CXCR4抑制剂AMD3100对非小细胞肺癌细胞侵袭转移的影响

目的探讨CXCR4抑制剂AMD3100对非小细胞肺癌95D细胞侵袭转移能力的影响。方法分别采用不同浓度(0、10、25、50、100 nmol/L)AMD3100处理高表达CXCR4的非小细胞肺癌95D细胞24、48、72和96 h后用四甲基偶氮唑盐(MTT)比色法检测增殖抑制率变化,并用Transwell小室内法检测不同浓度AMD3100处理48、96 h后的95D细胞侵袭及迁移能力变化。结果 AMD3100可不同程度抑制非小细胞肺癌95D细胞增殖,且抑制效应呈浓度依赖性;AMD3100可缩短95D细胞迁移距离和减少穿膜细胞数(P<0.05),其效应亦呈浓度依赖性。结论 CXCR4抑制剂AMD3100可抑制非小细胞肺癌95D细胞的侵袭、转移能力。     

SDF-1/CXCR4对大肠癌细胞株SW480增殖、迁移及侵袭的影响及意义

【摘要】目的探讨基质细胞衍生因子-1(SDF-1)及其特异性受体CXC趋化因子受体4（CXCR4）对大肠癌细胞SW480增殖、迁移及侵袭能力的影响及意义。方法取对数生长期大肠癌细胞SW480分为对照组(未经任何处理)、SDF-1组（加入100μg/L SDF-1）、SDF-1＋AMD3100混合组(向细胞中加入1mg/L AMD3100,孵育2h后加入100μg/LSDF-1)、AMD3100组（加入1mg/L AMD3100）。免疫组化法检测SW480细胞中CXCR4蛋白表达情况;RT-PCR法检测SW480细胞中CXCR4mRNA的表达情况,以及外源性SDF-1和AMD3100作用后CXCR4mRNA表达水平的变化;MTT增殖实验、Transwell迁移及侵袭实验分别检测SDF-1以及AMD3100对SW480细胞增殖、迁移及侵袭能力的影响。结果SW480细胞中CXCR4蛋白呈阳性表达（阳性率80%）。SW480细胞中有CXCR4mRNA的表达,100μg/LSDF-1促使CXCR4mRNA表达水平进一步上调,且能被1mg/L AMD3100阻断。SDF-1组细胞增殖活性(0.847±0.039）高于对照组(0.624±0.011)和SDF-1＋AMD3100混合组(0.607±0.016),AMD3100组(0.456±0.032)低于对照组和SDF-1＋AMD3100混合组(F=108.030,P＜0.05)。Transwell小室迁移及侵袭实验中SDF-1组穿膜细胞数(个：98.7±5.8、33.7±6.2)均多于对照组(21.0±2.2、6.1±2.3)、SDF-1＋AMD3100混合组(18.5±8.4、8.5±2.8)和AMD3100组(12.1±3.2、2.1±1.0),后3组间比较差异无统计学意义。结论SDF-1/CXCR4生物轴可促进大肠癌细胞SW480的增殖、迁移及侵袭。     

GLP-1 通过 SDF-1/CXCR4 信号通路调控人脐血内皮祖细胞增殖、 分化和凋亡

摘要:目的 探讨胰高血糖素样肽 1（GLP-1）调控人脐血内皮祖细胞（EPCs）增殖、 分化与凋亡的分子机制。方法 从健康孕妇脐带血分离和培养 EPCs, 以 2×105 密度接种于 6 孔细胞板, 分别转染空载体质粒（对照组）、 pcD⁃ NA3-GLP-1 质粒（GLP-1 组）、 pcDNA3-GLP-1 质粒+AMD3100（GLP-1+AMD3100 组）及单纯 AMD3100（AMD3100 组）。将 pcDNA3-GLP-1 质粒转染 EPCs, 以 25 μmol/L AMD3100 阻断体外培养的 EPCs 的基质细胞衍生因子 （SDF- 1） /趋化因子受体 4（CXCR4）信号通路 1 h。采用 RT-PCR 检测分化和凋亡相关基因 PPARγ、 C/EBPα与 Caspase-3 基因表达, MTT 比色法检测细胞增殖能力, Caspase-3 活性检测试剂盒测定 Caspase-3 活性。结果 与对照组相比, GLP-1 过表达显著提高了 C/EBPα及 PPARγ mRNA 表达, 促进了 EPCs 细胞增殖, 降低了 Caspase-3 mRNA 表达和 Caspase-3 活性 （均 P < 0.05）。当 SDF-1/CXCR4 信号通路被阻断后, GLP-1 对 C/EBPα及 PPARγ mRNA 表达、 EPCs 细胞增殖的促进作用, 以及对 Caspase-3 mRNA 表达和 Caspase-3 活性的抑制作用均明显减弱（均 P < 0.05）。结论 GLP-1 可通过调节 SDF-1/CXCR4 信号通路促进 EPCs 增殖与分化, 抑制其凋亡。     

骨髓基质干细胞分化为神经元样细胞迁移能力变化的研究

目的研究体外定向诱导骨髓间充质干细胞(BMSCs)分化为神经元样细胞的方法,探讨其分化后迁移能力变化的分子机制。方法体外培养BMSCs,以β-巯基乙醇(BME)1 mmol·L-1诱导分化,于诱导后12 h、24 h分别观察细胞形态变化,RT-PCR鉴定神经元特异性烯醇化酶(NSE)、神经丝蛋白(Neurofilament)、胶质纤维酸性蛋白(β-tubulinⅢ)mRNA的变化。Western blot检测CXCR4表达变化,细胞迁移实验检测BMSCs诱导前后迁移能力的变化。结果诱导后,BMSCs胞体收缩,突起伸出;RT-PCR发现诱导出的神经元样细胞的NSE、Neurofilament、β-tubulinⅢmRNA明显增加;Western blot结果表明神经元样细胞表面CXCR4的表达明显增加,细胞迁移实验发现神经元样细胞向SDF-1α的迁移能力明显增加。用CXCR4特异的抑制剂AMD3100处理后细胞迁移被明显抑制。结论在体外骨髓基质干细胞以BME为诱导剂可定向分化为神经元样细胞,且发现其迁移能力明显增加,其机制可能与其表面CXCR4表达上调有关。     

CXCR4拮抗剂对三阴性乳腺癌MDA MB 231细胞增殖和凋亡的影响及机制研究

摘 要 目的：探讨趋化因子基质细胞衍生因子 1受体（CXCR4）拮抗剂（AMD3100）对三阴性乳腺癌MDA MB 231细胞增殖和凋亡的影响及机制。 方法: 设MCF10A细胞组、三阴性乳腺癌MDA MB 231细胞组、氟尿嘧啶组(8.0 μg·ml-1)、CXCR4拮抗剂低、高剂量组(4.0,8.0 μg·ml-1),测定各组癌细胞的细胞活力、单克隆形成数目、细胞凋亡率、穿膜孔数,及三阴性乳腺癌MDA MB 231细胞基质细胞衍生因子1（SDF 1）、CXCR4、半胱氨酸蛋白酶 3（caspase 3）、半胱氨酸蛋白酶 6（caspase 6）、血管内皮生长因子（VEGF ）mRNA、蛋白水平。 结果: 与MCF10A细胞组比较,MDA MB 231细胞组吸光度（A）值、存活率水平、细胞克隆形成数目、穿膜数、SDF 1、CXCR4、VEGF mRNA及蛋白表达水平显著升高,细胞凋亡率、caspase 3、caspase 6 mRNA及蛋白表达水平显著降低（P<0.05）。与MDA MB 231细胞组比较,氟尿嘧啶组、CXCR4拮抗剂低、高剂量组的A值、存活率水平、细胞克隆形成数目、穿膜数、SDF 1、CXCR4、VEGF mRNA及蛋白表达水平显著降低,细胞凋亡率、caspase 3、caspase 6 mRNA及蛋白表达水平显著升高（P<0.05）。与氟尿嘧啶组比较,CXCR4拮抗剂低剂量组的A值、存活率水平、细胞克隆形成数目、穿膜数、SDF 1、CXCR4、VEGF mRNA及蛋白表达水平显著升高,细胞凋亡率、caspase 3、caspase 6 mRNA及蛋白表达水平显著降低（P<0.05）;CXCR4拮抗剂高剂量组差异无统计学意义（P>0.05）。 结论: AMD3100能抑制三阴性乳腺癌MDA MB 231细胞增殖、侵袭,诱导其凋亡;其机制与AMD3100能特异性阻断三阴性乳腺癌MDA MB 231细胞 SDF 1、CXCR4 mRNA及蛋白的表达水平,导致其SDF 1/CXCR4信号传导受阻有关。     

毛冬青甲素对大鼠骨髓间充质干细胞增殖及迁移的影响

目的观察毛冬青甲素(ilexonin A,IA)对大鼠骨髓间充质干细胞(BMSCs)增殖的影响,并探讨IA是否通过上调CXCR4的表达促进大鼠BMSCs迁移。方法采用MTT比色法检测IA(3.125、6.25、12.5、25、50、100、200、400、800 mg·L^-1)预处理BMSCs 24、48、72 h,观察对BMSCs增殖的影响,确定最佳药物浓度和最佳作用时间。用最佳浓度的IA干预第3代BMSCs 48 h,Transwell检测BMSCs迁移能力,Western blot法检测CXCR4的表达水平。结果 MTT结果显示,IA孵育BMSCs 24、48 h,与对照组相比,100~800 mg·L^-1组的细胞增殖明显减少(P<0.05);IA孵育BMSCs 48h,与对照组相比,6.25、3.125 mg·L^-1组细胞增殖明显增加(P<0.05);IA孵育BMSCs 72 h,与对照组相比,12.5~800mg·L^-1组细胞增殖明显减少(P<0.05),提示IA 6.25、3.125 mg·L^-1剂量组于48 h预处理BMSCs是最优选择。Transwell细胞迁移实验表明,IA(6.25、3.125 mg·L^-1)预处理BMSCs 48 h明显增强BMSCs运动迁移能力(P<0.05),且迁移与IA浓度无关,CXCR4拮抗剂AMD3100可完全阻断其促迁移效应。Western blot显示,IA(6.25、3.125 mg·L^-1)预处理BMSCs 48 h,上调CXCR4蛋白表达(P<0.05)。结论 IA可以促进BMSCs的增殖,并通过上调CXCR4的表达,提高BMSCs的迁移能力。     

SDF-1对糖尿病外周血EPCs功能的影响及其与PI3K/AKT信号通路的关系

目的观察基质细胞衍生因子-1(SDF-1)对糖尿病外周血内皮祖细胞(EPCs)功能的影响,探讨SDF-1对EPCs的影响是否与PI3K/AKT信号通路有关。方法采集糖尿病患者(DM组)和健康对照者(HC组)外周血30 m L,提取并培养EPCs。(1)SDF-1干预组加入100μg/L SDF-1培养液,非干预组加入EGM-2MV培养基,采用Boyden小室和体外血管生成试剂盒观察EPCs的迁移和体外血管生成能力。(2)将培养的EPCs分为空白对照组、1μg/L SDF-1组、10μg/L SDF-1组、100μg/L SDF-1组、单纯AMD3100组及100μg/L SDF-1+AMD3100组,通过Western blot法检测各组EPCs中AKT蛋白的表达水平。结果 (1)无SDF-1干预时,DM组EPCs迁移和血管形成能力低于HC组,SDF-1干预后,2组的EPCs迁移和血管形成能力均较干预前增强,但DM组增强的幅度高于HC组。(2)同一浓度下,DM组的AKT蛋白表达水平均低于HC组(均P<0.01)。无论是DM组还是HC组,AKT蛋白的表达均随着加入SDF-1浓度的增加而呈递增趋势(P<0.05);100μg/L SDF-1+AMD3100组AKT蛋白的表达水平较100μg/L SDF-1组明显降低(P<0.05)。结论 SDF-1可增强外周血EPCs迁移和血管形成能力,对糖尿病患者效果更为明显,且SDF-1对EPCs的影响与PI3K/AKT信号通路有关。     

钙离子对骨髓间充质干细胞CXCR4表达的影响

目的 探讨钙离子对骨髓间充质干细胞(BMSCs) CXCR4表达的影响.方法 用含不同浓度钙离子的DMEM培养液体外培养大鼠BMSCs:A组为无钙离子培养基;B、C、D组分别加入1、2、4mM氯化钙;E、F和G组为C组中分别加入钙敏感受体特异性抗体、AMD3100和抗CXCR4抗体.采用RT-PCR和Western blot检测CXCR4 mRNA水平和蛋白表达,采用迁移实验测定BMSCs向基质细胞衍生因子1(SDF-1)的趋化效应.结果 CXCR4mRNA转录水平与蛋白表达水平:与A组相比,B、C、D组显著上升呈浓度依赖性(P＜0.05);与C组相比,E组显著下调(P＜0.05).与A组相比,C组向SDF-1的迁移细胞数增加;与C组相比,E、F、G组向SDF-1的迁移细胞数减少(P＜0.05).结论 钙离子可促进BMSCs的CXCR4转录和表达,并促进BMSCs向SDF-1的趋化迁移,从而可能促进BMSCs的靶向归巢.     

HIV-1协同受体及其抑制剂研究进展

协同受体CCR5和CXCR4分别是嗜巨噬细胞性HIV-1和嗜T细胞性TIV-1侵入靶细胞的主要受体。CCR5抑制剂如TAK-779、SCH—C等，和CXCR4抑制剂如AMD3100、T22等，能分别与CCR5和CXCR4结合，从而阻断HIV-1侵入靶细胞。本文综述了HIV-1与CCR5和CXCR4的结合机制及其抑制剂的研究进展。     

SDF-1 α／CXCR4对THP-1的趋化活性及其Ox-LDL的干预作用

目的探讨基质细胞衍生因子（SDF-1α）对单核细胞的趋化作用,ox-LDL对SDF-1α趋化单核细胞的影响及其对THP-1细胞表达CXCR4的影响。方法用Transwell迁移实验来研究SDF-1α对单核细胞的趋化作用,将单核细胞或经ox-LDL处理的单核细胞加入上室,在下室加入SDF-1α孵育10h后计数各组下室迁移的细胞数。RT-PCR检测CXCR4表达变化,Western blot检测其蛋白表达变化,观察ox-LDL对THP-1细胞CXCR4表达的剂量和时间效应。结果SDF-1α呈浓度依赖性诱导单核细胞的迁移,加入CXCR4抗体可明显抑制这种作用。经不同浓度OX-LDL处理48h后的THP-1细胞再用10ng／ml SDF-1α进行趋化时,结果发现ox-LDL呈浓度依赖性的增加单核细胞的迁移,50μg／ml ox-LDL组所迁移的细胞数是对照组的11倍。THP-1细胞有基础水平的CXCR4表达,50μg／ml ox-LDL可使CXCR4的表达上调4-5倍。CXCR4上调最早在6h内发生,12h达高峰。结论SDF-1α／CXCR4参与趋化单核细胞迁移,其作用被OX-LDL加强;ox-LDL上调CXCR4表达。     

AMD3100 mobilizes endothelial progenitor cells in mice, but inhibits its biological functions by blocking an autocrine/paracrine regulatory loop of stromal cell derived factor-1 in vitro

Endothelial progenitor cells (EPCs), a CXCR4-bearing cell line, are thought to positively influence reendothelialization, vascular repair, and angiogenesis. AMD3100, a highly selective antagonist of stromal cell derived factor (SDF)-1 that binds to its receptor, CXCR4, has been shown to induce rapid mobilization of hematopoietic stem cells in mice, dogs, and humans. Results of this study indicate that AMD3100 injection can induce a rapid and potent mobilization of EPCs in mice compared with saline injection. The study demonstrates that murine spleen-derived EPCs can produce high levels of SDF-1 in vitro. Blocking this endogenous SDF-1 with AMD3100 decreases the number of EPCs; inhibits the proliferation, migration, and adhesion of EPCs; and induces EPC apoptosis. In addition, AMD3100 inhibits ectogenous SDF-1alpha induced improvement of EPC functions. However, AMD3100 incubation does not change the functions of CXCR4-negative cell line HPDE6. In conclusion, AMD3100 can be regarded as a potent mobilizer of EPCs in mice. EPCs have an autocrine/paracrine SDF-1 loop, and breaking this loop with AMD3100 can inhibit the EPC functions in vitro.     

Noncyclam tetraamines inhibit CXC chemokine receptor type 4 and target glioma-initiating cells

The three stereoisomers of the noncyclam compound 1 (1(R,R), 1(S,S), and the meso form 1(S,R)) and their corresponding tetrahydrochlorides 11 were prepared from (S)- and (R)-2-methylpiperidine. We have evaluated their inhibitory activity on the CXC chemokine receptor type 4 (CXCR4), toxicity properties, and assessment of their effect on glioma initiating cells (GICs) in comparison with the prototype compound AMD3100. The IC(50) values determined on human recombinant (CHO) cells showed very similar inhibitory activities albeit a lower K(B) for AMD3100, with the 1(R,R) isomer being second in potency. All the compounds showed low cardiac toxicity but, contrary to AMD3100, gave maximum nonlethal doses of around 2.0 mg/kg. The CXCR4 inhibitors had an effect on the state of differentiation of GICs, decreasing the percentage of CD44+ cells in glioblastoma multiform neurospheres in vitro. Moreover, these CXCR4 inhibitors blocked the capacity of cells to initiate orthotopic tumors in immunocompromised mice.     

Characterization of the molecular pharmacology of AMD3100: a specific antagonist of the G-protein coupled chemokine receptor, CXCR4

The chemokine receptor CXCR4 is widely expressed on different cell types, is involved in leukocyte chemotaxis, and is a co-receptor for HIV. AMD3100 has been shown to be a CXCR4 receptor antagonist, and to block HIV infection of T-tropic, X4-using, virus in vitro and in vivo. AMD3100 is an effective mobilizer of hematopoietic stem cells and is being investigated in clinical trials in multiple myeloma and non-Hodgkins lymphoma patients. Using the CCRF-CEM T-cell line that constitutively expresses CXCR4 we confirmed that AMD3100 was an antagonist of SDF-1/CXCL12 ligand binding (IC50=651+/-37 nM). We have also shown that AMD3100 inhibits SDF-1 mediated GTP-binding (IC50=27+/-2.2 nM), SDF-1 mediated calcium flux (IC50=572+/-190 nM), and SDF-1 stimulated chemotaxis (IC50=51+/-17 nM). AMD3100 did not inhibit calcium flux against cells expressing CXCR3, CCR1, CCR2b, CCR4, CCR5 or CCR7 when stimulated with their cognate ligands, nor did it inhibit receptor binding of LTB4. AMD3100 did not, on its own, induce a calcium flux in the CCRF-CEM cells, which express multiple GPCRs including CXCR4, CCR4 and CCR7. Furthermore, AMD3100 neither stimulated GTP-binding, an assay for GPCR activation, in CEM cell membranes; nor chemotaxis of CCRF-CEM cells. These data therefore demonstrate that AMD3100 is a specific antagonist of CXCR4, is not cross-reactive with other chemokine receptors, and is not an agonist of CXCR4.     

Potential clinical applications of the CXCR4 antagonist bicyclam AMD3100

The bicyclam AMD3100 (originally called JM3100), in which the two cyclam rings are tethered by an aromatic bridge, emanated from JM2763, where the two cyclam moieties are tethered by an aliphatic linker - JM2763 in turn originated from JM1657, where the cyclam rings are directly linked to one another via a C-C bridge, and which was identified as an impurity, showing anti-HIV activity, in a commercial cyclam preparation. AMD3100 proved very effective against HIV-1 and HIV-2, inhibiting virus replication within the nM range, without toxicity for the host cells at concentrations that were > 100,000-fold higher than those required to inhibit HIV replication. The anti-HIV activity of AMD3100 appeared to be confined to the T-lymphotropic (X4) HIV strains, i.e. those strains that use the CXCR4 receptor to enter their target cells, and AMD3100 as of today still stands as one of the most potent and selective CXCR4 antagonists ever discovered. Hence, AMD3100 was found to interfere with a number of (patho)physiological processes which depend on the interaction of CXCR4 with its natural ligand, stromal derived factor (SDF-1) and which play an important role in rheumatoid, allergic and malignant diseases. AMD3100 has been shown to mobilize CD34+ stem cells from the bone marrow into the bloodstream and has also been shown to augment migration of bone marrow-derived endothelial progenitor cells into sites of neovascularization after myocardial infarction. Currently, AMD3100 is actively pursued as a stem cell mobilizer for transplantation in patients with multiple myeloma and non-Hodgkin's lymphoma.     

AMD3465, a monomacrocyclic CXCR4 antagonist and potent HIV entry inhibitor

The chemokine receptors CCR5 and CXCR4 function as coreceptors for human immunodeficiency virus (HIV) and are attractive targets for the development of anti-HIV drugs. The most potent CXCR4 antagonists described until today are the bicyclams. The prototype compound, AMD3100, exhibits potent and selective anti-HIV activity against CXCR4-using (X4) viruses and showed antiviral efficacy in X4 HIV-1-infected persons in a phase II clinical trial. However, AMD3100 lacks oral bioavailability due to its high overall positive charge. Initial structure-activity relationship studies with bicyclam analogues suggested that the bis-macrocyclic structure was a prerequisite for anti-HIV activity. Now, we report that the N-pyridinylmethylene cyclam AMD3465, which lacks the structural constraints mentioned above, fully conserves all the biological properties of AMD3100. Like AMD3100, AMD3465 blocked the cell surface binding of both CXCL12 (the natural CXCR4 ligand), and the specific anti-CXCR4 monoclonal antibody 12G5. AMD3465 dose-dependently inhibited intracellular calcium signaling, chemotaxis, CXCR4 endocytosis and mitogen-activated protein kinase phosphorylation induced by CXCL12. Compared to the bicyclam AMD3100, AMD3465 was even 10-fold more effective as a CXCR4 antagonist, while showing no interaction whatsoever with CCR5. As expected, AMD3465 proved highly potent against X4 HIV strains (IC50: 1-10 nM), but completely failed to inhibit the replication of CCR5-using (R5) viruses. In conclusion, AMD3465 is a novel, monomacrocyclic anti-HIV agent that specifically blocks the interaction of HIV gp120 with CXCR4. Although oral bioavailability is not yet achieved, the monocyclams, with their decreased molecular charge as compared to the bicyclams, embody an important step forward in the design of oral CXCR4 antagonists that can be clinically used as anti-HIV drugs.