Cyclooxygenase-2 and tissue inhibitor of matrix metalloproteinases-1 confer the antimigratory effect of cannabinoids on human trabecular meshwork cells

Cannabinoids have received considerable attention as potential antiglaucomatous drugs. Recently, prostaglandins (PG) have been suggested to contribute to this effect. Within the factors conferring the development of glaucoma, depletion of the aqueous humor outflow-regulating trabecular meshwork (TM) cells elicited by migration from the outflow system is considered to play a pivotal role. This study therefore investigates the impact of two cannabinoids, Δ⁹-tetrahydrocannabinol (THC) and R(+)-methanandamide (MA), on the migration of human TM cells and the involvement of the PG-synthesizing enzyme cyclooxygenase-2 (COX-2) and one of its potential downstream targets, the tissue inhibitor of matrix metalloproteinases-1 (TIMP-1), to this response. Using Boyden chamber assays cannabinoids were shown to elicit an antimigratory effect that was reversed by antagonists for CB₁ as well as CB₂ receptors and accompanied by upregulation of COX-2 and TIMP-1 expression and PGE₂ synthesis. Knockdown of cannabinoid-induced COX-2 or TIMP-1 expression by siRNA or inhibition of COX-2 activity by NS-398 led to a significant suppression of this antimigratory action. Migration was also diminished by the major COX-2 product PGE₂ and by recombinant TIMP-1. Experiments using selective E prostanoid (EP) receptor agonists and antagonists revealed that decreased migration by PGE₂, THC and MA was mediated via EP₂ and EP₄ receptors. Finally, the cannabinoid-mediated increases of TIMP-1 levels were abolished by NS-398, and PGE₂ was shown to elicit a concentration-dependent increase of TIMP-1. Collectively, this data demonstrate a COX-2-dependent upregulation of TIMP-1 conferring the antimigratory action of cannabinoids. A decreased migration reducing TM cell loss in glaucoma might be involved in the antiglaucomatous action of cannabinoids.     

选择性COX-2抑制剂NS-398对人胃腺癌细胞增殖和凋亡的影响

目的 研究环氧化酶2(COX-2)抑制剂NS-398对胃癌培养细胞系BGC-823增殖及凋亡的影响.方法 应用MTT法检测NS-398对胃癌细胞BGC-823细胞增殖的影响;应用流式细胞术、逆转录聚合酶链反应(RT-PCR)和Western blot法检测NS-398对胃癌细胞BGC-823细胞凋亡的影响.结果 NS-398随剂量的增加及作用时间延长对胃癌细胞呈抑制作用;体外NS-398能减少BGC-823细胞株COX-2的表达,对BGC-823有细胞毒作用,可增加细胞凋亡率.结论 NS-398可抑制胃癌BGC-823细胞的增殖,促进胃癌BGC-823细胞的凋亡.其可能机制是与抑制COX-2表达有关.     

Pro-inflammatory effects of commercial alpha-lactalbumin on RAW 264.7 macrophages is due to endotoxin contamination

This study investigated the effects of alpha-lactalbumin (α-LA) on cellular signaling molecules associated with inflammatory responses in RAW 264.7 macrophages. The results indicated that commercial α-LA could increase prostaglandin E₂ (PGE₂) and the expression of COX-2 via increased phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), p38 mitogen-activated protein kinase (MAPK) and jun N-terminal kinase (JNK), and increase nitric oxide (NO) and the expression of iNOS via the activation of ERK1/2 and JNK. Furthermore, commercial α-LA could increase nuclear translocation of p65 nuclear factor-kappa B (p65 NF-κB) through stimulation on inhibitor kappa B-alpha (IκB-α) degradation. Since endotoxin also has these effects, we assayed the content of endotoxin in the commercial α-LA. We found to our surprise that endotoxin was there and that α-LA-induced NO and PGE₂ production could be suppressed by polymyxin B, a specific inhibitor of endotoxin. Thus, the pro-inflammatory effects of commercial α-LA might be caused by endotoxin contamination through activation and expression of iNOS and COX-2 which were upregulated by MAPKs or nuclear translocation of p65 NF-κB in RAW 264.7 cells. It is therefore crucial to assess the possibility of endotoxin contamination within any biological product being studied for immune augmenting activities before a meaning result can be obtained.     

羧甲基壳聚糖微球固定化AS1.398中性蛋白酶的制备和性质研究

以戊二醛为交联剂,制备羧甲基壳聚糖微球固定AS1．398中性蛋白酶。研究了羧甲基壳聚糖固定化酶微球的制备条件、固定化酶的适宜温度、pH、米氏常数和储存稳定性等性质。结果表明。在pH=7．0的中性环境下；交联剂与羧甲基壳聚糖摩尔比为0．4：1；干酶与羧甲基壳聚糖的质量比为0．125：1；在室温下交联至少4h。便可得到活力相对较高的固定化酶。自由酶和固定化酶的最适宜温度分别为40℃和65℃。在70℃下，固定化酶的热稳定性比自由酶要好。自由酶和固定化酶的最佳pH分别为7．0和6．0，固定化酶具有较宽的酸碱稳定性，在pH6～8都保持较高活力。固定化酶在5℃和35℃下储存1周后酶相对活力分别为97．6％和85．1％，而没有被固定的自由酶在5℃下储存1周酶相对活力仅为61．3％。自由酶和固定化酶的Km分别为5．66g／L和1．04g／L。     

Investigational Janus kinase inhibitors in development for myelofibrosis

Introduction: Since the discovery of the activating V617F mutation in Janus kinase 2 (JAK2), a number of pharmacologic inhibitors of JAK2 have entered clinical trials for patients with myelofibrosis. However, ruxolitinib, approved in 2011, remains the only one currently available for treatment of myelofibrosis, with many others having been discontinued for toxicity, and considerable uncertainty surrounding the future of those still in development.

Areas covered: The available clinical data on pacritinib and momelotinib, the two agents in the most advanced phases of clinical testing in myelofibrosis, are examined in detail. NS-018 and INCB039110, selective inhibitors of JAK2 and JAK1, respectively, are also discussed. Finally, the JAK2 inhibitors no longer in clinical development are summarized in tabular form.

Expert opinion: The different agents evaluated clearly differ in their kinomes, toxicity profiles and potential for myelosuppression. If approved, the JAK2-specific non-myelosuppressive inhibitor pacritinib could fulfill a major unmet need, that of patients with significant cytopenias. However, toxicity concerns persist. The data from the pivotal trials of momelotinib do not support its approval, although improvement of anemia is an important benefit. Selective JAK1 inhibition alone is unlikely to succeed in myelofibrosis. In these circumstances, rational ruxolitinib-based combinations may represent the best way forward.     

Molecular subclasses of hepatocellular carcinoma predict sensitivity to fibroblast growth factor receptor inhibition

A recent gene expression classification of hepatocellular carcinoma (HCC) includes a poor survival subclass termed S2 representing about one‐third of all HCC in clinical series. S2 cells express E‐cadherin and c‐myc and secrete AFP. As the expression of fibroblast growth factor receptors (FGFRs) differs between S2 and non‐S2 HCC, this study investigated whether molecular subclasses of HCC predict sensitivity to FGFR inhibition. S2 cell lines were significantly more sensitive (p < 0.001) to the FGFR inhibitors BGJ398 and AZD4547. BGJ398 decreased MAPK signaling in S2 but not in non‐S2 cell lines. All cell lines expressed FGFR1 and FGFR2, but only S2 cell lines expressed FGFR3 and FGFR4. FGFR4 siRNA decreased proliferation by 44% or more in all five S2 cell lines (p < 0.05 for each cell line), a significantly greater decrease than seen with knockdown of FGFR1‐3 with siRNA transfection. FGFR4 knockdown decreased MAPK signaling in S2 cell lines, but little effect was seen with knockdown of FGFR1‐3. In conclusion, the S2 molecular subclass of HCC is sensitive to FGFR inhibition. FGFR4‐MAPK signaling plays an important role in driving proliferation of a molecular subclass of HCC. This classification system may help to identify those patients who are most likely to benefit from inhibition of this pathway.     

NS398通过环氧化酶-2依赖途径诱导肾癌细胞786-O凋亡

目的探讨环氧化酶-2(COX-2)抑制剂NS398对肾癌细胞786-O增殖和凋亡的影响及其机制。方法同浓度NS398作用体外培养786-O细胞后,采用噻唑蓝(MTT)法检测24、48、72、96h后肾癌细胞786-O的增殖活性。30、180μmol/L的NS398作用786-O细胞72h后,RT-PCR法检测COX-2 mRNA表达;免疫细胞化学检测COX-2蛋白表达;酶联免疫吸附测定法(ELISA)检测前列腺素E2(PGE2)释放水平;流式细胞仪检测细胞凋亡情况。结果NS398可以抑制786-O细胞的增殖,呈时间和剂量依赖型。RT-PCR法和免疫细胞化学检测显示NS398作用后能降低COX-2 mRNA和蛋白表达。ELISA检测显示NS398作用后PGE2释放水平呈下调趋势;流式细胞仪检测表明,30、180μmol/L NS398处理组凋亡率分别为(14.3±1.4)%、(31.5±2.1)%,与对照组凋亡率(2.1±0.4)%比较,凋亡率显著上升(P<0.05)。结论NS398可能通过COX-2依赖性途径抑制肾癌细胞增殖和诱导其凋亡。     

非甾体类抗炎药NS398对胃癌细胞株SCG7901的影响

目的：探讨环氧合酶抑制剂NS398对胃癌细胞株SCG7901的生物学行为的影响。方法2014年6—12月于河北医科大学第二医院实验室,用MTT、DNA梯度降解试验及流式细胞仪等方法,研究NS398对胃癌细胞株SCG7901的抑制增殖、促进凋亡作用。结果 NS398对胃癌细胞株SCG7901产生明显的生长抑制作用,且表现为剂量依赖性(F=35.984,P<0.01);DNA梯度降解试验、流式细胞仪分析NS398浓度依赖性地诱导了胃癌细胞株SCG7901凋亡(F=12.414,P<0.01),且凋亡与细胞周期受阻有关(F=21.315,P<0.01),主要阻止在S/G2期。结论NS398对胃癌细胞株SCG7901具有显著浓度依赖性的体外生长抑制、诱导凋亡、阻滞DNA合成作用。     

FGFR2 genomic aberrations: Achilles heel in the management of advanced cholangiocarcinoma

Cholangiocarcinoma is the most common aggressive biliary tract malignancy with dismal prognosis. Though surgical resection of the primary tumors yields better prognosis, majority of patients present at advanced, inoperable stages rendering systemic therapy as the only option. A significant progress has been made in understanding the cholangiocarcinoma tumorigenesis and molecular markers over the last decade, which opens doors to precision medicine in this dismal cancer. Intrahepatic cholangiocarcinomas are most likely to harbor mutations in isocitrate dehydrogenase genes (IDH1, IDH2), fibroblast growth factor receptors (FGFR1, FGFR2, FGFR3), Eph receptor 2 (EPHA2), and BAP1 (gene involved in chromatin remodeling) genes, whereas ARID1B, ELF3, PBRM1, cAMP dependent protein kinase (PRKACA, and PRKACB) genetic mutations were implicated more commonly in distal and perihilar subtypes. Genomic studies have shown that FGFR2 aberrations are implicated in approximately 15% of intrahepatic cholangiocarcinomas, which make FGFR2 aberrations (Achilles heel) as potential novel targets in the management of cholangiocarcinoma. The current review comprehensively focuses on the role of FGFR2 inhibition either alone or in combination with other targeted therapy that act on down-stream and alternate kinase pathways in cholangiocarcinoma.     

Evidence that PGE2 in the dorsal and median raphe nuclei is involved in LPS-induced anorexia in rats

Anorexia is an element of the acute-phase immune response. Its mechanisms remain poorly understood. Activation of inducible cyclooxygenase-2 (COX-2) in blood-brain-barrier endothelial cells and subsequent release of prostaglandins (e.g., prostaglandin E2, PGE2) may be involved. Therefore, we sought to relate the effects of prostaglandins on the anorexia following gram-negative bacterial lipopolysaccharide treatment (LPS) to neural activity in the dorsal and median raphe nuclei (DRN and MnR) in rats. COX-2 antagonist (NS-398, 10 mg/kg; IP) administration prior to LPS (100 μg/kg; IP) prevented anorexia and reduced c-Fos expression the DRN, MnR, nucleus tractus solitarii and several related forebrain areas. These data indicate that COX-2-mediated prostaglandin synthesis is necessary for LPS anorexia and much of the initial LPS-induced neural activation. Injection of NS-398 into the DRN and MnR (1 ng/site) attenuated LPS-induced anorexia to nearly the same extent as IP NS-398, suggesting that prostaglandin signaling in these areas is necessary for LPS anorexia. Because the DRN and MnR are sources of major serotonergic projections to the forebrain, these data suggest that serotonergic neurons originating in the midbrain raphe play an important role in acute-phase response anorexia.