多肽AP25抗肿瘤活性研究

目的 研究多肽AP25在组织水平和整体动物水平的抗肿瘤作用,为临床试验提供依据.方法 采用大鼠动脉环和鸡胚尿囊膜实验检测AP25对血管新生的抑制作用,采用动物体内实验评价AP25抗肿瘤活性.结果 大鼠动脉环实验表明多肽AP25可以抑制大鼠动脉环微血管生成,并且在0.25 mg·L-1时抑制率达到62.19%;鸡胚尿囊膜实验表明多肽AP25对血管新生有明显的抑制作用,且具有剂量依赖关系,20 mg·L-1抑制率达到80.37%;动物体内实验表明多肽AP25在裸鼠移植瘤模型上可以明显地抑制HCT116细胞的生长.结论 多肽AP25对新生血管具有明显的抑制作用,主要通过抑制新生血管达到抑制肿瘤的生长,进而发挥抗肿瘤的活性.     

MEK抑制剂CQN系列化合物对A549细胞体外抗肿瘤作用

目的观察丝裂原激活蛋白激酶的激酶(MEK)抑制剂CQN系列化合物对A549细胞体外抗肿瘤作用。方法采用非小细胞肺癌A549细胞(K-ras突变)体外抗肿瘤增殖抑制实验对9个CQN系列MEK抑制剂进行筛选,得到具有显著抗肿瘤活性的化合物。通过流式细胞术检测化合物对细胞周期和凋亡的影响;并通过ELISA法检测化合物对肿瘤发生关键蛋白ERK1/2磷酸化的抑制作用;通过细胞划痕实验检测化合物对细胞迁移能力的影响。结果细胞增殖实验筛选出高活性化合物CQN-4和CQN-5,两者的体外抗A549细胞增殖抑制活性与对照药曲美替尼(trametinib)相当。CQN-4和CQN-5可将A549细胞阻滞于G1期,与对照相比可使细胞凋亡率明显增加(P<0.01),且呈浓度依赖性。与对照相比,CQN-4和CQN-5对A549细胞ERK1/2磷酸化均有抑制作用(P<0.01),且具有浓度依赖性。细胞划痕愈合实验显示CQN-4和CQN-5对A549细胞迁移有显著的抑制作用,且具有浓度依赖性。结论 MEK抑制剂化合物CQN-4和CQN-5对A549细胞具有显著的体外抗肿瘤活性。     

靶向抗肿瘤肽的构建及其生物学活性研究

目的构建重组靶向抗肿瘤肽(Citrostatin),研究其生物学活性。方法构建Citrostatin重组基因,经原核生物表达和纯化获得Citrostatin融合蛋白,通过内皮细胞杀伤实验、细胞毒活性实验、体外管状机构生成抑制实验等分析Citrostatin的生物学活性。结果Citrostatin融合蛋白经表达、酶切纯化后,目的蛋白纯度达90%以上。Citrostatin能明显抑制内皮细胞ECV304的增殖(IC50=2.28μmol/L),有效杀伤肿瘤细胞1990及NCI-H640(IC50分别为9.24,2.74μmol/L),并明显抑制体外管状结构的生成。结论成功构建了重组靶向抗肿瘤肽Citrostatin,细胞和血管水平研究表明该融合肽同时具有抑制肿瘤新生血管的形成和直接杀伤肿瘤的双重活性。     

蝎及蝎毒抗肿瘤作用研究进展

目的介绍蝎、蝎毒及其中活性组分抗肿瘤作用研究进展。方法根据近年来国内外公开发表的有关文献,从蝎及蝎毒的抗肿瘤作用、蝎毒抗肿瘤成分及其生物学机制、全蝎及蝎毒抗肿瘤的临床应用等方面综述。结果与结论全蝎粗提物可使体外培养的人体子宫颈癌细胞全部死亡脱壁,全蝎的醇制剂在体外能显著抑制人肝癌细胞呼吸,并对结肠和人肝癌细胞的生长均有明显的抑制作用,蝎尾提取物对肿瘤兼有预防和治疗的双重作用;蝎毒粗毒对肿瘤有确切的抑制作用,且优于化疗药物;蝎毒中具有抗肿瘤活性的单组分活性肽为Chlorotoxin和东亚钳蝎镇痛抗肿瘤活性肽;Chlorotoxin特异性抑制神经胶质瘤细胞的迁移与浸润,而不影响正常脑细胞;东亚钳蝎镇痛抗肿瘤活性肽是Na+离子通道抑制剂,其抗肿瘤作用机理可能与Chlorotoxin不同。     

异甘草素抗肿瘤活性及初步机制研究

目的探讨异甘草素(isoliquiritigenin,ISL)抗肿瘤活性及初步机制。方法采用SRB法检测ISL对A549、SW620和HMEC-1细胞增殖的抑制作用;Transwell小室检测ISL对HMEC-1细胞迁移能力的影响;明胶酶谱法检测ISL对HMEC-1细胞MMP-2和MMP-9的表达;管样结构形成实验测定ISL对HMEC-1细胞管样结构形成能力;细胞内活性氧(ROS)通过荧光探针DCFH-DA进行测定;流式细胞术检测ISL对HMEC-1的细胞周期。鸡胚绒毛尿囊膜血管生成实验检测ISL体内抗血管生成作用。结果 ISL可明显抑制A549、SW620和HMEC-1细胞的增殖、HMEC-1细胞的迁移、管样结构形成以及细胞内MMP-2和MMP-9的表达,且均呈现浓度依赖关系。同时,ISL还可抑制HMEC-1细胞内由VEGF诱导产生的ROS量,且存在浓度依赖关系。流式细胞术检测发现高浓度ISL可将HMEC-1细胞阻滞于S期,而低浓度ISL通过诱导细胞凋亡来抑制HMEC-1细胞的增殖。ISL能明显抑制鸡胚尿囊膜新生毛细血管的生成。结论ISL具有明显的抗肿瘤活性,能抑制血管生成,其机制可能与清除HMEC-1细胞内ROS生成,诱导细胞凋亡有关。     

山楂叶总黄酮对胶质瘤U87细胞的抑制作用

目的研究山楂叶总黄酮对胶质瘤U87细胞的抑制作用。方法体外培养U87细胞,采用25、50、100 mg·L~(-1)的山楂叶总黄酮,通过CCK-8实验、划痕实验、Transwell法、黏附实验,检测山楂叶总黄酮对U87细胞增殖、迁移能力、侵袭能力、黏附能力的影响;克隆形成实验检测细胞克隆形成能力。结果与空白对照组相比,山楂叶总黄酮呈浓度依赖性抑制U87细胞的增殖、迁移、侵袭及黏附能力,差异具有统计学意义(P<0.05),山楂叶总黄酮50 mg·L~(-1)时抑制胶质瘤U87细胞生长作用最为明显。结论山楂叶总黄酮对胶质瘤U87细胞有一定的抑制作用。     

覆盆子苷—F3抗血管生成作用的研究

目的：考察覆盆子分离的化合物覆盆子苷-F3抗血管生成作用。方法：采用MTT法筛选出具有明显差异细胞毒性的活性化合物,并进一步通过划痕实验检测细胞迁移能力和体外血管生成实验检测细胞成管能力。结果：覆盆子苷-F3对Bel-7402细胞和人脐静脉内皮细胞(HUVECs)有明显的差异细胞毒性作用。与对照组相比,覆盆子苷-F3对HUVECs的迁移和血管生成具有显著抑制作用(P<0.01)。结论：覆盆子苷-F3在体外能有效抑制血管生成,其作用机制可能与抑制血管内皮细胞的增殖和迁移,从而达到抑制血管新生有关。     

人肝癌HepG-2细胞对人淋巴管内皮细胞增殖及管状结构形成的影响

目的探讨人肝癌HepG-2细胞对人淋巴内皮细胞(HLEC)增殖、迁移和管状结构形成的影响。方法采用transwell方法建立HepG-2细胞与HLEC共培养系统;通过绘制细胞生长曲线,观察HepG-2细胞对HLEC增殖的影响;通过细胞迁移实验,观察HepG-2细胞对HLEC迁移的影响;通过基质胶实验,观察HepG-2细胞对HLEC管状结构形成的影响。结果 HepG-2细胞能够显著促进HLEC增殖(P<0.05),迁移(P<0.05)及管状结构形成(P<0.05),呈剂量依赖性。结论 HepG-2细胞促进HLEC增殖、迁移及管状结构形成可能是肿瘤淋巴道转移的机制之一。     

孕激素受体拮抗剂ZXH951对乳腺癌细胞生长抑制作用及对端粒酶活性的影响

目的　研究ZXH951在体外对乳腺癌细胞的生长抑制作用及对端粒酶活性的影响。方法　分别用细胞生长曲线、集落形成及竞争性配体与受体结合法,测定ZXH951的体外抗肿瘤作用及其与孕激素受体（PR）、雌激素受体(ER)结合活性;用流式细胞术及多聚酶链反应-端粒重复序列扩增法探讨ZXH951对人乳腺癌T47D细胞周期及端粒酶活性的影响。结果　ZXH951对ER和PR双阳性的乳腺癌细胞T47D体外增殖有较强的抑制作用,而对ER和PR双阴性的人乳腺癌细胞MDA-MB-231无明显抑制作用;ZXH951与PR有较强结合活性,而与ER无明显结合活性;可将T47D细胞阻滞在G1期,并对端粒酶活性有一定抑制作用。 结论　ZXH951是一个有发展前景的新型抗孕激素类化合物,在体外对乳腺癌细胞有较强的抑制作用,其作用机制可能与其通过PR介导的细胞增殖及对端粒酶活性抑制有关。     

酪氨酸激酶受体抑制剂AL3810对人肺腺癌细胞A549的抗肿瘤作用

目的 评价酪氨酸激酶受体抑制剂AL3810对人肺腺癌细胞A549的体外及体内抗肿瘤作用.方法 ①采用MTT法评价AL3810对13种人体肿瘤细胞株和人胚肺细胞的体外增殖抑制作用.②采用流式细胞仪检测AL3810诱导细胞凋亡的能力及其对细胞周期的影响.③采用流式细胞仪检测AL3810诱导Caspase-3活化的作用.④划痕试验评价AL3810对A549细胞体外迁移的影响.⑤以人肺腺癌A549裸鼠异种移植瘤模型评价AL3810对肿瘤生长抑制作用的量效关系.结果 AL3810对大部分细胞均有很强的生长抑制作用.其能诱导A549细胞凋亡且具有时效性,能将细胞周期阻滞于G0-G1期;能将无活性的Caspase-3前体活化且具有时效关系;能时间依赖性地显著抑制A549细胞的体外迁移;对A549异种移植瘤生长具有良好的抑制作用,且具有量效关系.结论 酪氨酸激酶受体抑制剂AL3810具有显著的抗肿瘤活性,能剂量依赖性地抑制A549的生长.可能机制为诱导肿瘤细胞凋亡和阻滞细胞周期于G0-G1期、诱导细胞内Caspase-3活化、抑制细胞迁移等,值得进一步开发.     

钙网织蛋白促进新生血管形成在类风湿关节炎发病机制中的作用

目的探讨钙网织蛋白（CRT）促进新生血管形成在类风湿关节炎（RA）发病机制中的作用。方法采用酶联免疫吸附试验（ELISA）法检测活动期RA 患者、骨性关节炎（OA）患者和健康对照组（HC）血清以及RA 和OA 患者关节滑膜液中CRT 的含量。采用免疫组织化学（IHC）法检测CRT 在RA 和OA 患者关节滑膜组织中的表达。应用人脐静脉内皮细胞（HUVECs）体外模型,分别采用MTT 实验、细胞划痕实验及构建体外血管生成的三维模型检测 CRT 在HUVECs 增殖、迁移及管腔形成中的作用。结果RA 组血清中CRT 含量（[ 6.4±3.1）μg/L]明显高于OA 组（[ 3.7±0.9）μg/L]及HC 组（[ 3.4±1.0）μg/L];RA 组滑膜液中CRT 含量（[ 6.9±3.4）μg/L]明显高于OA 组（[ 3.9±0.7）μg/L],差异有统计学意义（P < 0.01）。CRT 在RA 滑膜组织中高表达,主要位于滑膜衬里层和衬里下层的血管内皮细胞及血管周围、炎性细胞内外等,而OA 滑膜组织中CRT 仅见极少量的表达。MTT 实验、细胞划痕实验及体外血管生成三维模型检测结果显示,CRT 具有明显促进HUVECs 增殖、迁移及管腔形成的作用。结论CRT 在RA 患者血清、滑膜液及滑膜组织中表达均升高,CRT 可能通过促进新生血管形成参与RA 滑膜炎和血管翳形成的发病机制。     

卡立泊来德对K562细胞诱导的血管生成的影响

目的研究卡立泊来德(cariporide)处理对K562细胞诱导的血管生成能力的影响。方法应用MTT检测K562细胞上清液对脐静脉内皮细胞增殖能力的影响;transwell检测K562细胞上清液对脐静脉内皮细胞迁移能力的影响;基质胶血管形成法检测K562细胞上清液对脐静脉内皮细胞体外血管形成能力的影响;激光扫描共聚焦显微镜测定K562细胞的细胞内的pH;酶联免疫吸附实验(ELISA)检测K562细胞上清中血管内皮生长因子的表达水平。结果cariporide处理可以明显降低K562细胞上清液对脐静脉内皮细胞增殖,迁移和体外成管能力的诱导;cariporide处理后K562细胞的细胞内pH明显下降,分泌VEGF能力也受到抑制。结论 cariporide能抑制K562细胞的血管生成诱导能力,这种抑制是通过细胞内pH下降以及VEGF分泌减少引起的。     

Structural modification of honokiol, a biphenyl occurring in Magnolia officinalis: the evaluation of honokiol analogues as inhibitors of angiogenesis and for their cytotoxicity and structure-activity relationship

Honokiol, widely known as an antitumor agent, has been used as an antiangiogenesis drug lead. In this paper, 47 honokiol analogues and derivatives were investigated for their antiangiogenic activity by application of the transgenic zebrafish screening model, antiproliferative and cytotoxic activity against HUVECs, and three tumor cell lines by MTT assay. 3',5-Diallyl-2,4'-dihydroxy-[1,1'-biphen-yl]-3,5'-dicarbaldehyde (8c) was found to suppress the newly grown segmental vessels from the dorsal aorta of zebrafish and prevent inappropriate vascularization as well as exhibit more potent inhibitory effects on the proliferation of HUVECs, A549, HepG2, and LL/2 cells (IC(50) = 15.1, 30.2, 10.7, and 21.7 μM, respectively) than honokiol (IC(50) = 52.6, 35.0, 16.5, and 65.4 μM, respectively). Analogue 8c also effectively inhibited the migration and capillary-like tube formation of HUVECs in vitro. The antiangiogenic effect and antiproliferative activity of these structurally modified honokiol analogues and derivatives have led to the establishment of a structure-activity relationship.     

Antiangiogenic activity of Diallyl Sulfide (DAS)

Antiangiogenic activity of Diallyl sulfide (DAS) was studied using in vivo as well as in vitro models. In vivo antiangiogenic activity was studied using B16F-10 melanoma cell induced capillary formation in C57BL/6 mice. DAS significantly inhibited tumour directed capillary formation. Studies of serum cytokine profile of angiogenesis induced animals clearly showed that DAS significantly reduced the production of proinflammatory cytokines such as IL-1beta, IL-6, TNF-alpha and GM-CSF which are known proangiogenic factors. The serum level of VEGF, an important proangiogenic factor, in angiogenesis induced animals was found to be significantly reduced upon treatment with DAS which may be due to its efficacy in the down regulation of VEGF mRNA expression. Administration of DAS significantly enhanced the production of antiangiogenic factors such as IL-2 and TIMP. In vitro studies using rat aortic ring assay showed that administration of DAS at no n-toxic concentrations significantly inhibited microvessel sprouting. Studies using Human umbilical vein endothelial cells (HUVECs) clearly demonstrated that administration of DAS significantly retarded endothelial cell proliferation, migration, invasion and tube formation. These data clearly suggests that antiangiogenic activity of DAS can be related to its negative regulation of proangiogenic factors such as VEGF and proinflammatory cytokines and positive regulation of antiangiogenic factors such as IL-2 and TIMP.     

Anti-angiogenic activities of gliotoxin and its methylthio-derivative, fungal metabolites

In the search for new naturally occurring angiogenic inhibitor, we found that culture broths from two unidentified fungal strains exerted potent inhibitory activities on capillary-like tube formation of human umbilical vein endothelial cells (HUVEC) in vitro. Two active compounds were isolated by bioassay-guided separation and their structures were identified as gliotoxin (1) and its derivative methylthiogliotoxin (2) by spectroscopic analyses. These compounds significantly inhibited the migration of HUVEC assessed by in vitro wounding migration assay and exhibited at least 10 times more potent inhibition of proliferation of HUVECs as compared with that of cancer cell lines such as HeLa, MCF-7, and KB 3-1 cells. Especially, gliotoxin having disulfide group exerted more potent activities than methylthiogliotoxin, suggesting that gliotoxin could be a useful compound for further study as an anti-angiogenic agent.     

芸香宁碱抑制血管生成作用的研究

目的：研究芸香宁碱对血管生成的抑制作用,并研究其初步的作用机制。方法用人脐静脉内皮细胞（HUVEC）的生长、迁移实验及体内动物模型－鸡胚绒毛尿囊膜法（CAM 法）研究化合物的体内外抗血管生成作用;用酶联免疫吸附法检测芸香宁碱在非凋亡剂量时对肿瘤细胞培养上清液中 VEGF 蛋白分泌量的影响。结果芸香宁碱对血管内皮细胞具有优先抑制作用,对 HUVEC 的半数抑制剂量为（33．2±0．4）μmol·L －1,而对其他肿瘤细胞的 IC50值均大于这一数值;芸香宁碱在体外能够明显抑制内皮细胞的迁移和对细胞外基质黏附作用,并呈剂量依赖性,体内实验显示30μmol·L －1剂量浓度时显著抑制 CAM新生血管形成;芸香宁碱在15μmol·L －1和30μmol·L －1的浓度剂量时显著抑制人肝癌 HepG2细胞培养上清液中VEGF 蛋白的分泌,具有显著性差异。结论芸香宁碱在非凋亡浓度剂量具有抑制新生血管形成作用,其机制与抑制血管内皮细胞增殖以及抑制肿瘤细胞表达 VEGF 有关。     

Anti-angiogenic activity of triptolide in anaplastic thyroid carcinoma is mediated by targeting vascular endothelial and tumor cells

Triptolide is confirmed to suppress angiogenesis of anaplastic thyroid carcinoma. Here we further expound the precise mechanism involved in this activity. Triptolide downregulated nuclear factor kappa B (NF-κB) pathway and its targeting genes associated with endothelial cell mobilization in human umbilical vein endothelial cells (HUVECs) and impaired VEGF expression in thyroid carcinoma TA-K cells. Furthermore, both triptolide and the conditioned medium from triptolide-treated TA-K cells (CMT) significantly attenuated proliferation, migration and tube formation of HUVECs. In vivo, triptolide inhibited TA-K cell-induced tumor growth, vascular formation and VEGF expression. Our data establish that triptolide inhibits tumor angiogenesis by the dual action on vascular endothelial cells and tumor cells, thus providing a novel and overall explanation for the anti-angiogenesis action of triptolide. The multicellular targets emphasize triptolide as a high-performance and potential angiogenesis inhibitor.     

Anti-tumor and anti-angiogenic effects of Phyllanthus urinaria in mice bearing Lewis lung carcinoma

Phyllanthus urinaria, a widely used herb medicine in Asia, was tested for its anti-tumor effect in vivo for the first time. The anti-tumor activity in P. urinaria extract was evaluated by its effect on tumor developed in C57BL/6J mice with implantation of Lewis lung carcinoma cells. The oral administration of P. urinaria to mice caused significant inhibition of tumor development with lower occurrence rate and markedly reduced tumor size. Neither the total body weight of mouse nor the weights of organs including heart, lung, liver, spleen and kidney revealed any difference between two groups, suggesting limited in vivo cytotoxic effect of P. urinaria in mice. TUNEL assay demonstrated the increase of apoptosis in tumor sections prepared from P. urinaria-treated mice compared with control mice. It is worth of note that the neovascularization in tumor was inhibited in P. urinaria-treated mice, which implicated the potential anti-angiogenic effect of P. urinaria. Further study using an in vitro matrix-induced tube formation of HUVECs again confirmed the anti-angiogenic action of P. urinaria. P. urinaria exerted no inhibitory effect on the growth of HUVECs, however, the migration of HUVECs as analyzed using transwell assay was suppressed markedly by P. urinaria in a dose-dependent manner. All together, the present study indicated that P. urinaria extract is an anti-tumor and anti-angiogenic agent, which can be used safely in animals.     

PI3K/Akt抑制剂CCT128930抑制人脐静脉血管内皮细胞增殖与血管生成的实验研究

目的探讨一类新的PI3K/Akt抑制剂CCT128930对人脐静脉血管内皮细胞(HUVEC)增殖与血管生成的影响。方法采用MTT法检测CCT128930对HUVEC存活的影响;流式细胞术分析细胞周期变化;AnnexinⅤ-FITC试剂盒检测细胞凋亡;体外小管形成实验观察CCT128930对HUVEC体外小管形成的影响;免疫印迹法检测蛋白表达水平。结果CCT128930可通过阻滞细胞于G1期而抑制HUVEC的增殖,且抑制作用呈剂量依赖性,但对HUVEC的凋亡无影响;HUVEC经CCT128930处理后,体外小管形成能力受到明显抑制;低浓度的CCT128930抑制内皮细胞中VEGF的表达,但对Akt的磷酸化水平无影响。结论 CCT128930能够抑制HUVEC的增殖与血管生成,其抑制血管生成活性可能与其调控VEGF的表达水平相关。     

Phosphatidylinositol inhibits vascular endothelial growth factor-A--induced migration of human umbilical vein endothelial cells

Phosphatidylinositol (PI), a phospholipid in component of cell membranes, is widely distributed in animals, plants, and microorganisms. Here, we examined in vitro whether PI inhibits the angiogenesis induced by vascular endothelial growth factor-A (VEGF-A). PI concentration-relatedly and significantly (at 10 and 30 microg/ml) inhibited VEGF-A-induced tube formation in a co-culture of human umbilical vein endothelial cells (HUVECs) and fibroblasts. PI also inhibited the migration, but not proliferation, induced in HUVECs by VEGF-A. Furthermore, PI at 30 microg/ml inhibited the VEGF-A-induced phosphorylation of serine/threonine protein kinase family protein kinase B (Akt) and p38 mitogen activate kinase (p38MAPK), key molecules in cell migration, but not phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), a key molecule in cell proliferation. These findings indicate that PI inhibits VEGF-induced angiogenesis by inhibiting HUVECs migration and that inhibition of phosphorylated-Akt and -p38MAPK may be involved in the mechanism. Therefore, PI may be expected to prevent some diseases caused by angiogenesis.