Human Ribonuclease with a Pendant Poly(Ethylene Glycol) Inhibits Tumor Growth in Mice

Human pancreatic ribonuclease (RNase 1) is a small secretory protein that catalyzes the cleavage of RNA. This highly cationic enzyme can enter human cells spontaneously but is removed rapidly from circulation by glomerular filtration. Here, this shortcoming is addressed by attaching a poly(ethylene glycol) (PEG) moiety to RNase 1. The pendant has no effect on ribonucleolytic activity but does increase persistence in circulation. The RNase 1-CPEG conjugates inhibit the growth of tumors in a xenograft mouse model of human lung cancer. Both retention in circulation and tumor growth inhibition correlate with the size of the pendant PEG. A weekly dose of the 60-kDa conjugate at 1 μmol/kg inhibited nearly all tumor growth without affecting body weight. Its molecular efficacy is ~5000-fold greater than that of erlotinib, which is a small molecule in clinical use for the treatment of lung cancer. These data demonstrate that the addition of a PEG moiety can enhance the in vivo efficacy of human proteins that act within cells and highlight a simple means of converting an endogenous human enzyme into a cytotoxin with potential clinical utility.     

Novel Immunocytokine IL12-SS1 (Fv) Inhibits Mesothelioma Tumor Growth in Nude Mice

Mesothelin is a glycosylphosphatidylinositol-anchored glycoprotein that is highly expressed on the cell surface of malignant mesothelioma. Monoclonal antibodies against mesothelin are being evaluated for the treatment of mesothelioma. Immunocytokines represent a novel class of armed antibodies. To provide an alternative approach to current mesothelin-targeted antibody therapies, we have developed a novel immunocytokine based on interleukin-12 (IL12) and the SS1 Fv specific for mesothelin. IL12 possesses potent anti-tumor activity in a wide variety of solid tumors. The newly-developed recombinant immunocytokine, IL12-SS1 (Fv), was produced in insect cells using a baculovirus-insect cell expression system. The SS1 single-chain Fv was fused to the C terminus of the p35 subunit of IL12 through a short linker (GSADGG). The single-chain IL12-SS1 (Fv) immunocytokine bound native mesothelin proteins on malignant mesothelioma (NCI-H226) and ovarian (OVCAR-3) cells as well as recombinant mesothelin on A431/H9 cells. The immunocytokine retained sufficient bioactivity of IL12 and significantly inhibited human malignant mesothelioma (NCI-H226) grown in the peritoneal cavity of nude mice and showed comparable anti-tumor activity to that of the SS1P immunotoxin. IL12-SS1 (Fv) is the first reported immunocytokine to mesothelin-positive tumors and may be an attractive addition to mesothelin-targeted cancer therapies.     

SIRT3过表达通过调控细胞代谢转换抑制肾肿瘤细胞的增殖

目的:研究SIRT3对肾透明细胞癌(clear cell renal cell carcinoma, ccRCC)769-P细胞增殖和抗氧化能力的影响,并进一步探究其作用机制。方法:在769-P细胞的基础上构建SIRT3过表达稳转细胞系;利用CCK-8试剂检测769-P SIRT3过表达细胞的增殖速度;利用CellROX~Deep Red染料并结合流式细胞分析检测SIRT3过表达对769-P细胞中ROS水平的影响;利用定量蛋白质组学和代谢组学的方法,探究SIRT3对769-P细胞的作用机制。结果:CCK-8实验结果表明,769-P SIRT3过表达细胞的生长速度与对照细胞相比下降了约48%;定量蛋白质组学分析显示,769-P SIRT3过表达细胞中ALDOA、ALDOA、ENO2、PKM、LDHA、LDHB表达量下调约0.4至0.7倍,SDHB和CS上调约1.3倍;代谢组学分析显示,PEP、pyruvic acid、lactate、carnitine水平下降约0.4至0.7倍,isocitric acid和acetyl-CoA水平升高分别约1.3和2.8倍;分析还显示SIRT3过表达上调SOD2、TXN、GPX4和GLRX5的表达量约1.3至2倍,降低ROS水平约40%,增强细胞对过氧化氢的耐受力。结论:SIRT3过表达引起769-P细胞的代谢转换,从而抑制其增殖;且上调769-P细胞中抗氧化酶的表达,降低ROS水平,增强细胞的抗氧化能力。     

Molecular and Cellular Mechanisms of Ecstasy-Induced Neurotoxicity: An Overview

“Ecstasy” [(±)-3,4-methylenedioxymethamphetamine, MDMA, XTC, X, E] is a psychoactive recreational hallucinogenic substance and a major worldwide drug of abuse. Several reports raised the concern that MDMA has the ability to induce neurotoxic effects both in laboratory animals and humans. Despite more than two decades of research, the mechanisms by which MDMA is neurotoxic are still to be fully elucidated. MDMA induces serotonergic terminal loss in rats and also in some mice strains, but also a broader neuronal degeneration throughout several brain areas such as the cortex, hippocampus, and striatum. Meanwhile, in human “ecstasy” abusers, there are evidences for deficits in seronergic biochemical markers, which correlate with long-term impairments in memory and learning. There are several factors that contribute to MDMA-induced neurotoxicity, namely, hyperthermia, monoamine oxidase metabolism of dopamine and serotonin, dopamine oxidation, the serotonin transporter action, nitric oxide, and the formation of peroxinitrite, glutamate excitotoxicity, serotonin 2A receptor agonism, and, importantly, the formation of MDMA neurotoxic metabolites. The present review covered the following topics: history and epidemiology, pharmacological mechanisms, metabolic pathways and the influence of isoenzyme genetic polymorphisms, as well as the acute effects of MDMA in laboratory animals and humans, with a special focus on MDMA-induced neurotoxic effects at the cellular and molecular level. The main aim of this review was to contribute to the understanding of the cellular and molecular mechanisms involved in MDMA neurotoxicity, which can help in the development of therapeutic approaches to prevent or treat the long-term neuropsychiatric complications of MDMA abuse in humans.     

PHA-767491抑制多种肿瘤细胞生长

Cdc7激酶抑制剂PHA-767491是最新发现的一类抗肿瘤新药.本实验利用不同浓度的PHA-767491对肿瘤细胞进行抑制研究.实验结果显示,PHA-767491对肿瘤细胞有很强的生长抑制作用,且抑制效果随着药物浓度或时间的增加而增强;通过和化疗药物5-氟尿嘧啶对比发现,PHA-767491只需较低剂量就能发挥出抑制肿瘤的作用,且疗效远高于5-氟尿嘧啶.研究进一步还发现,PHA-767491可通过促使PARP和casepase3蛋白的剪切诱导肿瘤细胞凋亡,同时PHA-767491还可以引起肿瘤细胞自噬.综上研究表明,PHA-767491可以通过诱导细胞凋亡和引起细胞自噬作用对多种肿瘤细胞有较好的治疗效果,而对正常细胞毒性很低.因此该实验研究为今后抗肿瘤新药PHA-767491的进一步应用于癌症的临床治疗提供了重要的实验依据.     

Hereditary Catalepsy: Genetic and Molecular Mechanisms of Catalepsy in Mice

The results of experiments on the inheritance and neurobiological mechanism of high predisposition to tonic immobility (catalepsy) in CBA mice are discussed. Genetic analysis has demonstrated a monogenic inheritance of the predisposition to catalepsy. A set of polymorphic microsatellite markers has been used to demonstrate that the predisposition to catalepsy is linked to the distal fragment of mouse chromosome 13, which contains the gene of the 5-HT_1A serotonin receptor. Pharmacological and biochemical evidence for the association between hereditary catalepsy and 5-HT_1A receptor dysfunction are presented. The use of CBA mice for studying the mechanisms of depression and the effects of antidepressants is discussed.     

Molecular Mechanisms of Cellular Senescence in Neurodegenerative Diseases

Neurodegenerative diseases, such as Alzheimer’s and Parkinson’s, are characterized by several pathological features, including selective neuronal loss, aggregation of specific proteins, and chronic inflammation. Aging is the most critical risk factor of these disorders. However, the mechanism by which aging contributes to the pathogenesis of neurodegenerative diseases is not clearly understood. Cellular senescence is a cell state or fate in response to stimuli. It is typically associated with a series of changes in cellular phenotypes such as abnormal cellular metabolism and proteostasis, reactive oxygen species (ROS) production, and increased secretion of certain molecules via senescence-associated secretory phenotype (SASP). In this review, we discuss how cellular senescence contributes to brain aging and neurodegenerative diseases, and the relationship between protein aggregation and cellular senescence. Finally, we discuss the potential of senescence modifiers and senolytics in the treatment of neurodegenerative diseases.     

Molecular and Cellular Mechanisms of Lamina-specific Axon Targeting

The specificity of synaptic connections is directly related to the functional integrity of neural circuits. Long-range axon guidance and topographic mapping mechanisms bring axons into spatial proximity of target cells and thus limit the number of potential synaptic partners. Synaptic specificity is then achieved by extracellular short-range guidance cues and cell-surface recognition cues. Neural activity may enhance the precision and strength of specific circuit connections. Here, we focus on one of the final steps of synaptic matchmaking: the targeting of synaptic layers and the mutual recognition of axons and dendrites within these layers.Perception and behavior are critically dependent on synaptic communication between specific neurons. Understanding how neurons achieve such “synaptic specificity” is therefore one of the most fundamental issues in developmental neuroscience. Langley’s notion of “chemical relations” between synaptically connected neurons (Langley 1892) and Sperry’s “chemoaffinity” hypothesis (Sperry 1963) provided a conceptual framework for the development of precise synaptic connections in the central nervous system. Sperry postulated that molecular interactions between neurons and their extracellular environment (including between and amongst axons and dendrites) ensure that connections form only between “appropriate” synaptic partners (Sperry 1963). This hypothesis has been confirmed by experimental work over the last four decades, most importantly by the identification of molecular cues that provide synaptic specificity (see Sanes and Yamagata 2009 for a recent comprehensive review). However, within this broad framework, a number of alternate mechanisms have been shown or proposed to play roles in specific aspects of such targeting processes. Here, we focus on mechanisms that underlie the formation of synaptic layers, a prominent anatomical feature of the visual system as well as many other areas of the CNS.As reviewed previously (O''Leary 2010), the chemoaffinity principle underlies the developmental process of topographic mapping. Indeed, the precision with which neurons preserve the spatial relationships between the visual world and its representation in the brain is remarkable: Across animals ranging from flies to vertebrates, axons that bear signals from adjacent points in visual space invariably choose adjacent targets in the brain (Braitenberg 1967; Lemke and Reber 2005; Sperry 1963). Thus, position-dependent guidance of axons ensures that a visuotopic map develops. However, position in space is just one attribute of a visual stimulus; others include color, brightness, edge detection, and movement. If position in visual space is encoded by localized activation within a two-dimensional field of neurons, then these other features are encoded by local circuits that act both in series and in parallel and are reiterated many times across the field (Fig. 1). These local circuit modules are often envisioned as “columns” that lie orthogonal to the topographic map, with each column corresponding to a pixel in visual space and each level of the column representing a different, specific visual feature within that pixel, such as brightness, color, etc. (Fig. 1). How these columns acquire their laminated structure represents a developmental challenge of extraordinary scale. Although long-range axon guidance and topographic mapping no doubt contribute to restricting the astronomical number of potential synaptic partners, these mechanisms are clearly not sufficient; additional mechanisms must (and do) exist that act on a local scale to provide an additional level of positional information and cell-type-specific “chemoaffinity.”Open in a separate windowFigure 1.Laminae are a fundamental organizing unit of neural circuits. Each column corresponds to a single topographic position (e.g., location on the retina). Within each column, different cell types (shown type A: blue, and type B: red) respond to different features in the visual world, such as motion or luminance. These pixels are repeated many times over and thus cover all of visual space. A simple rule of “Cell type A connects to Layer A, etc.” ensures that functional segregation is maintained in the connections from the retina to the target (parallel processing). Each pixel P1, P2, and P3 connects to a single column (C1, C2, and C3), establishing serial processing. Within each column, there are local circuits that, too, are layer-specific. Thus, laminae ensure functional specificity of both afferent-target connections and local circuit connections.A prominent principle, which guides the formation of connections between specific cell types and is a characteristic feature of CNS architecture, is the concentration of synapses in small areas. These synapse clusters can take the form of planar layers or spherical glomeruli. Although glomeruli are a specialization that appears most prominent in the olfactory system, layers, or laminae, are an almost ubiquitous feature of central nervous system architecture. Indeed, even crude histological stains reveal that axons and dendrites often accumulate in neuropil (cell-body-free areas). Cell-type-specific or single-cell labeling has shown that, within individual neuropil layers, neurites and synapses are not distributed randomly. Rather, synaptic connections arising between neurons with the same or similar functional properties are localized to particular sublaminae that distinguish synapses with different properties (Fig. 1). The structural underpinnings of this functional principle are provided by mechanisms that ensure the lamina-specific branching of the corresponding neurites. How this enormous precision is achieved is the subject of intense investigations in the Drosophila, zebrafish, chick, and mouse visual systems. We will begin by describing three anatomical regions in these model organisms. Then, we will discuss three broad principles of layer-specific targeting in the visual system, namely cell–cell recognition, guidance by matrix cues, and activity-dependent sorting of axon terminals.     

水通道 AQP1 敲除小鼠肿瘤血管生成障碍及肿瘤生长减缓

血管生成是肿瘤生长、浸润和转移的必要步骤. 肿瘤血管生成涉及瘤旁组织血管内皮细胞增殖、向肿瘤细胞团内迁移以及管腔形成,目前机理尚不完全清楚. 水通道 AQP1 在多种肿瘤血管内皮高表达,提示其可能参与肿瘤血管的生成过程. 应用 AQP1 敲除小鼠荷瘤实验证实了 AQP1 在黑色素瘤生长和血管新生中的作用. 结果表明,皮下接种的黑色素瘤在 AQP1 敲除小鼠的生长较之在野生型小鼠延迟近 30％ (P<0.01). 免疫组化与肿 瘤病理形态学分析显示, AQP1 在野生型小鼠黑色素瘤血管内皮细胞上高表达,而在 AQP1 敲除小鼠黑色素瘤血管内皮细胞呈阴性表达. 在病理结构上,黑色素瘤细胞围绕血管分支呈岛状分布. 野生型小鼠黑色素瘤内血管管腔较细小,而 AQP1(－/－)小鼠黑色素瘤内血管床显著膨大. AQP1(－/－)小鼠肿瘤内平均微血管密度 (47/mm²) 较之 AQP1(＋/＋) 肿瘤 (142/mm²) 减少 67％ (P<0.01). 围绕 AQP1(－/－) 肿瘤血管的肿瘤细胞岛周边坏死区域明显大于 AQP1(＋/＋)肿瘤. 上述结果提出确切证据表明, AQP1 缺失使肿瘤血管生成发生障碍,从而影响了肿瘤血液供应和肿瘤生长. AQP1参与肿瘤血管生成的机理值得深入研究.     

肿瘤浸润转移分子机制的研究进展

肿瘤浸润转移是多因素参与、多步骤完成的生物化学变化过程。人们已经逐渐认识到浸润转移不仅与肿瘤细胞有关,更是肿瘤细胞和肿瘤组织微环境复杂的相互作用的结果,其过程涉及多个分子作用机制和信号转导途径,包括细胞和细胞的黏附分子、细胞外基质降解、生长因子、趋化因子和淋巴血管生成因子等。本文综述了肿瘤浸润转移的分子机制。     

肝纤维化发病机制中细胞和分子机制的研究进展

关于肝纤维化形成的复杂的细胞和分子联系已经有了相当多的研究进展。最近的数据表明,纤维化进程的终止和纤维分解途径的恢复可以逆转晚期肝纤维化甚至肝硬化。因此,需要更好地阐明参与肝纤维化的细胞和分子机制。HSC(肝星状细胞)的激活是肝纤维化发生的中心事件,此外还有产生基质的其他细胞来源,包括肝门区的成纤维细胞,纤维细胞和骨髓来源的肌纤维母细胞。这些细胞与其邻近细胞通过多种联系聚集产生纤维疤痕并造成持续性损伤。阐明不同类型的细胞的相互作用,揭示细胞因子对这些细胞的影响,理清活化HSC基因表达的调控,将有助于我们探索新的肝纤维化治疗靶点。此外,不同的病因有不同的致病途径,弄清这一点有助于针对特异性疾病治疗方法的发现。本文概述了肝纤维化的细胞和分子机制的最新研究进展,可能为未来治疗方法带来新的突破。     

Dietary Supplementation with Methylseleninic Acid Inhibits Mammary Tumorigenesis and Metastasis in Male MMTV-PyMT Mice

Male breast cancer, which makes up approximately 1% of all breast cancers, is an aggressive disease with poor prognosis. We investigated the effects of dietary supplementation with selenium in the form of methylseleninic acid [(MSeA) 2.5 mg selenium/kg] on mammary tumorigenesis in male MMTV-PyMT mice. The mammary tumor latency was 14.6 weeks for the MSeA-fed group and 13.8 weeks for the controls fed the AIN93G diet (p < 0.05). Dietary supplementation with MSeA, versus the control, resulted in a 72% reduction in tumor progression, a 46% reduction in both final volume and weight of mammary tumors, and a 70% reduction in the number of lung metastases. Mammary tumorigenesis in MMTV-PyMT mice, versus non-tumor-bearing wild-type mice, resulted in significant increases in concentrations of plasminogen activator inhibitor-1, urokinase plasminogen activator, monocyte chemotactic protein-1, and vascular endothelial growth factor, but not aromatase and estrogen, in the plasma. Concentrations of all variables mentioned above in both plasma and mammary tumors were lower in MSeA-fed mice. Mammary tumorigenesis reduced plasma levels of adiponectin compared to non-tumor-bearing controls. Adiponectin concentrations in mammary tumors, but not in plasma, were higher in MSeA-fed mice than in controls. In summary, dietary supplementation with selenium in the form of MSeA inhibits mammary tumorigenesis and its pulmonary metastasis in male MMTV-PyMT mice.

     

肿瘤细胞抗TRAIL凋亡诱导的分子机制

肿瘤坏死因子相关的凋亡诱导配体(tumornecrosisfactor-relatedapoptosis-inducingligand,TRAIL)是肿瘤坏死因子(tumornecrosisfactor,TNF)超家族的成员之一,它能选择性诱导肿瘤细胞凋亡,对大多数正常细胞无杀伤作用。研究表明,某些恶性肿瘤抵抗TRAIL诱导的凋亡,且TRAIL重复作用使一些TRAIL敏感的细胞产生获得性抗性,这是TRAIL应用于肿瘤治疗的重大障碍。现对与TRAIL凋亡诱导通路直接相关的抗TRAIL机制及由Akt等途径介导的抗性分子机制进行综述。