Nobiletin,a Polymethoxylated Flavone,Inhibits Glioma Cell Growth and Migration via Arresting Cell Cycle and Suppressing MAPK and Akt Pathways

Nobiletin, a bioactive polymethoxylated flavone (5,6,7,8,3^',4^'‐hexamethoxyflavone), is abundant in citrus fruit peel. Although nobiletin exhibits antitumor activity against various cancer cells, the effect of nobiletin on glioma cells remains unclear. The aim of this study was to determine the effects of nobiletin on the human U87 and Hs683 glioma cell lines. Treating glioma cells with nobiletin (20–100 µm ) reduced cell viability and arrested the cell cycle in the G0/G1 phase, as detected using a 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide assay and propidium iodide (PI) staining, respectively; however, nobiletin did not induce cell apoptosis according to PI‐annexin V double staining. Data from western blotting showed that nobiletin significantly attenuated the expression of cyclin D1, cyclin‐dependent kinase 2, cyclin‐dependent kinase 4, and E2 promoter‐binding factor 1 (E2F1) and the phosphorylation of Akt/protein kinase B and mitogen‐activated protein kinases, including p38, extracellular signal‐regulated kinase, and c‐Jun N‐terminal kinase. Our data also showed that nobiletin inhibited glioma cell migration, as detected by both functional wound healing and transwell migration assays. Altogether, the present results suggest that nobiletin inhibits mitogen‐activated protein kinase and Akt/protein kinase B pathways and downregulates positive regulators of the cell cycle, leading to subsequent suppression of glioma cell proliferation and migration. Our findings evidence that nobiletin may have potential for treating glioblastoma multiforme. Copyright © 2015 John Wiley & Sons, Ltd.     

Toxicology of 3-epi-deoxynivalenol,a deoxynivalenol-transformation product by Devosia mutans 17-2-E-8

Microbial detoxification of deoxynivalenol (DON) represents a new approach to treating DON-contaminated grains. A bacterium Devosia mutans 17-2-E-8 was capable of completely transforming DON into a major product 3-epi-DON and a minor product 3-keto-DON. Evaluation of toxicities of these DON-transformation products is an important part of hazard characterization prior to commercialization of the biotransformation application. Cytotoxicities of the products were demonstrated by two assays: a MTT bioassay assessing cell viability and a BrdU assay assessing DNA synthesis. Compared with DON, the IC₅₀ values of 3-epi-DON and 3-keto-DON were respectively 357 and 3.03 times higher in the MTT bioassay, and were respectively 1181 and 4.54 times higher in the BrdU bioassay. Toxicological effects of 14-day oral exposure of the B6C3F₁ mouse to DON and 3-epi-DON were also investigated. Overall, there were no differences between the control (free of toxin) and the 25 mg/kg bw/day or 100 mg/kg bw/day 3-epi-DON treatments in body and organ weights, hematology and organ histopathology. However, in mice exposed to DON (2 mg/kg bw/day), white blood cell numbers and serum immunoglobulin levels were altered relative to controls, and lesions were observed in adrenals, thymus, stomach, spleen and colon. Taken together, in vitro and in vivo studies indicate that 3-epi-DON is substantially less toxic than DON.     

Okra polysaccharide-2 plays a vital role on the activation of RAW264.7 cells by TLR2/4-mediated signal transduction pathways

Polysaccharide is the main active component of okra (Abelmoschus esculentus L.) and it can effectively stimulate the activation of macrophages. However, the immune regulatory mechanism is still not clear. Therefore, the present study aimed to reveal the possible mechanism by investigating the effect of okra polysaccharide-2 (RPS-2) on Toll-like receptor (TLR) 2/4-mediated signal transduction pathways in RAW264.7 murine macrophage cells. In order to confirm whether RPS-2 stimulated macrophages activation via TLR2 or TLR4, RAW264.7 murine macrophage cells were pretreated with TLR2/4 inhibitors for 1 h before RPS-2 treatment, and then the NO, IL-10, TNF-α levels were tested. The results indicated that both TLR2 and TLR4 were the keys of immune regulatory effect of RPS-2. Afterwards, the effect of RPS-2 on NF-κB and MAPKs signaling pathways were studied by western blot analysis. It showed RPS-2 induced the phosphorylation of p65, IκBα, p38, ERK1/2 and JNK. At the same time, the specific inhibitors reduced these phosphorylation levels as well as NO, IL-10 and TNF-α amounts. In a word, RPS-2 activated macrophages by NF-κB and MAPKs signal transduction pathways.     

Cinobufagin exerts anti-proliferative and pro-apoptotic effects through the modulation ROS-mediated MAPKs signaling pathway

Abstract

Cinobufagin (CBG) is a cardiotoxic bufanolide steroid secreted by the skin and parotid venom glands of the Asiatic toad Bufo bufo gargarizans (called Chan-Su). Although CBG is known to exhibit anti-cancer activities, very little is known about its potential mechanism(s) of action. In this study, we investigated whether CBG mediates its effect through the modulation of the mitogen-activated protein kinases (MAPKs) signaling pathway in human multiple myeloma (MM) U266 cells. We found that CBG caused the significant activation of ERK, JNK and p38 MAPK in U266 cells. CBG showed much higher cytotoxicity against U266 cells as compared to peripheral blood mononuclear cells (PBMC). Induction of CBG increased reactive oxygen species (ROS) generation from mitochondria, which is associated with the induction of apoptosis as characterized by increased sub-G1 DNA contents of cell cycle, positive Annexin V binding, activation of caspase-3 and cleavage of PARP. Inhibition of ROS generation by N-acetyl-l-cysteine (NAC) significantly prevented CBG-induced ERK, JNK and p38 MAPK activation and apoptosis. CBG also down-regulated the expression of various downstream gene products that mediate cell proliferation, survival, angiogenesis and metastasis. Interestingly, ERK, JNK and p38MAPK pharmacological inhibitors blocked CBG-induced MAPKs activation and ERK inhibitor (PD98059) also prevented the CBG-induced caspase-3 activation and PARP cleavage in U266 cells. Taken together, these findings suggest that CBG can act as a potent anticancer agent against MM and possibly exerts its effects through the ROS-mediated activation of ERK, JNK and p38 MAPK leading to the activation of caspase-3 in U266 cells.     

1,3,5,8‐Tetrahydroxy‐9H‐xanthen‐9‐one exerts its antiageing effect through the regulation of stress‐response genes and the MAPK signaling pathway

The antioxidative effects of 30 xanthone derivatives (XDs) ( XD‐ n , n = 1–30) in HepG2 cells were evaluated by the cellular antioxidant activity assay. Results showed that all XDs were antioxidants and 1,3,5,8‐tetrahydroxy‐9H‐xanthen‐9‐one ( XD‐2 ) was the most active antioxidant. The all‐oxygenated substituted xanthones extended the lifespan of wild‐type N2 nematodes under normal culture conditions and XD‐2 was the best one. XD‐2 eliminated excessive intracellular reactive oxygen species and enhanced the expression levels and activities of the antioxidant enzymes superoxide dismutase, catalase, and glutathione peroxidase. XD‐2 inhibited the H₂O₂‐increased phosphorylation levels of c‐JUN N‐terminal kinase, extracellular signal‐regulated kinase, and p38 in HepG2 cells. In vivo, XD‐2 also extended the lifespan of wild‐type N2 nematodes under oxidative stress induced by paraquat, but failed in extending the lifespan of CF1038 (daf‐16 deletion) and AY102 (pmk‐1 deletion) mutant nematodes. It was revealed by real‐time polymerase chain reaction that the genes daf‐16, sir‐2.1, akt‐1, and age‐1 were all inhibited by paraquat stimuli, while XD‐2 reversed these inhibitions; in contrast, paraquat stimuli upregulated both the skn‐1 and pmk‐1 genes. However, treatment by  XD‐2 further increased the levels of both genes. These pieces of evidence implied that XD‐2 promotes longevity through endogenous signaling pathways rather than through the antioxidative activity alone. Taken all together, it may be concluded that XD‐2 is a promising antiageing agent.     

莫沙必利对阿司匹林致大鼠急性胃黏膜损伤的保护作用

研究显示促胃肠动力药物莫沙必利对胃黏膜损伤具有一定的保护作用。目的：研究不同剂量莫沙必利对阿司匹林致大鼠急性胃黏膜损伤的保护作用及其机制。方法：将50只大鼠随机分为阴性对照组、单纯损伤组以及不同剂量莫沙必利干预组（0．25mg／kg、0．50mg／kg、0．75mg／kg）。干预组大鼠以不同剂量莫沙必利灌胃行预处理,以150mg／kg阿司匹林灌胃制备急性胃黏膜损伤模型。实验第4d,处死大鼠。评估大鼠胃黏膜损伤指数和组织学变化,以免疫组化法检测Occludin蛋白分布,蛋白质印迹法检测Occludin、ZO．1以及磷酸化ERK（p-ERK）、磷酸化JNK（p-JNK）和磷酸化p38（p-p38）蛋白表达。结果：与单纯损伤组相比,各莫沙必利干预组胃黏膜损伤指数均明显降低（P〈0．05）;胃黏膜组织学明显改善;胃黏膜Occludin、ZO-1蛋白表达呈剂量依赖性升高（P〈0．05）;胃黏膜p-ERK、p-p38蛋白表达呈剂量依赖性降低（P〈0．05）;而胃黏膜p-JNK蛋白表达无明显差异。结论：莫沙必利对阿司匹林致大鼠急性胃黏膜损伤具有明显保护作用,其机制可能为降低MAPKs信号通路中ERK和p38蛋白磷酸化程度,并上调胃黏膜紧密连接蛋白Occludin和ZO-1表达,从而改善胃黏膜屏障的功能。     

MAPKs通路在MSCs的增殖及向成骨细胞分化过程中的作用

间充质干细胞（MSCs）是一种多潜能成体干细胞,在体外诱导剂的作用下能向成骨细胞分化。在MSCs向成骨细胞分化过程中,受到MAPKs、BMPs、Notch和Wnt等多种信号通路的调控。其中MAPKs信号通路研究比较深入,近年来研究表明在MAPKs信号通路的五种途径中,ERKs、p38MAPK和JNKs途径参与了成骨细胞增殖和分化的信号转导。现对MAPKs通路与其参与的MSCs增殖和成骨分化过程简要综述。     

MK2 plays an important role for the increased vascular permeability that follows thermal injury

We previously reported Rho kinase is involved in vessel hyper-permeability caused by burns. Here we further explore the Rho kinase downstream signaling, it is found that its specific inhibitor Y27632 significantly diminishes the activation of JNK and p38 MAPKs but not ERK that induced by serum from burned rats (burn-serum). JNK activation was found involved in the expression of HUVEC adhesion molecules following thermal injury, although not in the process of stress fiber formation. Inhibition of various MAPKs by specific inhibitors showed that SB203580 (inhibitor of p38), but neither SP600125 (inhibitor of JNK) nor PD98059 (inhibitor of ERK), abolish activation of the p38 downstream kinase MK2. Demonstration of stress fibers by fluorescent-labeled phalloidin showed that inhibition of MK2, either by its specific inhibitor or by dominant negative adeno-viral-carried constructs, significantly reduced burn-serum-induced HUVEC stress-fiber formation, while inhibition of another downstream p38 MAPK kinase, PRAK, had no such effects. Transfection of dominant negative adeno-viral MK2 (Ad-MK2(A)) significantly inhibited thermal injury-induced blood vessel hyper-permeability in rats and, moreover, prolonged the survival of burned rats beyond 72 h following thermal injury. One of the mechanisms behind these phenomena is that Ad-MK2(A) causes a significant depression of burn-serum-induced HSP27-phosphorylation, while the adeno-viral transported dominant negative PRAK (Ad-PRAK(A)) does not block. Although the effect of blockade of MK2 through its adeno-viral approach requires further study and investigation of alternatives to know for sure, we may have found a new pathway behind thermal-injury-induced blood vessel hyper-permeability, namely: Rho kinase > p38 > MK2 > HSP27.     

伊班膦酸钠干预骨关节炎模型大鼠丝裂原活化蛋白激酶信号通路的变化

背景:丝裂原活化蛋白激酶信号通路参与成骨细胞与破骨细胞的分化,与软骨下骨重建过程密切相关,在骨关节炎的发生发展中起重要作用。双膦酸盐作为骨吸收抑制剂,主要用于骨质疏松的治疗。目的:探讨伊班膦酸钠对大鼠骨关节炎的治疗效果,以及其对丝裂原活化蛋白激酶信号通路的影响。方法:实验方案经南华大学附属第一医院动物实验伦理委员会批准。30只雌性SD大鼠随机分为假手术组、模型组、治疗组。模型组和治疗组行双侧去卵巢及前交叉韧带切断术,假手术组大鼠只切除卵巢周围与卵巢大小相仿的脂肪组织,切开双侧膝关节腔,但不切断前交叉韧带。术后1周,治疗组予以腹腔注射伊班膦酸钠10μg/kg,模型组予以腹腔注射等量生理盐水,假手术组不作干预。12周以后,处死实验动物,进行关节软骨的组织形态学观察及Mankin评分,软骨下骨的Micro-CT扫描及骨组织显微结构定量分析,检测丝裂原活化蛋白激酶信号通路中细胞外信号调节蛋白激酶(ERK1)和c-Jun氨基酸末端激酶(JNK)的mRNA表达量及蛋白表达水平。结果与结论:①模型组软骨结构明显破坏,Mankin评分较假手术组明显增高,而治疗组的Mankin评分较模型组显著降低(P<0.01);②与假手术组比较,模型组中的骨密度、骨体积分数、骨小梁数量降低,骨小梁分离度显著增高(P<0.01);与模型组相比,治疗组的骨密度、骨体积分数、骨小梁数量增加,骨小梁分离度明显下降(P<0.01);③模型组中的ERK1、JNK mRNA表达和蛋白表达较假手术组明显增加(P<0.05,P<0.01);与模型组相比,治疗组中的ERK1、JNK mRNA表达和蛋白表达显著降低(P<0.05);④结果说明,伊班膦酸钠可能通过抑制ERK1、JNK丝裂原活化蛋白激酶信号通路的表达改善膝骨关节炎大鼠的软骨下骨的微结构,抑制软骨的退变。     

卡泊三醇对TNF-α诱导的人角质形成细胞中CCL20表达的影响

目的：明确卡泊三醇体外对TNF-α诱导的人角质形成细胞CCL20表达的影响。方法：体外培养人角质形成细胞,分为TNF-α 组,卡泊三醇组,TNF-α+卡泊三醇组,TNF-α+卡泊三醇+SB202190(p38 MAPK抑制剂)组,TNF-α+卡泊三醇+SP600125(JNK抑制剂)组,TNF-α+卡泊三醇+U0126(ERK抑制剂)组,TNF-α+卡泊三醇+CAPE(NF-κB抑制剂)组。实时荧光定量PCR法和酶联免疫吸附试验检测CCL20 mRNA及蛋白表达水平。Western blot法检测p38 MAPK,ERK和NF-κBp65磷酸化情况。结果：TNF-α+卡泊三醇组CCL20的表达水平及ERK,p38 MAPK,NF-κBp65磷酸化水平低于TNF-α组,差异均有统计学意义（均P<0.05）。TNF-α+卡泊三醇组CCL20的表达水平低于TNF-α+卡泊三醇+U0126(ERK抑制剂)组,高于TNF-α+卡泊三醇+SB202190(p38 MAPK抑制剂)组和TNF-α+卡泊三醇+CAPE(NF-κB抑制剂)组,差异均有统计学意义（均P<0.05）。CCL20的表达水平在TNF-α+卡泊三醇组与TNF-α+卡泊三醇+SP600125(JNK抑制剂)组间比较差异无统计学意义（P>0.05）。结论：卡泊三醇可能通过调节TNF-α诱导的ERK,p38 MAPK,NF-κBp65磷酸化,从而参与调节人角质形成细胞中CCL20表达。