蛇毒金属蛋白酶抑制剂重组蛋白抗肿瘤血管生成作用及其机制

目的研究蛇毒金属蛋白酶抑制剂重组蛋白(recombinant snake venom metalloproteinase inhibitor,r SVMPI)对血管生成的影响及其分子机制。方法应用鸡胚绒毛尿囊膜(chicken chorioallantoic membrane,CAM)血管生成模型观察SVMPI重组蛋白对血管生成的影响;采用Alamar Blue分析方法检测人脐静脉内皮细胞(human umbilical veins endothelial cells,HUVECs)增殖能力、Annexin V-FITC双标记流式细胞术检测细胞凋亡、划痕标记法检测细胞体外迁移能力、Boyden小室分析方法检测细胞体外趋化能力及管腔形成法检测体外血管新生能力;通过real-time PCR及Western blot检测重组蛋白处理后的HUVECs KDR、FGFR-1表达。结果r SVMPI减少鸡胚尿囊膜新生血管密度指数,减弱由VEGF诱导的HUVECs趋化能力,抑制HUVECs体外新生小管的形成,降低HUVECs细胞KDR和FGFR-1的表达水平。结论r SVMPI可能通过阻断VEGF-KDR或b FGF-FGFR信号转导途径,发挥抑制血管生成的作用。     

姜黄素具有抑制血管生成与诱导肿瘤细胞凋亡双重作用

目的　探讨姜黄素的抗肿瘤机制。方法　采用鸡胚绒毛尿囊膜模型观察姜黄素对血管生成的影响 ;利用培养的肿瘤细胞SMMC 772 1,采用电子显微镜及流式细胞仪观察姜黄素诱导SMMC 772 1细胞凋亡的作用。结果　姜黄素能明显抑制鸡胚绒毛尿囊膜内的血管生成 ,2 0 μmol·L-1的姜黄素即可诱导SMMC 772 1细胞凋亡。结论　姜黄素的抗肿瘤机制是多方面的 ,既可抑制血管生成又可诱导肿瘤细胞凋亡     

基因重组心肌肌钙蛋白Ⅰ融合蛋白的抗肿瘤效应

目的研究基因重组心肌肌钙蛋白I与人工短肽的融合蛋白(CIS)对肿瘤生长的作用。方法用MTT法观察CIS体外对人脐静脉内皮细胞(HUVEC)生长的作用。利用鸡胚绒毛尿囊膜模型观察CIS对新生血管生长的影响。用6种小鼠肿瘤异位可移植模型观察CIS在体内对肿瘤生长的作用。结果 CIS对HUVEC细胞增殖具有明显抑制作用,并呈剂量依赖关系;鸡胚绒毛尿囊膜实验显示,CIS浓度为5、10 mg·L~(-1)时,新生血管生成的数量明显减少;荷瘤鼠体内异位移植模型实验显示:CIS(10 mg·kg~(-1))处理组肿瘤生长缓慢,瘤体明显小于模型对照组,对S180肿瘤瘤重抑制率85.3%,对Lewis肺癌肿瘤瘤重抑制率87.0%,对H22肝癌肿瘤瘤重抑制率84.2%,对人小细胞肺癌H446肿瘤瘤重抑制率60.42%,对人可移植性肝癌SMMC7721肿瘤瘤重抑制率61.62%,对人胃低分化腺癌BGC823肿瘤瘤重抑制率为41.84%。结论 CIS在体外抑制HUVEC细胞的生长,在鸡胚绒毛尿囊膜实验中,CIS对新生血管生成有明显的抑制作用。在体内,CIS融合蛋白可有效抑制小鼠可移植肿瘤细胞的生长。CIS抗肿瘤效应很可能是通过抑制肿瘤组织中血管内皮细胞的增殖,进而减少肿瘤组织中新生血管生成的数量而达到的。     

沙蟾毒精抑制血管生成的作用

探讨沙蟾毒精(arenobufagin)对体内外血管生成的抑制作用。采用MTT法检测沙蟾毒精对人鼻咽癌细胞(CNE-2)、人喉癌细胞(Hep2)、人神经母细胞瘤(SH-SY5Y)、人结肠癌细胞(LOVO)、人前列腺癌细胞(PC-3及DU145)和人脐静脉内皮细胞(HUVECs)的细胞毒性及运用光学显微镜观察细胞形态的变化,发现沙蟾毒精剂量依赖性抑制CNE-2、Hep2、SH-SY5Y、LOVO、PC-3、DU145和HUVECs增殖,且在对人癌细胞亚细胞毒浓度下能够有效抑制HUVECs的增殖。采用鸡胚尿囊膜(chick embryo chorioallantoic membrane,CAM)模型观察新生血管的生成,结果发现沙蟾毒精作用24 h后,尿囊膜新生血管生成明显减少。采用细胞周期分析法观察到沙蟾毒精阻滞HUVECs于G2/M期,且随着浓度增加,细胞出现sub-G1凋亡峰。运用流式细胞术检测沙蟾毒精作用HUVECs后的凋亡现象以及线粒体膜电位的变化,确证沙蟾毒精呈时间和剂量依赖性诱导HUVECs凋亡,同时检测到线粒体膜电位下降。Western blotting检测发现,沙蟾毒精作用HUVECs后,凋亡标志...     

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.     

蝎毒多肽提取物抗肿瘤血管生成作用的实验研究

目的探讨东亚钳蝎蝎毒的多肽提取物PESV的抗血管生成活性和对肿瘤生长的抑制作用。方法①用不同浓度的PESV(4～20mg·L-1)作用于人脐静脉内皮细胞(HUVEC),采用BrdU参入的ELISA法观察HUVEC增殖活性和凋亡水平变化,流式细胞术检测凋亡细胞比例,免疫组化法检测Bal和Bax表达。②观察PESV对鸡胚尿囊膜(CAM)新生血管生成的影响。③皮下注射PESV(0.3mg·kg-1),观察对S180肉瘤和H22肝癌荷瘤小鼠肿瘤生长、肿瘤血管生成和血管生成因子(VEGF和bFGF)表达的影响。结果①体外实验显示,PESV在8～20mg·L-1范围明显抑制HUVEC的增殖活性(与对照组比较,P<0.01),而对乳腺癌细胞MDAMB231的增殖无影响;PESV作用后HUVEC凋亡细胞比例较对照组增加,P<0.05,Bax表达增加;Bcl2表达降低。②0.5mg/CAM和0.8mg/CAM的PESV能明显抑制CAM新生血管的形成。③体内实验显示PESV能明显抑制小鼠S180肉瘤和H22肝癌的肿瘤生长和血管生成水平,并降低肿瘤组织内血管生成相关因子VEGF和bFGF的表达。结论PESV具有良好的体内和体外抗肿瘤血管生成活性,并籍此抑制肿瘤的生长。     

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。     

片仔癀体外抗肿瘤作用研究

目的研究片仔癀对体外肿瘤细胞增殖的影响,以及对鸡胚尿囊膜血管生成的抑制作用。方法采用MTT法检测片仔癀对体外培养肿瘤细胞(肝癌SMMC-7721细胞、胃癌SGC-7901细胞、宫颈癌HeLa细胞)的抗肿瘤作用;以鸡胚尿囊膜血管生成(CAM)实验,研究片仔癀对新生血管生成的抑制作用。结果片仔癀在0.2～1.0mg/mL对各肿瘤细胞的增殖有显著抑制作用,1.0mg/mL片仔癀对肝癌细胞增殖抑制率达到81.55%;片仔癀在0.1～1000μg/mL对鸡胚尿囊膜血管生成有显著抑制作用,给药鸡胚的新生血管数减少,血管变细,其中10μg/mL抑制率最高,一级血管抑制率为61.1%,二级血管抑制率为59.1%。结论在实验给药浓度下,片仔癀对受试肿瘤细胞的增殖具有较强抑制作用,对鸡胚尿囊膜血管生成有显著抑制作用。     

青蒿琥酯抑制Twist1蛋白表达和血管再生以及降低口腔鳞癌细胞Tca8113转移的作用

目的 研究青蒿琥酯抑制Twist 1表达、降低口腔鳞癌细胞的转移及对血管生成的抑制作用.方法 采用50、100、150、200 μg·mL-1的青蒿琥酯体外培养的口腔鳞癌细胞,然后分别采用Western blot法、MTT法、划痕实验及流式细胞仪检测不同浓度的青蒿琥酯干预对口腔鳞癌细胞Twist1表达、增殖、迁移及凋亡的影响;采用鸡胚尿囊膜检测青蒿琥酯对鸡胚尿囊膜血管生成的影响.结果 与对照组对比,青蒿琥酯可抑制Twist1蛋白的表达、口腔鳞癌细胞的增殖和迁移,且存在时间和剂量依赖性;青蒿琥酯可引起细胞的凋亡,处理组的细胞凋亡率显著高于对照组;青蒿琥酯对鸡胚尿囊膜血管新生有一定的抑制作用(P＜0.05).结论 中药青蒿琥酯可抑制Twist 1蛋白的表达、细胞增殖、迁移,同时可有效抑制血管再生.     

新生霉素抑制血管生成及其与长春新碱的协同作用

目的研究新生霉素的抑制血管生成作用及其机制。方法以鸡胚尿囊膜模型测定对血管生成的作用，并分别用MTT法、明胶酶谱法等观察新生霉素对于内皮细胞和肺癌PG细胞的影响。结果新生霉素200 μg/egg对鸡胚尿囊膜血管生成的抑制率为68.7％，且呈浓度依赖性抑制内皮细胞的增殖、运动、MMP-2分泌以及管腔的形成；新生霉素和长春新碱对血管生成及PG细胞的增殖均有明显的协同作用。结论本研究首次确定新生霉素具有抑制血管生成活性,与长春新碱联合可增强对血管生成的抑制作用。     

Thalidomide inhibits growth of tumors through COX-2 degradation independent of antiangiogenesis

Thalidomide is an antiangiogenic drug and is clinically useful in a number of cancers. However, the molecular mechanism by which thalidomide exerts its antitumor effects is poorly understood. This study was designed to clarify the relationship between antiangiogenesis and antitumor effects of thalidomide and to explore the molecular mechanism for its antitumor activity. We evaluated the effects of thalidomide on the growth of human tumor cells expressing (MCF-7 and HL-60) or not expressing (HeLa and K562) COX-2 in vitro. We also studied the effects of thalidomide on COX-1, COX-2 or bcl-2 expression, TNFalpha, VEGF, GSH and cytochrome c in these cells. Thalidomide could inhibit tumor growth in a concentration-dependent manner in MCF-7 and HL-60; its IC50s for them were 18.36+/-2.34 and 22.14+/-2.15 microM, respectively, while this effect was not observed in HeLa and K562. Thalidomide reduced COX-2 expression accompanied by a decrease of bcl-2 protein, TNFalpha, VEGF, GSH and an increased cytochrome c, but had no effect on that of COX-1, in MCF-7 and HL-60. Moreover, cells not expressing COX-2 were insensitive to the growth-inhibitory and effects on cytokines of thalidomide. In our mouse xenograft model of OVCAR-3 and HCT-8, we found that thalidomide could decrease intratumoral microvessel density in both tumors; it exerted antitumor effects only on OVCAR-3 expressing COX-2 but did not on HCT-8 not expressing COX-2. Effect of thalidomide on COX-1 and COX-2 in vivo was consistent with that of in vitro. These results demonstrated that thalidomide might inhibit growth of tumors through COX-2 degradation independent of antiangiogenesis.     

桦木酸的抗肿瘤作用及其诱导KB细胞凋亡的研究

目的研究桦木酸(betulinic acid,BetA)体内对S180肉瘤的抑制作用和体外对人口腔上皮癌(KB)细胞系凋亡的诱导活性。方法建立小鼠体内荷S180肉瘤模型,测定BetA的体内抑瘤率;采用MTT分析、细胞形态学观察、原位末端标记和流式细胞仪检测等方法检测BetA对KB细胞生长状态和细胞周期的影响以及诱导细胞凋亡的作用。结果BetA 800和1 200 mg.kg-1给荷瘤小鼠灌胃,对S180肉瘤有显著的抑制作用;体外对KB细胞生长呈剂量依赖性抑制作用(其IC50为11.60μmol.L-1),并出现大量凋亡细胞。结论BetA体内对S180肉瘤、体外对KB细胞增殖均有抑制作用,诱导肿瘤细胞死亡的主要途径可能是凋亡。     

Antiangiogenesis response of endothelial cells to the antitumour drug 10-methoxy-9-nitrocamptothecin.

10-Methoxy-9-nitrocamptothecin (MONCPT), a topoisomerase I inhibitor, exhibited high anticancer activity in solid tumour xenograft animal models in our previous study. We hypothesized that this phenomenon was associated with antiangiogenesis response. In the present study, we found that MONCPT exhibited high antiproliferation action in human EA.hy926 endothelial cells and the IC(50) value was 0.13+/-0.04microM (MTT assay). With AO/EB stain, MONCPT (50-5000nM)-mediated apoptosis was observed in EA.hy926 cells, and the similar results were shown in flow cytometry assay, the percentage of apoptotic cells induced by MONCPT (50-5000nM) was 9.2-58.5%. In Chick embryo chorioallantoic membrane (CAM) assay, MONCPT (1-5microg) resulted in a dose-dependent angiogenic inhibition. In addition, MONCPT significantly inhibited chemotactic-migration invasion on gelatin and tube formation on Matrigel of HUVECs. These results suggest that MONCPT has potential property for inhibiting angiogenesis which is involved in its antitumour activity.     

Effects of putative hydroxylated thalidomide metabolites on blood vessel density in the chorioallantoic membrane (CAM) assay and on tumor and endothelial cell proliferation

Angiogenesis, in particular anti-angiogenesis, is an area of particular therapeutic interest in cancer treatment. Several anti-angiogenic agents are in the final stages of clinical trials. One of these agents, thalidomide, best known for its teratogenic potential, is showing promise against several tumor types. Thalidomide has been shown previously to require bio-activation to exert its anti-angiogenic effect in isolated blood vessels and endothelial cells. In this work, we confirmed these findings using the in utero chicken embryo chorioallantoic membrane (CAM) system. In particular, the anti-angiogenic effect of thalidomide is significantly enhanced by activation by human but not by rat liver microsomes. We also showed in the CAM assay that hydroxylation of thalidomide at either the 1'- or 5-position retained anti-angiogenic activity whereas its hydroxylation at the 4-position led to an inactive compound. We further demonstrated that thalidomide shows weak anti-proliferative activity against MDA-MB-231 human breast cancer cells in culture. Thalidomide showed slightly more anti-proliferative activity, however, against the SH-SY5Y human neuroblastoma and human umbilical vein endothelial cell (HUVEC) types. Furthermore, incubation of thalidomide with human liver microsomes added no additional anti-proliferative effect in these cell types versus thalidomide given alone. Finally, we report that none of the thalidomide metabolites tested had any anti-proliferative effect against the breast or neuroblastoma cells, but do possess appreciable anti-proliferative activity against the endothelial cells. In summary, this work suggests that hydroxylated thalidomide analogs based on putative metabolites of the drug possess significant anti-angiogenic activity and that exploring further derivatives of these as potential anti-angiogenic agents warrants further merit.     

沙利度胺对人慢性粒细胞白血病细胞株K562细胞凋亡的影响研究

目的:研究沙利度胺对人慢性粒细胞白血病细胞株K562细胞凋亡的影响。方法:将K562细胞分为对照(未加药)组和沙利度胺低、中、高剂量(0·5、1·0、2·0mmol·L-1)组,加入相应药物分别作用24、48、72、96h后,MTT法检测各组细胞生长抑制率,Wright-Giemsa染色观察各组细胞形态变化,AnnexinⅤ-FITC/PI双染法流式细胞仪检测各组细胞凋亡率。结果:K562细胞的生长抑制率和凋亡率与沙利度胺浓度和作用时间均呈正相关;与对照组比较,沙利度胺3个剂量组作用72、96h后细胞的生长抑制率明显升高(P<0·05),4个时间点的细胞凋亡率均明显升高(P<0·01)。沙利度胺3个剂量组作用72h后,K562细胞出现细胞体积缩小等凋亡形态。结论:沙利度胺能够抑制K562细胞的增殖,呈一定的浓度和时间依赖性,并能够诱导K562细胞的凋亡。     

沙利度胺的抗肝癌细胞株SMMC7721作用

目的 通过体外细胞培养研究沙利度胺(Tha)作用于肝细胞癌(HCC)细胞株 SMMC7721,探讨Tha抗HCC的作用机制.方法根据参考文献~([1]) 确定各组药物的最适作用浓度后,通过不同浓度Tha分别与HCC细胞SMMC7721体外培养,以MTT法检测细胞增殖活性,免疫组化法测血管内皮生长因子(VEGF)蛋白表达变化,流式细胞仪检测细胞凋亡率.结果不同浓度、不同时间Tha组细胞抑制率比较,差异有统计学意义(P<0.01).不同浓度Tha组作用于SMMC7721细胞48 h后,VEGF蛋白表达和细胞凋亡率随浓度增高而增加(P<0.05).结论Tha是有效的辅助抗肝癌药物.     

Isoindolone derivative QSN-IOc induces leukemic cell apoptosis and suppresses angiogenesis via PI3K/AKT signaling pathway inhibition

Aim： 2-（4,6-Dimethoxy-l,3-dioxoisoindolin-2-yl） ethyl 2-chloroacetate （QSN-IOc） is one of isoindolone derivatives with antiproliferative activity against human umbilical vein endothelial cells （HUVECs）. The aim of this study was to investigate its antitumor activity in vitro and anti-angiogenic effects in vitro and in vivo. Methods： K562 leukemic cells and HUVECs were used for in vitro studies. Cell viability was examined using MTT assay. Cell apoptosis and mitochondrial transmembrane potential （Δψm） were detected with flow cytometry. Tube formation and migration of HUVECs were studied using two-dimensional Matrigel assay and wound-healing migration assay, respectively. VEGF levels were analyzed with RT-PCR and Western blotting. A zebrafish embryo model was used for in vivo anti-angiogenic studies. The molecular mechanisms for apoptosis in K562 cells and antiangiogenesis were measured with Western blotting. Results： In antitumor activity studies, QSN-IOc suppressed the viability of K562 cells and induced apoptosis in dose- and time- dependent manners. Furthermore, QSN-IOc dose-dependently decreased the Δψm in K562 cells, increased the release of cytochrome c and the level of Bax, and decreased the level of Bcl-2, suggesting that QSN-10c-induced apoptosis of K562 cells was mediated via the mitochondrial apoptotic pathway. In anti-angiogenic activity studies, QSN-IOc suppressed the viability of HUVECs and induced apoptosis in dose dependent manners. QSN-IOc treatment did not alter the Δψm in HUVECs, but dose-dependently inhibited the expression of VEGF, inhibited the tube formation and cell migration in vitro, and significantly suppressed the number of ISVs in zebrafish embryos in vivo. Furthermore, QSN-IOc dose-dependently suppressed the phosphorylation of AKT and GSK313 in both HUVECs and K562 cells. Conclusion： QSN-IOc is a novel antitumor compound that exerts both antitumor and anti-angiogenic effects via inhibiting the PI3K/ AKT/GSK313 signaling pathway.     

Thalidomide in cancer treatment: a potential role in the elderly?

There is increased interest in the treatment of cancer with thalidomide because of its antiangiogenic, immunomodulating and sedative effects. In animal models, the antitumour activity of thalidomide is dependent on the species, route of administration and coadministration of other drugs. For example, thalidomide has shown antitumour effects as a single agent in rabbits, but not in mice. In addition, the antitumour effects of the conventional cytotoxic drug cyclophosphamide and the tumour necrosis factor inducer 5,6-dimethylxanthenone-4-acetic acid (DMXAA) were found to be potentiated by thalidomide in mice bearing colon 38 adenocarcinoma tumours. Further studies have revealed that thalidomide upregulates intratumoral production of tumour necrosis factor-alpha 10-fold over that induced by DMXAA alone. Coadministration of thalidomide also significantly reduced the plasma clearance of DMXAA and cyclophosphamide. All these effects of thalidomide may contribute to the enhanced antitumour activity. Recent clinical trials of thalidomide have indicated that it has minimal anticancer activity for most patients with solid tumours when used as a single agent, although it was well tolerated. However, improved responses have been reported in patients with multiple myeloma. Palliative effects of thalidomide on cancer-related symptoms have also been observed, especially for geriatric patients with prostate cancer. Thalidomide also eliminates the dose-limiting gastrointestinal toxic effects of irinotecan. There is preliminary evidence indicating that the clearance of thalidomide may be reduced in the elderly. The exact role of thalidomide in the treatment of cancer and cancer cachexia in the elderly remains to be elucidated. However, it may have some value as part of a multimodality anticancer therapy, rather than as a single agent.