μmol/L NS398作用后,细胞球Do、SF2减小(P<0.05),SER为1.12。照射使ECA109细胞成球率增加(P<0.05)。NS398联合X线照射组与单纯照射组相比,细胞成球率显著降低(t=7.01,P<0.01),亲本细胞和细胞球β-catenin的表达水平均降低(t=10.15,P<0.01;t=3.225,P<0.05)。结论 :细胞球的增敏效应小于亲本细胞,具有抗辐射特性,其机制可能与抑制细胞β-catenin蛋白表达有关。     

Chronic inhibition of cyclooxygenase-2 induces dendritic hypertrophy and limited functional improvement following motor cortex stroke

The cyclooxygenase-2 (COX-2) enzyme is part of the inflammatory pathway and is induced within the brain by a variety of pathological events, including ischemia. Pharmacological agents that inhibit COX-2 have been found to be neuroprotective in a number of injury models, and long-term administration of these drugs has been shown to induce plastic changes in the brain. In the current experiment, we investigated the effectiveness of stimulating cortical plasticity following stroke injury through the administration of the COX-2 inhibitor drug NS398. Furthermore, we determined whether the induced plastic changes improved functional outcome following motor cortex stroke. Chronic drug administration was found to induce dendritic hypertrophy in cells in the parietal cortex, and this anatomical change was associated with the animals making significantly more reach attempts, as well as successful reaches during a skilled reaching task. Additional motor tests however revealed that the treatment did not affect the level of motor recovery, as the animals showed chronic impairments in the Schallert cylinder, and the forepaw inhibition tasks. Short-term administration of the drug, immediately following the stroke did not induce any dendritic changes, nor was it found to improve behavioral performance on any of the motor tasks. Based on these results we conclude that the plastic changes that are induced by long-term COX-2 inhibitor administration provide some benefit to functional outcome following ischemic cortical injury.     

NS-398对HepG2细胞增殖的抑制作用及机制探讨

目的探讨氮-2,环己氧-4,硝基苯—甲基磺胺（NS-398）对肝癌细胞株HepG2细胞的生长抑制作用及其机制。方法分别用100、200、300、400μmol/L的NS-398处理HepG2细胞,用MTT法测算肿瘤细胞抑制率,流式细胞仪检测细胞周期及细胞凋亡率,用免疫组化和Westernblot检测细胞的血管生长因子（VEGF）。结果NS-398呈剂量依赖性的方式抑制HepG2细胞增殖,并诱导其凋亡,随着NS-398浓度增大,S期细胞明显减少,有G1期细胞累积现象,其24h半数有效剂量（IC50）为300μmol/L。NS-398浓度为200μmol/L以上时HepG2细胞VEGF的表达与对照组相比,P〈0.01。结论NS-398对肝癌细胞增殖有抑制作用,并诱导其凋亡,可能与G1阻滞以及VEGF的表达受抑制有关。     

Discovery and Characterization of ML398, a Potent and Selective Antagonist of the D4 Receptor with in Vivo Activity

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选择性COX-2抑制剂NS398干预后对HIBD新生大鼠海马CA1区存活锥体细胞计数的变化

目的研究新生大鼠缺氧缺血性脑损伤(HIBD)时应用选择性COX-2抑制剂NS398干预后海马CA1区存活锥体细胞计数的变化。方法于制备新生大鼠左脑的HIBD模型前30min,应用腹部注射NS39820mg/kg,同时建立未注射HIBD组及对照组,于HIBD后7d,测试左侧海马CA1区存活锥体细胞数。结果统计学比较,HIBD组CA1区存活锥体细胞计数显著低于对照组;NS398干预组存活锥体细胞数显著高于HIBD组。结论应用选择性COX-2抑制剂NS398可以减少新生鼠HIBD时锥体细胞缺失。