TRP通道蛋白异源组装的研究进展

TRP通道是一类在神经系统分布广泛的阳离子通道,参与了生物体内许多重要的生理功能,包括感觉信息传递、调节胞内Ca²⁺平衡及发育过程等。近年来的研究发现,TRP通道不仅以同源四聚体形式行使功能,还可以组装成异源四聚体。不同亚基所形成的异源通道具有不同的生物物理学功能和药理学特性,因此TRP通道的组装机理和异源组装通道的功能研究成为该领域的热点而日益得到关注。文章对TRP通道家族中选择性异源组装及组装的分子基础研究的最新现状进行了概述。     

K+ Channel Expression in Human Breast Cancer Cells: Involvement in Cell Cycle Regulation and Carcinogenesis

K⁺ channels are a most diverse class of ion channels in the plasma membrane and are distributed widely throughout a variety of cells including cancer cells. Evidence has been accumulating from fundamental studies indicating that tumour cells possess various types of K⁺ channels and that these K⁺ channels play important roles in regulating tumor cell proliferation, cell cycle progression and apoptosis. Moreover, a significant increase in K⁺ channel expression has been correlated with tumorigenesis, suggesting the possibility of using these proteins as transformation markers and perhaps reducing the tumor growth rate by selectively inhibiting their functional activity. Significant progress has been made in defining the properties of breast K⁺ channels, including their biophysical and pharmacological properties and distribution throughout different phases of the cell cycle in breast cell line MCF-7. This review aims to provide a comprehensive overview of the current state of research into K⁺ channels/currents in breast cancer cells. The possible mechanisms by which K⁺ channels affect tumor cell proliferation and cell cycle progression are discussed.     

尿激酶受体反义RNA抑制人乳腺癌细胞的侵袭作用

将尿激酶受体 u PAR反义 RNA表达质粒 p URAS以脂质体法转染高侵袭性人乳腺癌细胞株 MDA- MB- 2 31 ,G41 8筛选抗性克隆 .Northern印迹法检测 u PAR反义 RNA的表达 ,RT- PCR法检测 u PAR的表达 ,牛奶板法测定细胞培养上清中纤溶活性 .改良 Boyden小室模型和裸小鼠乳房脂肪垫接种试验分别检测肿瘤细胞体外和体内侵袭能力 .反义克隆细胞能表达 u PAR反义RNA,其 u PAR表达水平及培养上清中纤溶活性明显降低 .反义细胞克隆体外侵袭能力比原代细胞 MDA- MB- 2 31和转染载体细胞克隆显著降低 .裸小鼠体内侵袭实验表明 ,反义细胞克隆的成瘤性、生长性和侵袭性均显著受到抑制 .u PAR至少在一部分恶性乳腺癌侵袭行为中发挥重要作用 ,反义 RNA可望成为抗肿瘤侵袭治疗的一种有效手段 .     

Targeting Breast Cancer Stem Cells

The potential roles of breast cancer stem cells (BCSCs) in tumor initiation and recurrence have been recognized for many decades. Due to their strong capacity for self-renewal and differentiation, BCSCs are the major reasons for poor clinical outcomes and low therapeutic response. Several hypotheses on the origin of cancer stem cells have been proposed, including critical gene mutations in stem cells, dedifferentiation of somatic cells, and cell plasticity remodeling by epithelial-mesenchymal transition (EMT) and the tumor microenvironment. Moreover, the tumor microenvironment, including cellular components and cytokines, modulates the self-renewal and therapeutic resistance of BCSCs. Small molecules, antibodies, and chimeric antigen receptor (CAR)-T cells targeting BCSCs have been developed, and their applications in combination with conventional therapies are undergoing clinical trials. In this review, we focus on the features of BCSCs, emphasize the major factors and tumor environment that regulate the stemness of BCSCs, and discuss potential BCSC-targeting therapies.     

ATP-Activated Nonselective Cation Current in NG108-15 Cells

Abstract: ATP (1 mM) induced a biphasic increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i), i.e., an initial transient increase decayed to a level of sustained increase, in NG108-15 cells. The transient increase was inhibited by a phospholipase C inhibitor, 1-[6-[[17β-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione (U73122), whereas the sustained increase was abolished by removal of external Ca²⁺. We examined the mechanism of the ATP-elicited sustained [Ca²⁺]_i increase using the fura-2 fluorescent method and the whole-cell patch clamp technique. ATP (1 mM) induced a membrane current with the reversal potential of 12.5 ± 0.8 mV (n = 10) in Tyrode external solution. The EC₅₀ of ATP was ～0.75 mM. The permeability ratio of various cations carrying this current was Na⁺ (defined as 1) > Li⁺ (0.92 ± 0.01; n = 5) > K⁺ (0.89 ± 0.03; n = 6) > Rb⁺ (0.55 ± 0.02; n = 6) > Cs⁺ (0.51 ± 0.01; n = 5) > Ca²⁺ (0.22 ± 0.03; n = 3) > N-methyl-d -glucamine (0.13 ± 0.01; n = 5), suggesting that ATP activated a nonselective cation current. The ATP-induced current was larger at lower concentrations of external Mg²⁺. ATP analogues that induced the current were 2-methylthio-ATP (2MeSATP), benzoylbenzoic-ATP, adenosine 5′-thiotriphosphate (ATPγS), and adenosine 5′-O-(2-thiodiphosphate), but not adenosine, ADP, α,β-methylene-ATP (AMPCPP), β,γ-methylene-ATP (AMPPCP), or UTP. Concomitant with the current data, 2MeSATP and ATPγS, but not AMPCPP or AMPPCP, increased the sustained [Ca²⁺]_i increase. We conclude that ATP activates a class of Ca²⁺-permeable nonselective cation channels via the P_2z receptor in NG108-15 cells.     

乳腺癌肿瘤干细胞的研究进展

干细胞(stem cell,SC)是一类具有自我更新、多向分化潜能的特殊细胞群体.而肿瘤干细胞(cancer stem cell,CSC)不仅具备了干细胞方面的特征,同时还有其自身特殊性.乳腺癌中肿瘤干细胞的发现为乳腺癌的治疗提供了创新性策略.近年来,有关乳腺癌肿瘤干细胞的筛选标记和方法以及信号转导通路方面的研究颇多,但其中也不乏争议.就乳腺癌干细胞研究的最新进展作一端述.     

TRPC channel lipid specificity and mechanisms of lipid regulation

TRPC channels are a subset of the transient receptor potential (TRP) proteins widely expressed in mammalian cells. They are thought to be primarily involved in determining calcium or sodium entry and have broad-ranging functions that include regulation of cell proliferation, motility and contraction. The channels do not respond to a single stimulator but rather are activated or modulated by a multiplicity of factors, potentially existing as integrators at the plasma membrane. This review considers the sensitivity of TRPCs to lipid factors, with focus on sensitivities to diacylglycerols, lysophospholipids, arachidonic acid and its metabolites, sphingosine-1-phosphate (S1P), cholesterol and derivatives, and other lipid factors such as gangliosides. Promiscuous and selective lipid-sensing are apparent. In many cases the lipids stimulate channel function or increase insertion of channels in the membrane. Both direct and indirect (receptor-dependent) lipid effects are evident. Although information is limited, the lipid profiles are consistent with TRPCs having close working relationships with phospholipase C and A2 enzymes. We need much more information about lipid-sensing by TRPCs if we are to fully appreciate its significance, but the available data suggest that lipid-sensing is a key, but not exclusive, aspect of TRPC biology.     

P2Y1 Receptor Activation of the TRPV4 Ion Channel Enhances Purinergic Signaling in Satellite Glial Cells

Transient receptor potential (TRP) ion channels of peripheral sensory pathways are important mediators of pain, itch, and neurogenic inflammation. They are expressed by primary sensory neurons and by glial cells in the central nervous system, but their expression and function in satellite glial cells (SGCs) of sensory ganglia have not been explored. SGCs tightly ensheath neurons of sensory ganglia and can regulate neuronal excitability in pain and inflammatory states. Using a modified dissociation protocol, we isolated neurons with attached SGCs from dorsal root ganglia of mice. SGCs, which were identified by expression of immunoreactive Kir4.1 and glutamine synthetase, were closely associated with neurons, identified using the pan-neuronal marker NeuN. A subpopulation of SGCs expressed immunoreactive TRP vanilloid 4 (TRPV4) and responded to the TRPV4-selective agonist GSK1016790A by an influx of Ca²⁺ ions. SGCs did not express functional TRPV1, TRPV3, or TRP ankyrin 1 channels. Responses to GSK1016790A were abolished by the TRPV4 antagonist HC067047 and were absent in SGCs from Trpv4^−/− mice. The P2Y₁-selective agonist 2-methylthio-ADP increased [Ca²⁺]_i in SGCs, and responses were prevented by the P2Y1-selective antagonist MRS2500. P2Y₁ receptor-mediated responses were enhanced in TRPV4-expressing SGCs and HEK293 cells, suggesting that P2Y₁ couples to and activates TRPV4. PKC inhibitors prevented P2Y₁ receptor activation of TRPV4. Our results provide the first evidence for expression of TRPV4 in SGCs and demonstrate that TRPV4 is a purinergic receptor-operated channel in SGCs of sensory ganglia.     

Raloxifene Induces Autophagy-Dependent Cell Death in Breast Cancer Cells via the Activation of AMP-Activated Protein Kinase

Raloxifene is a selective estrogen receptor modulator (SERM) that binds to the estrogen receptor (ER), and exhibits potent anti-tumor and autophagy-inducing effects in breast cancer cells. However, the mechanism of raloxifene-induced cell death and autophagy is not well-established. So, we analyzed mechanism underlying death and autophagy induced by raloxifene in MCF-7 breast cancer cells.Treatment with raloxifene significantly induced death in MCF-7 cells. Raloxifene accumulated GFP-LC3 puncta and increased the level of autophagic marker proteins, such as LC3-II, BECN1, and ATG12-ATG5 conjugates, indicating activated autophagy. Raloxifene also increased autophagic flux indicators, the cleavage of GFP from GFP-LC3 and only red fluorescence-positive puncta in mRFP-GFP-LC3-expressing cells. An autophagy inhibitor, 3-methyladenine (3-MA), suppressed the level of LC3-II and blocked the formation of GFP-LC3 puncta. Moreover, siRNA targeting BECN1 markedly reversed cell death and the level of LC3-II increased by raloxifene. Besides, raloxifene-induced cell death was not related to cleavage of caspases-7, -9, and PARP. These results indicate that raloxifene activates autophagy-dependent cell death but not apoptosis. Interestingly, raloxifene decreased the level of intracellular adenosine triphosphate (ATP) and activated the AMPK/ULK1 pathway. However it was not suppressed the AKT/mTOR pathway. Addition of ATP decreased the phosphorylation of AMPK as well as the accumulation of LC3-II, finally attenuating raloxifene-induced cell death.Our current study demonstrates that raloxifene induces autophagy via the activation of AMPK by sensing decreases in ATP, and that the overactivation of autophagy promotes cell death and thereby mediates the anti-cancer effects of raloxifene in breast cancer cells.     

Calcium-Sensitive Nonselective Cation Channel Identified in the Epithelial Cells Isolated from the Endolymphatic Sac of Guinea Pigs

We identified a Ca²⁺-sensitive cation channel in acutely dissociated epithelial cells from the endolymphatic sac (ES) of guinea pigs using the patch-clamp technique. Single-channel recordings showed that the cation channel had a conductance of 24.0 ± 1.3 pS (n= 8) in our standard solution. The relative ionic permeability of the channel was in the order K⁺= Na⁺ > Ca²⁺≫ Cl⁻. This channel was weakly voltage-dependent but was strongly activated by Ca²⁺ on the cytosolic side at a concentration of around 1 mm in inside-out excised patches. With cell-attached patches, however, the channel was activated by much lower Ca²⁺ concentrations. Treatment of the cells, under cell-attached configuration, with ionomycin (10 μm), carbonyl cyanide 3-chlorophenylhydrazone (CCCP, 20 μm), or ATP (1 mm), which increased intracellular Ca²⁺ concentration ([Ca²⁺]_i), activated the channel at an estimated [Ca²⁺]_i from 0.6 μm to 10 μm. It is suggested that some activators of the channel were deteriorated or washed out during the formation of excised patches. Based on this Ca²⁺ sensitivity, we speculated that the channel contributes to the regulation of ionic balance and volume of the ES by absorbing Na⁺ under certain pathological conditions that will increase [Ca²⁺]_i. This is the first report of single-channel recordings in endolymphatic sac epithelial cells. Received: 24 October 2000/Revised: 10 April 2001     

Calcium-sensing receptor activation contributed to apoptosis stimulates TRPC6 channel in rat neonatal ventricular myocytes

Capacitative calcium entry (CCE) refers to the influx of calcium through plasma membrane channels activated on depletion of endoplasmic sarcoplasmic/reticulum (ER/SR) Ca²⁺ stores, which is performed mainly by the transient receptor potential (TRP) channels. TRP channels are expressed in cardiomyocytes. Calcium-sensing receptor (CaR) is also expressed in rat cardiac tissue and plays an important role in mediating cardiomyocyte apoptosis. However, there are no data regarding the link between CaR and TRP channels in rat heart. In this study, in rat neonatal myocytes, by Ca²⁺ imaging, we found that the depletion of ER/SR Ca²⁺ stores by thapsigargin (TG) elicited a transient rise in cytoplasmic Ca²⁺ ([Ca²⁺]_i), followed by sustained increase depending on extracellular Ca²⁺. But, TRP channels inhibitor (SKF96365), not L-type channels or the Na⁺/Ca²⁺ exchanger inhibitors, inhibited [Ca²⁺]_i relatively high. Then, we found that the stimulation of CaR with its activator gadolinium chloride (GdCl₃) or by an increased extracellular Ca²⁺([Ca²⁺]_o) increased the concentration of intracelluar Ca²⁺, whereas, the sustained elevation of [Ca²⁺]_i was reduced in the presence of SKF96365. Similarly, the duration of [Ca²⁺]_i increase was also shortened in the absence of extracellular Ca²⁺. Western blot analysis showed that GdCl₃ increased the expression of TRPC6, which was reversed by SKF96365. Additionally, SKF96365 reduced cardiomyocyte apoptosis induced by GdCl₃. Our results suggested that CCE exhibited in rat neonatal myocytes and CaR activation induced Ca²⁺-permeable cationic channels TRPCs to gate the CCE, for which TRPC6 was one of the most likely candidates. TRPC6 channel was functionally coupled with CaR to enhance the cardiomyocyte apoptosis.     

A Novel Large Conductance, Nonselective Cation Channel in Pheochromocytoma (PC12) Cells

A new type of nonselective cation channel was identified and characterized in pheochromocytoma (PC12) cells using inside-out and cell-attached patch-clamp recordings. The channel shows a large unitary conductance (274 pS in symmetric 145 mm K⁺) and selectivity for Na⁺≈ K⁺ > Li⁺, and is practically impermeable to Cl⁻. The channel activity-voltage relationship is bell-shaped, showing maximal activation at ≈−10 mV. The overall activity of this channel is unmodified by [Na⁺] _ic , or [Ca⁺⁺] _ic . However, increases in [Ca⁺⁺] _ic lead to a decrease in the unitary current amplitude. In addition, overall activity is mildly increased when suction is applied to the back of the patch pipette. Together, these characteristics distinguish the present channel from all other large conductance nonselective cation channels reported so far in a variety of preparations. The frequency of appearance of this channel type is similar in undifferentiated and NGF-treated PC12 cells (≈8–27% of patches). The combination of large conductance, permeability to Na⁺, and existence of conducting states at negative potentials, may provide a significant pathway for inward current and depolarization in PC12 cells. Received: 14 February 1997/Revised: 28 July 1997     

人乳腺癌干细胞异种移植动物模型的制备和应用

越来越多的证据显示乳腺癌干细胞是导致乳腺癌发生、发展、复发和转移的根源。因此,模拟出与人乳腺癌发病机制相似的动物模型将对乳腺癌的治疗起着至关重要的作用。本文旨在介绍乳腺癌干细胞异种移植动物模型的制备方法、应用以及近年来的研究进展。     

胰岛素样生长因子-1受体（IGF—IR）在乳腺癌组织中的表达及其临床意义初探

目的：检测分析胰岛素样生长因子-1受体（IGF-IR）在乳腺癌组织中的表达状况及其临床意义。方法：应用半运用半定量RT-PCR方法分析84例乳腺癌和癌旁正常乳腺组织中IGF—IR基因mRNA的表达水平,并分析其表达与患者临床病理特征及预后之间的关系。结果：乳腺癌组织中IGF-IR基因mRNA表达水平显著高于癌旁乳腺组织,二者具有统计学差别（P〈0．001）。乳腺癌组织中IGF-IR基因mRNA表达水平与肿瘤组织分化程度及乳腺癌患者的TNM分期和淋巴结转移情况显著相关（P值分别是0．005,0．025和0．041）。另外,高表达IGF-IR的乳腺癌患者的五年总体生存率（38．3％）显著高于低表达IGF-1R的患者（49．7％;P=0．009）。多因素COX模型分析结果表明：IGF-IR基因mRNA表达水平是乳腺癌患者的一个独立预后分子（HR=2．78,95％CI：1．94-3．94,P=0．041）。结论：IGF-IR基因表达水平上调在乳腺癌发展过程中起着重要的作用。IGF-IR基因mRNA表达水平有望成为临床乳腺癌患者预后判断的一个重要分子标志物。     

胰岛素样生长因子-1 受体(IGF-IR)在乳腺癌组织中的表达及其 临床意义初探

目的:检测分析胰岛素样生长因子-1受体(IGF-IR)在乳腺癌组织中的表达状况及其临床意义。方法:应用半运用半定量RT-PCR方法分析84例乳腺癌和癌旁正常乳腺组织中IGF-1R基因mRNA的表达水平,并分析其表达与患者临床病理特征及预后之间的关系。结果:乳腺癌组织中IGF-1R基因mRNA表达水平显著高于癌旁乳腺组织,二者具有统计学差别(P<0.001)。乳腺癌组织中IGF-1R基因mRNA表达水平与肿瘤组织分化程度及乳腺癌患者的TNM分期和淋巴结转移情况显著相关(P值分别是0.005,0.025和0.041)。另外,高表达IGF-1R的乳腺癌患者的五年总体生存率(38.3%)显著高于低表达IGF-1R的患者(49.7%;P=0.009)。多因素COX模型分析结果表明:IGF-1R基因mRNA表达水平是乳腺癌患者的一个独立预后分子(HR=2.78,95%CI:1.94-3.94,P=0.041)。结论:IGF-1R基因表达水平上调在乳腺癌发展过程中起着重要的作用。IGF-1R基因mRNA表达水平有望成为临床乳腺癌患者预后判断的一个重要分子标志物。     

NDRG2 通过影响CD24 调控乳腺癌细胞的粘附能力

目的:阐明NDRG2(N-Myc downstream-regulated gene 2)在乳腺癌细胞中对CD24的调控及其对乳腺癌细胞粘附能力的影响。方法:RT-PCR和Western blot方法检测乳腺癌细胞MCF-7及Bcap-37中NDRG2和CD24的表达;通过腺病毒上调MCF-7细胞中NDRG2的表达,或利用siRNA下调Bcap-37细胞中NDRG2的表达,检测CD24基因和蛋白的变化。粘附实验检测改变NDRG2表达水平后对MCF-7及Bcap-37细胞粘附能力的影响。结果:MCF-7细胞中NDRG2基因和蛋白的表达水平低于Bcap-37细胞,而CD24的表达水平高于Bcap-37细胞;在MCF-7细胞中通过腺病毒载体上调NDRG2可以抑制CD24的表达并抑制其粘附能力,而在Bcap-37细胞中利用siRNA下调NDRG2的表达可以提高CD24的水平及细胞的粘附能力;结论:NDRG2通过影响CD24参与调控乳腺癌细胞的粘附能力。     

植物雌激素与乳腺癌发病发展的研究进展

总结植物雌激素(phytoestrogen,PE)在乳腺癌发病、发展过程中的研究进展。应用Medline、Embase、CNKI及VIP数据库检索"植物雌激素、乳腺癌"相关基础临床试验流行病学调查。PE作为非甾体结构的雌激素类似物,能够与雌激素受体结合产生作用。目前研究主要集中于PE通过雌激素受体对乳腺癌细胞的增殖、凋亡、自噬、周期的影响和信号传导通路的研究,体外实验PE有抑制乳腺癌细胞增殖、促进凋亡、自噬的作用,但是对转移的作用不明确,可通过PI3K/AKT和ROS/p38MAPK途径诱导凋亡,PE与ER结合或调整ERα/ERβ达到抑制乳腺癌细胞的作用,流行病学调查未发现PE对人体有害,鲜有临床报道。因此,本文对PE对乳腺癌的研究进展进行总结分析,旨在为乳腺肿瘤患者治疗提供新方向。植物雌激素有希望成为乳腺肿瘤患者治疗的新方向。     

TRPM3表达上调通过调控Beclin1的表达促进卵巢癌细胞自噬

目的:探讨瞬时受体电位离子通道3(TRPM3)和Beclin1在卵巢癌中的表达和对卵巢癌细胞自噬的影响。方法:收集6例正常卵巢组织标本和20例卵巢癌组织标本,应用免疫组织化学染色法检测TRPM3和Beclin1在卵巢癌组织中的表达,采用western blotting法检测TRPM3和Beclin1在正常卵巢上皮细胞Moody和卵巢癌细胞Hey、ES-2中的表达差异。用TRPM3-siRNA瞬时转染细胞Hey和ES-2,通过western blotting法检测TRPM3基因沉默情况及Beclin1、p62和LC3的蛋白表达变化。结果:在正常卵巢组织和卵巢癌组织中,TRPM3的阳性表达率分别为33.3%、80.0%(P0.05),而Beclin1的阳性表达率分别为33.3%、65.0%(P0.05)。与正常卵巢上皮细胞Moody相比,卵巢癌细胞Hey和ES-2中TRPM3、Beclin1蛋白表达水平明显较高(P0.05)。沉默TRPM3基因表达的卵巢癌细胞中Beclin1和LC3蛋白表达与对照组相比明显降低,而p62表达升高(P0.05)。结论:卵巢癌组织和细胞中TRPM3蛋白呈高表达,可能通过调控Beclin1促进卵巢癌细胞的自噬。