肿瘤抑素抗肿瘤相关肽治疗人喉鳞状细胞癌裸鼠模型的实验研究

目的:研究肿瘤抑素抗肿瘤相关肽对裸鼠喉癌皮下移植模型的抑瘤作用,探讨其抑瘤机制及人喉癌生物治疗新方法。方法:建立人喉癌Hep-Ⅱ细胞株裸鼠皮下接种模型,应用肿瘤抑素抗肿瘤相关肽治疗,观察肿瘤生长情况。对治疗后的肿瘤组织进行病理学检查、免疫组织化学检查及电镜观察,记数微血管密度。结果:在治疗组与对照组之间,平均鼠净重、瘤重、瘤体积及瘤重/鼠净重比,差异均有统计学意义(均P<0.01),治疗组抑瘤率为51.58%。病理学检查及电镜观察表明,治疗组肿瘤生长受到抑制,细胞分裂少见,可见肿瘤细胞的坏死凋亡,新生血管明显减少,其微血管密度明显低于对照组,差异有统计学意义(P<0.01)。结论:肿瘤抑素抗肿瘤相关肽可显著抑制人喉癌裸鼠模型肿瘤的生长和发展。     

人肿瘤抑素基因真核表达载体的构建及其活性研究

目的构建携带人肿瘤抑素(Tumstatin)基因的重组腺相关病毒载体,研究其对人脐静脉内皮细胞(HUVECs)增殖的影响。方法构建重组载体pAAV-Tum质粒,并建立一株稳定表达Tumstatin基因的HU-VECs细胞株。用逆转录聚合酶链反应检测该细胞系中Tumstatin基因的表达,四甲基唑蓝法检测该细胞增殖情况。结果构建的pAAV-Tum重组腺相关病毒载体能在HUVECs细胞中表达,其表达产物能抑制该细胞增殖(0.335±0.011)。结论成功构建了pAAV-Tum重组载体,构建的重组载体能有效表达,为其后的抗肿瘤血管生成治疗研究奠定了基础。     

肿瘤抑素相关肽抗肿瘤活性研究

[目的]评价肿瘤抑素相关肽19肽和21肽抗肿瘤活性.[方法]已构建好的19肽和21肽重组质粒在大肠杆菌BL-21(DE3)中表达,经几丁质亲和层析柱纯化出19肽和21肽.用SDS-PAGE聚丙烯酰胺电泳对表达及纯化结果进行初步鉴定,进行19肽、21肽和19肽 21肽的腹水型转移型小鼠H22肝癌实体瘤模型大体抑瘤实验,并做组织病理学切片分析.[结果]经一步亲和层析可获得可溶性19肽和21肽.19肽,21肽,19肽 21肽联合用药组在小鼠抑瘤实验中抑瘤率分别达48.46%,42.86%,53.94%.组织病理学切片结果可见3种给药方式都可促进小鼠肿瘤组织坏死,血管数量减少.联合用药组抗肿瘤作用强于单独用药组.[结论]肿瘤抑素相关肽19肽和21肽,具有抗肿瘤活性.     

Effects of cloned tumstatin-related and angiogenesis-inhibitory peptides on proliferation and apoptosis of endothelial cells

Background Tumstatin is a recently developed endogenous vascular endothelial growth inhibitor that can be applied as an anti-angiogenesis and antineoplastic agent. The study aimed to design and synthesize the small molecular angiogenesis inhibition-related peptide （peptide 21）, to replicate the structural and functional features of the active zone of angiogenesis inhibition using tumstatin and to prove that synthesized peptide 21 has a similar activity： specifically inhibiting tumor angiogenesis like tumstatin. Methods Peptide 21 was designed and synthesized using biological engineering technology. To determine its biological action, the human umbilical vein endothelial cell line ECV304, the human ovarian cancer cell line SKOV-3 and the mouse embryo-derived NIH3T3 fibroblasts were used in in vitro experiments to determine the effect of peptide 21 on proliferation of the three cell lines using the MTT test and growth curves. Transmission electron microscopy （TEM） and flow cytometry （FCM） were applied to analyze the peptide 21-induced apoptosis of the three cell lines qualitatively and quantitatively. In animal experiments, tumor models in nude mice subcutaneously grafted with SKOV-3 were used to observe the effects of peptide 21 on tumor weight, size and microvessel density （MVD）. To initially investigate the role of peptide 21, the effect of peptide 21 on the expression of vascular endothelial growth factors （VEGFs） by tumor tissue was semi-quantitatively analyzed. Results The in vitro Ml-r test and growth curves all indicated that cloned peptide 21 could specifically inhibit ECV304 proliferation in a dose-dependent manner （P 〈0.01）; TEM and FCM showed that peptide 21 could specifically induce ECV304 apoptosis （P 〈0.01）. Results of in vivo experiments showed that tumors in the peptide 21 group grew more slowly. The weight and size of the tumors after 21 days of treatment were smaller than those in the control group （P 〈0.05）, with a mean tumor inhibition rate of 67.86%; MVD     

Patent focus on cancer chemotherapeutics. II Angiogenesis agents: April 2000 - September 2000

Angiogenesis refers to the formation of capillary blood vessels from existing blood vessels: a process that is believed to be critical for tumour growth and metastasis. Angiogenesis inhibition represents a new approach to cancer chemotherapy with several agents and approaches now entering late clinical development. This review summarises the key aspects of recent patent applications referring to inhibitors of angiogenesis that have been published between April and September 2000. The review covers the main mechanism-based approaches such as MMPI, integrin antagonists, urokinase inhibitors and inhibitors of the growth factor signalling pathways of fibroblast growth factor (FGF), platelet derived growth factor (PDGF), vascular endothelial growth factor (VEGF) and Tie-2/Tek. Applications referring to endogenous inhibitors such as endostatin or angiostatin are also included, as are selected natural products that have data suggesting a link to angiogenesis-specific mechanisms of action.     

微小RNA-21和肿瘤抑素在膀胱癌中的表达及其相关性的研究

目的探讨膀胱癌中微小RNA-21和肿瘤抑素表达与临床病理特征之间的关系。方法采用SABC法对98例膀胱癌患者分别进行微小RNA-21和肿瘤抑素的免疫组化染色,其与肿瘤临床分期和细胞分级间的关系及微小RNA-21和肿瘤抑素在瘤细胞中的共表达情况。结果膀胱癌患者中微小RNA-21和肿瘤抑素的阳性表达率分别是69.4%和43.9%。与浅表性和分化较好的膀胱癌相比,侵袭性和分化差的癌细胞中微小RNA-21表达上调明显,而肿瘤抑素表达则显著下调,组间比较（P〈0.01）,差异有统计学意义。有68例患者存在微小RNA-21和肿瘤抑素共表达,两者之间呈负相关（P〈0.05）,差异也具有统计学意义。结论微小RNA-21和肿瘤抑素共表达失调可能促进膀胱癌的发生发展,抑制微小RNA-21表达,上调肿瘤抑素水平有希望成为有效治疗膀胱癌的一种新方法 。     

肿瘤抑素Tum5对人脐静脉血管内皮细胞血管生成活性以及碱烧伤诱导大鼠角膜新生血管的抑制作用

目的　研究肿瘤抑素Tum5对人脐静脉血管内皮细胞血管生成活性以及碱烧伤诱导的大鼠角膜新生血管的抑制作用。方法　取对数生长期人脐静脉血管内皮细胞分为4组:正常对照组:无任何处理的人脐静脉血管内皮细胞;rAd-GFP组:人脐静脉血管内皮细胞经rAd-GFP感染并培养;rAd-Tum5组:人脐静脉血管内皮细胞经rAd-Tum5感染并培养;rAd-Tum5+VEGF组:人脐静脉血管内皮细胞经rAd-Tum5感染后经外源性VEGF处理。通过CCK-8活性检测试剂盒、Transwell实验、Matrigel实验分别检测细胞增殖率、迁移细胞数及管腔形成情况。取雄性健康清洁级SD大鼠64只采用随机数字表法将大鼠分为:正常对照组、碱烧伤组、碱烧伤+rAd-GFP组、碱烧伤+rAd-Tum5组,每组16只,除正常对照组外其余各组建立角膜碱烧伤模型。其中正常对照组无任何处理,碱烧伤组、碱烧伤+rAd-GFP组、碱烧伤+rAd-Tum5组于烧伤后分别经结膜下注射等量无菌生理盐水、rAd-GFP、rAd-Tum5病毒,记录碱烧伤后第1天、第7天、第14天角膜新生血管相对面积及浸润炎性细胞数。结果　CCK-8实验结果显示rAd-Tum5组较正常对照组、rAd-GFP组细胞增殖率降低（均为P<0.01）;而rAd-Tum5+VEGF组细胞增殖率较rAd-Tum5组显著升高（P=0.004）,rAd-Tum5+VEGF组与正常对照组和rAd-GFP组相比差异均无统计学意义（均为P>0.05）。Transwell实验结果显示rAd-Tum5组相对于正常对照组、rAd-GFP组每个视野细胞迁移数显著降低（均为P<0.01）;而rAd-Tum5+VEGF组相对于rAd-Tum5组显著升高（P=0.000）,但其迁移能力尚未恢复到正常水平,与正常对照组和rAd-GFP组相比差异均有统计学意义（均为P<0.05）。Matrigel实验结果显示rAd-Tum5组相对于正常对照组、rAd-GFP组每个视野细胞成管数显著降低（均为P<0.01）;而rAd-Tum5+VEGF组相对于rAd-Tum5组显著升高（P=0.001）。大鼠角膜碱烧伤后第1-14天,各组角膜新生血管密度及数量逐渐增高,但是碱烧伤+rAd-Tum5组第7天、第14天角膜新生血管相对面积均显著低于碱烧伤组和碱烧伤+rAd-GFP组,提示Tum5能降低角膜新生血管生长速率并减少新生血管密度,抑制大鼠碱烧伤诱导的新生血管的形成。碱烧伤后第7天、第14天,正常对照组角膜完整、细胞排列有序,无炎性细胞浸润;碱烧伤后第7天,碱烧伤组、碱烧伤+rAd-GFP组上皮结构紊乱,角膜水肿,炎性细胞浸润明显,而碱烧伤+rAd-Tum5组每个视野炎性细胞浸润数较碱烧伤组、碱烧伤+rAd-GFP组明显减少,差异均有统计学意义（均为P<0.01）。碱烧伤后第14天,碱烧伤组、碱烧伤+rAd-GFP组和碱烧伤+rAd-Tum5组每个视野炎性细胞浸润数较第7天显著下降,同时碱烧伤+rAd-Tum5组角膜上皮结构整齐,水肿消退,每个视野炎性细胞浸润数显著低于碱烧伤组以及碱烧伤+rAd-GFP组,差异均有统计学意义（均为P<0.01）。结论　Tum5可能经VEGF通路抑制人脐静脉血管内皮细胞血管生成活性;Tum5可显著抑制碱烧伤诱导的角膜新生血管生成及炎性细胞浸润。     

Identification of amino acids essential for the antiangiogenic activity of tumstatin and its use in combination antitumor activity

Tumstatin is an angiogenesis inhibitor that binds to αvβ3 integrin and suppresses tumor growth. Previous deletion mutagenesis studies identified a 25-aa fragment of tumstatin (tumstatin peptide) with in vitro antiangiogenic activity. Here, we demonstrate that systemic administration of this tumstatin peptide inhibits tumor growth and angiogenesis. Site-directed mutagenesis identified amino acids L, V, and D as essential for the antiangiogenic activity of tumstatin. The tumstatin peptide binds to αvβ3 integrin on proliferating endothelial cells and localizes to select tumor endothelium in vivo. Using 3D molecular modeling, we identify a putative interaction interface for tumstatin peptide on αvβ3 integrin. The antitumor activity of the tumstatin peptide, in combination with bevacizumab (anti-VEGF antibody), displays significant improvement in efficacy against human renal cell carcinoma xenografts when compared with the single-agent use. Collectively, our results demonstrate that tumstatin peptide binds specifically to the tumor endothelium, and its antiangiogenic action is mediated by αvβ3 integrin, and, in combination with an anti-VEGF antibody it exhibits enhanced tumor suppression of renal cell carcinoma.     

Implication of tumstatin in tumor progression of human bronchopulmonary carcinomas

The NC1 domain of alpha3 chain of type IV collagen, namely tumstatin, has been shown to display specific anti-angiogenic properties by inhibiting endothelial cells' proliferation and inducing their apoptosis via an interaction with alphavbeta3 integrin. Until now, the tumstatin anti-angiogenic effect has only been shown by in vitro studies or mouse xenograft experiments. In the present study, we examined the expression of tumstatin in relationship with tumor vascularization in 34 bronchopulmonary human carcinomas. We observed a clear association between tumstatin expression and tumor vascularization. Indeed, a strong expression of tumstatin in the tumor environment correlated with a mildly developed vascular network. In contrast, tumstatin was absent or poorly detected in highly vascularized tumors. Moreover, alphavbeta3 integrin and tumstatin colocalized in capillary endothelial cells, suggesting a potential interaction between these 2 molecules. Thus, our results plead in favor of an in vivo anti-angiogenic effect of tumstatin. This factor, largely expressed in well-differentiated lung carcinomas, could indeed reduce tumor vascularization and thereby limit tumor progression.     

The YSNSG cyclopeptide derived from tumstatin inhibits tumor angiogenesis by down‐regulating endothelial cell migration

We previously demonstrated that the CNYYSNS peptide derived from tumstatin inhibited in vivo tumor progression. The YSNS motif formed a β‐turn crucial for biological activity. More recently, a YSNSG cyclopeptide with a constrained β‐turn on the YSNS residues was designed. Intraperitoneal administration of the YSNSG cyclopeptide inhibited in vivo melanoma progression more efficiently than the native linear peptide. In the present article, we showed that the YSNSG cyclopeptide also triggered an inhibition of in vivo tumor neovascularization and we further analyzed its in vitroantiangiogenic effect. The YSNSG cyclopeptide did not alter endothelial cell proliferation but inhibited cell migration by 83% in an in vitro wound healing model. The inhibition was mediated by a decrease in active MT1‐MMP at the migration front as well as a decrease in u‐PA and u‐PAR expression. The cyclopeptide also altered β1‐integrin distribution in endothelial cell lamellipodia, induced a strong decrease in the phosphorylated focal adhesion kinase (p125^FAK), disorganized F‐actin stress fibers and decreased the number of lamellipodia, resulting in a non migratory phenotype. Our results confirm the YSNSG cyclopeptide as a potent antitumor agent, through both the inhibition of invasive properties of tumor cells and the antiangiogenic activity.