局部应用内皮抑素对小鼠Lewis肺癌抑瘤作用的研究

目的我们通过局部应用内皮抑素治疗小鼠Lewis肺癌,观察内皮抑素对小鼠Lewis肺癌的抑瘤作用,及内皮抑素对VEGF表达的影响.方法用小鼠Lewis肺癌细胞种植于15只C57小鼠体内,建立小鼠Lewis肺癌移植瘤模型,当移植瘤体积生长至400～600mm3时分组并开始给药.小鼠随机分为三组,A组为空白对照组,B组每日尾静脉注射内皮抑素20μg,C组每日于肿瘤部位局部注射内皮抑素20μg,共11天.测定抑瘤率及微血管密度评价疗效,免疫组化(S-P)法测定VEGF在各组的表达水平.结果试验组较对照组抑瘤率增高(P＜0.05),微血管密度降低(P＜0.05),试验组VEGF水平较对照组低(P＜0.01).结论内皮抑素对小鼠Lewis肺癌移植瘤的生长有明显抑制作用,局部用药较全身用药的作用增强,内皮抑素可以通过降低VEGF在肿瘤组织中的表达抑制肿瘤生长.     

血管抑素基因和内皮抑素基因抑制肝癌作用的比较

日的:比较血管抑素(angiostatin)和内皮抑素(endostain)基因抑制肝癌作用的差异.方法:建立大鼠肝癌模型,随机分组,在肝癌局部分别注入血管抑素基因、内皮抑素基因、血管抑素 内皮抑素基因及0.9%氯化钠溶液,观察各组肿瘤微血管密度(microvessel density,MVD)、肿瘤凋亡率、肺转移瘤的数量、大鼠生存期及肿瘤生长率等指标.结果:所有治疗组的肿瘤体积生长率均受到抑制,以双基因联合的作用最为明显.各治疗组肺内转移灶数、MVD和凋亡指数与对照组相比,差异均有统计学意义(P<0.01,P<0.05和P<0.05);双基因联合应用组与其他治疗组相比,差异具有统计学意义(P<0.05);而单一基因治疗组之间比较差异无统计学意义(P>0.05).双基因联合应用后大鼠存活时间最长,而对照组生存时间最短.结论:血管抑素、内皮抑素均通过抑制肿瘤新生血管的生长、迁移而达到抑制肿瘤生长和转移的目的,2种抑素基因的协同作用更加明显.     

血管内皮抑素对小鼠体内Lewis肺癌血管生成及转移影响的研究

目的：观察血管内皮抑素（Endostatin）对C57小鼠体内Lewis肺癌生长、血管生成及转移的影响。方法：将荷Lewis肺癌的C57鼠进行不同剂量组的内皮抑素和顺铂干预，观察肿瘤生长、移植瘤及转移瘤体内的血管内皮生长因子（VEGF）和微血管密度（MVD）的表达及转移发生率，并作统计学分析。结果：内皮抑素对鼠Lewis肺癌的生长有明显的抑制作用。内皮抑素处理组（内含400μg、300μg、200μg和200μg＋DDP组）和模型组转移率间的差异有统计学意义（P=0．0303）。内皮抑素能显著下调移植瘤及转移瘤内的VEGF。移植瘤中模型组与其余各组之间均存在显著差异（P〈0．01）；400μg组与200μg组、DDP组、联合用药组之间均存在统计学差异（P〈0．05）；300μg组与200组之间存在统计学差异（P〈0．05）；转移瘤中模型组与其余各组之间均存在统计学差异（P〈0．05）。300μg与200μg之间有统计学差异（P〈0．05），200μg与DDP组和200μg＋DDP组之间有统计学差异（P〈0．05）。原位移植瘤与肺转移肿瘤组织中VEGF表达呈正相关（r=0．977，P=0．001）。内皮抑素能显著下调移植瘤及转移瘤内和MVD。在移植瘤中400μg和300μg组肿瘤微血管密度最小，彼此无差异；200μg加DDP组肿瘤微血管密度次之，200μg组肿瘤微血管密度再次，DDP组肿瘤微血管密度在各实验组中最多，模型组肿瘤微血管密度最大。在转移瘤中400μg、300μg和200μg组微血管密度最小，这三组彼此无差异。200μg加DDP组肺转移瘤组织微血管密度次之，DDP组肺转移瘤组织微血管密度在各实验组中最多；模型组肿瘤微血管密度最大。内皮抑素可以减少肿瘤肺转移，作用程度在一定范围内与内皮抑素剂量呈正相关。结论：血管内皮抑素可以通过下调瘤组织中的VEGF和MVD抑制肿瘤生长及转移。     

腺病毒介导的内皮抑素基因治疗小鼠肺癌

目的 探讨内皮抑素对小鼠肺癌生长和转移的抑制作用及其对肿瘤内部新生血管的影响.方法 在C57BL/6小鼠背部皮下注射2×106 lewis肺癌(LLC)细胞,建立小鼠肺癌种植瘤模型,2周后,瘤内注射2×109 pfu内皮抑素腺病毒载体,观察内皮抑素对肿瘤生长、转移及生存率的影响,检测内皮抑素在肿瘤组织的原位表达和血液循环中的表达水平及持续时间.用免疫组化方法,检测肿瘤内部血管密度,观察治疗对肿瘤血管的影响.用透射电镜观察肿瘤细胞的凋亡情况.结果 免疫组化检测结果显示,内皮抑素蛋白在内皮抑素组的肿瘤组织中呈强阳性表达,而在空载体对照组和阴性对照组中呈阴性表达或很少量表达.用酶联免疫吸附实验(ELISA)法检测内皮抑素组血清内皮抑素浓度,第2周可达1540±560 ng/ml;1个月后,血清内皮抑素浓度降至对照水平.内皮抑素组的肿瘤体积和生存率,与空载体对照和阴性对照组比较,差异有统计学意义(P＜0.05).抗CD31抗体标记的肿瘤内血管密度(MVD)在内皮抑素组、空载体对照组和阴性对照组中,分别为37.5±4.6、65.2±5.8和68.5±4.5个/200倍视野,抗CD105抗体标记的肿瘤内MVD分别为10.5±3.2、39.7±5.6和42.4±4.8个/200倍视野,内皮抑素组与空载体对照组和阴性对照组比较,差异有统计学意义(P＜0.05).内皮抑素组的组织在电镜下呈凋亡相的肿瘤细胞多见.结论 腺病毒介导的内皮抑素基因可在体内高效、较长时间表达内皮抑素蛋白,对小鼠皮下种植瘤有一定的治疗作用,其作用的靶点是抑制新生血管的生成.     

重组腺病毒介导小鼠内皮抑素对荷骨肉瘤MG-63细胞裸鼠肺转移的抑制

目的: 构建携小鼠内皮抑素(endostatin， ES)基因的重组腺病毒载体，观察其对荷骨肉瘤裸鼠肺转移的抑制，探讨内皮抑素表达水平与骨肉瘤肺转移的关系。方法:构建pDC315mEndo表达质粒，同源重组产生重组腺病毒AdmEndo。裸鼠右前肢皮下注射骨肉瘤MG63细胞建立移植瘤裸鼠模型；随机分为4组：小鼠内皮抑素腺病毒（AdmEndo）组，携带EGFP基因腺病毒（AdEGFP）组， PBS组，未接种肿瘤细胞裸鼠空白对照组。各组裸鼠每周分别注射相应药物200 μl，连续5次，观察各组动物移植瘤体积、瘤组织病理，ELISA法检测各组裸鼠血ES水平；7周后处死动物，观察有无肺转移及肺转移灶病理。结果：AdEGFP组肿瘤体积为（1.53±0.05） cm3,PBS组为（1.56±0.07） cm3, AdmEndo组为（0.91±0 .03） cm3，AdmEndo治疗的抑瘤率达40.7％。AdmEndo组裸鼠血内皮抑素表达水平明显高于AdEGFP组和PBS组（P<0.05）。AdmEndo组裸鼠肺部未发现肿瘤转移灶，其他两组肺部见大量散在转移灶，肺转移率分别为80%和90%。未发生肺转移裸鼠的ES水平显著高于发生肺转移的裸鼠（P<0.05）。结论：腺病毒介导的小鼠内皮抑素显著抑制了荷骨肉瘤裸鼠肺转移，内皮抑素表达水平与肺转移有着直接的关系。     

内皮抑素基因治疗对小鼠肿瘤组织内新生血管形成抑制作用的实验研究

目的:研究腺病毒介导的内皮抑素基因治疗对小鼠肺肿瘤组织内部血管的抑制作用。方法:皮下注射2×106LLC细胞至C57BL/6小鼠背部建立小鼠肺癌种植瘤模型,2周后瘤内注射2×109pfu内皮抑素腺病毒载体治疗。免疫组化检测内皮抑素在肿瘤组织的原位表达,观察治疗对肿瘤生长的影响。免疫组化标记计数肿瘤内部血管密度,观察治疗对肿瘤血管的影响。结果:Admendostatin治疗从第2周起就可明显减缓肿瘤生长速度,P<0.05。免疫组化显示治疗组肿瘤组织内皮抑素蛋白强阳性表达,对照组阴性或痕量表达。Admendostatin治疗组、空载体对照组和阴性对照组肿瘤血管密度用CD31和CD105抗原标记分别为37.5±4.6、65.2±5.8、68.5±4.5和10.5±3.2、39.7±5.6、42.4±4.8。治疗组与两对照组相比差异有统计学意义,P<0.05;空载体对照与阴性对照相比差异无统计学意义,P>0.05。结论:腺病毒介导的内皮抑素基因治疗可抑制小鼠皮下种植瘤的生长,其作用机制是抑制肿瘤内部新生血管的生成,应用CD105抗原标记可准确反映新生血管的变化。     

重组腺相关病毒介导人内皮抑素抑瘤作用的实验研究

背景与目的：研究表明,内皮抑素能抑制肿瘤血管的生成,进而抑制肿瘤的生长和转移,然而有关内皮抑素治疗的研究报道却很少。本研究初步探讨了重组腺相关病毒介导内皮抑素对肿瘤生长和转移的作用。方法：用RT-PCR方法获得人内皮抑素基因,构建内皮抑素可分泌表达的通用型重组腺相关病毒（rAAV）载体,包装获得重组腺相关病毒rAAV-SS-Endostain。以动物实验分析其抗肿瘤作用。结果：C57BL/6小鼠肌肉注射10^11TUrAAC-SS-Endostatin一次,该重组病毒对小鼠黑色瘤B16F10移植瘤生长的抑制率为57.1％,在实验肺转移模型中,对转移灶的抑制率为70.7％。结论：重组腺相关病毒介导的内皮抑素能有效抑制肿瘤的生长和转移。     

人血管能抑素抑制小鼠Lewis肺癌移植瘤生长、转移及血管新生

目的:探讨人血管能抑素(canstatin)对小鼠Lewis肺癌移植瘤生长、转移和血管新生的影响.方法:将pCMV-Script/canstatin及空载体pCMV-Script通过电穿孔的方法转染A549细胞,G418筛选获得阳性克隆.RT-PCR检测转染后细胞中canstatin mRNA的表达,Western blotting检测转染后细胞中canstatin蛋白的表达.建立Lewis肺癌小鼠移植瘤模型,观察pCMV-Script/canstatin组A549细胞培养上清对小鼠Lewis肺癌移植瘤的治疗作用,免疫组化检测各治疗组荷瘤小鼠移植瘤的微血管密度.结果:pCMV-Script/canstatin转染A549细胞在G418筛选后成功形成克隆,转染的A549细胞能有效表达canstatin mRNA和蛋白.pCMV - Script/canstatin治疗组小鼠肿瘤体积明显小于pCMV-Script组和NS组[(1.47 ±0.21)cm3 vs(2.43 ±0.15) cm3、(2.53 ±0.18) cm3,P ＜0.01);pCMV-Script/canstatin组、pCMV - Script组和NS组的肺转移结节数分别为(3.00±1.00)、(7.80±1.48)、(7.60 ±2.41)个,pCMV-Script/canstatin组肿瘤转移受到显著的抑制(P＜0.01);pCMV-Script/canstatin组小鼠的肿瘤组织微血管数明显少于pCMV-Script组和NS组[(84.40 ±8.83) vs (188.68 ±11.15)、(190.24±12.91)个,P＜0.01].结论:pCMV-Script/canstatin能在A549细胞中表达并分泌至细胞外,canstatin可明显抑制Lewis肺癌移植瘤的生长、转移和血管新生.     

内皮抑素对小细胞肺癌移植瘤微血管生成的影响

[目的]探讨内皮抑素对小细胞肺癌裸鼠移植瘤微血管生成的影响。[方法]将人小细胞肺癌细胞株NCI—H446接种于裸鼠背部皮下．待移植肿瘤成形后．随机分为对照组、试验1组和试验2组,每日于肿瘤部位分别注射生理盐水200μl、内皮抑素20μg、内皮抑素40μg,观察三组小鼠移植瘤的生长情况．并检测各组肿瘤的微血管密度。[结果]不同剂量的内皮抑素对肿瘤生长的影响均呈现明显的时间-效应和剂量-效应关系：随着内皮抑素剂量升高．移植瘤中的微血管密度下降。[结论]内皮抑素通过阻断微血管生成而抑制小细胞肺癌移植肿瘤的生长．     

大剂量重组人血管内皮抑素对肺癌转移瘤的实验研究*

目的　探讨大剂量重组人血管内皮抑素（恩度）单药对肺癌的作用。方法　建立肺癌裸鼠转移瘤模型,检测腹腔给药和皮下注射两种途径恩度（５０ｍｇ／ｋｇ）在实验ＳＤ大鼠中的血药浓度,同时分别设生理盐水对照组和恩度１０、２０、５０、１００、２００ｍｇ／（ｋｇ·ｄ）组,观察小鼠的生存情况,计算生存期以及肺部转移灶数量。结果　恩度腹腔给药和皮下注射两种途径药代动力学相似,半衰期约为２４ｈ。１００ｍｇ／（ｋｇ·ｄ）恩度组小鼠的中位生存期达６１天,较对照组延长１０天。小鼠肺部转移病灶数量与恩度剂量有一定相关性,１００ｍｇ／（ｋｇ·ｄ）恩度组转移灶数量最少,与对照组比较有统计学上显著差异（Ｐ＜０.０５）。结论　在肺癌转移瘤模型中,大剂量恩度能够有效地抑制肺癌的生长,生存期延长,生存质量较好。     

Liposomes complexed to plasmids encoding angiostatin and endostatin inhibit breast cancer in nude mice.

Gene therapy transfer of angiostatin and endostatin represents an alternative method of delivering angiogenic polypeptide inhibitors. We examined whether liposomes complexed to plasmids encoding angiostatin or endostatin inhibited angiogenesis and the growth of MDA-MB-435 tumors implanted in the mammary fat pads of nude mice. We determined that plasmids expressing angiostatin (PCI-Angio) or endostatin (PCI-Endo) effectively reduced angiogenesis using an in vivo Matrigel assay. We then investigated the efficacy of these plasmids in reducing the size of tumors implanted in the mammary fat pad of nude mice. Both PCI-Angio and PCI-Endo significantly reduced tumor size when injected intratumorally (P < 0.05). Compared to the untreated control group, the mice treated with PCI-Angio and PCI-Endo exhibited a reduction in tumor size of 36% and 49%, respectively. In addition, we found that i.v. injections of liposomes complexed to PCI-Endo reduced tumor growth in the nude mice by nearly 40% when compared to either empty vector (PCI) or untreated controls (P < 0.05). These findings provide a basis for the further development of nonviral delivery of antiangiogenic genes.     

Inhibition of B16BL6 tumor progression by coadministration of recombinant angiostatin K1-3 and endostatin genes with cationic liposomes

Transfection of the antiangiogenic angiostatin and endostatin genes was shown to be an alternative to high-dose administration of angiostatin or endostatin proteins for cancer therapy. We have systematically investigated whether coadministration of the mouse angiostatin kringle 1-3 gene (pFLAG-AngioK1/3) and the endostatin gene (pFLAG-Endo) complexed with cationic liposomes exhibits enhanced therapeutic efficacy. In vitro, the coexpressed mixture of angiostatin K1-3 and endostatin more effectively reduced angiogenesis in chorioallantoic membranes than either angiostatin K1-3 or endostatin alone. In vivo, subcutaneous co-administration of pFLAG-AngioK1/3 and pFLAG-Endo lipoplexes more effectively inhibited vascularization in Matrigel plugs implanted in mice than either one alone. Additionally, subcutaneous administration of these genes inhibited the growth and formation of pulmonary metastases of B16BL6 melanoma cells in mice. Compared to treatment with an empty vector, treatment with pFLAG-AngioK1/3 plus pFLAG-Endo inhibited 81% of tumor growth, while treatment with pFLAG-AngioK1/3 or pFLAG-Endo inhibited tumor growth 70 and 69%, respectively. Cotreatment with the two plasmids after primary tumor excision induced a 90% inhibition of pulmonary metastases versus 79% for pFLAG-AngioK1/3 or 80% for pFLAG-Endo individually. These results suggest that combined administration of angiostatin K1-3 and endostatin genes complexed with cationic liposomes may be an innovated antiangiogenic strategy for cancer therapy.     

Inhibition of angiogenesis and tumor progression by hydrodynamic cotransfection of angiostatin K1-3, endostatin, and saxatilin genes

In vivo expression of angiostatin and endostatin, two different types of endothelial cell growth inhibitor, have been reported to inhibit vascularization in tumor tissues, resulting in tumor growth inhibition. Recently, in vivo expression of saxatilin, a novel disintegrin purified from snake (Gloydius saxatilis) venom, was able to strongly inhibit endothelial cell proliferation and smooth muscle cell migration, resulting in tumor growth inhibition. However, the antitumor efficacy of the individual antiangiogenic molecules expressed in vivo was not sufficiently potent to induce tumor regression in animal models. Therefore, in this study, we have systemically examined how combinational transfer of angiostatin, endostatin, and saxatilin genes affects neovascularization in tumor tissues and tumor progression in a mouse model. In Matrigel-implanted mice, cotransfection with plasmids encoding angiostatin K1-3 (pFLAG-Angio K1/3), endostatin (pFLAG-Endo), and saxatilin (pFLAG-Sax) resulted in the most effective inhibition of angiogenesis. In addition, hydrodynamic cotransfection of the three genes induced more inhibition of B16BL6 melanoma growth and pulmonary metastasis than other combinations of transfected genes. Compared with the empty vector-treated control group, cotreatment with the three plasmids reduced B16BL6 tumor growth by 89% and pulmonary metastasis by 90%. These results provide additional evidence supporting the combined systemic expression of antiangiogenic factors, such as angiostatin K1-3, endostatin, and saxatilin, as an alternative procedure for antiangiogenic cancer therapy.     

Intramuscular delivery of antiangiogenic genes suppresses secondary metastases after removal of primary tumors

The success of surgery to remove primary tumors can be compromised by the subsequent outgrowth of metastases. It is recognized that primary tumors secrete antiangiogenic factors that suppress the outgrowth of their daughter metastases. In accord we show here that surgical removal of primary EL-4 lymphomas led to a marked decrease in the levels of circulating angiostatin and endostatin, and promoted the growth of distant nodular tumors. Expression vectors encoding angiostatin and endostatin, formulated with poly-N-vinyl pyrrolidone (PVP), were injected into the tibialis and gastrocnemia muscles, leading to expression of angiostatin and endostatin in muscle fibers. High levels of biologically active exogenous proteins were secreted into the circulation. Intramuscular gene therapy with angiostatin and endostatin plasmids significantly inhibited tumor vascularity and induced tumor cell apoptosis, and thereby suppressed the growth of secondary subcutaneous and disseminated metastatic tumors in the lung and liver. Simultaneous intramuscular delivery of both angiostatin and endostatin plasmids significantly prolonged the survival of mice after removal of primary tumors. These results suggest that intramuscular gene transfer of angiostatin and endostatin might serve as a prophylactic cancer-prevention strategy to combat the recurrence of cancer after surgical resection of primary tumors.     

Antitumor effects of angiostatin K1-3 and endostatin genes coadministered by the hydrodynamics-based transfection method

Angiostatin and endostatin are potent endothelial cell growth inhibitors and have been carefully evaluated for antiangiogenic cancer therapy. Previously, we have shown that subcutaneous administration of angiostatin K1-3 and endostatin genes complexed with liposomal vectors is a more practical treatment procedure than administration of angiostatin and endostatin proteins. This study provides additional conclusive evidence supporting the effectiveness of antiangiogenic cancer gene therapy employing angiostatin K1-3 and endostatin genes. Plasmids encoding a mouse angiostatin K1-3 gene (pFLAG-AngioK1/3) and an endostatin gene (pFLAG-Endo) were introduced by the hydrodynamic transduction method into mice carrying Matrigel plugs or B16BL6 mouse melanoma tumors. A single systemic injection of the two genes exhibited potent antiangiogenic and antitumor activity in the mouse model. Hydrodynamic coadministration of the genes inhibited the B16BL6 mouse melanoma growth and pulmonary metastasis more effectively than administration of either gene alone. Compared with the untreated control group, the mice cotreated with pFLAG-AngioK1/3 and pFLAG-Endo exhibited 75% reduction of tumor growth while those treated with pFLAG-AngioK1/3 or pFLAG-Endo showed 46% and 52% reduction, respectively. The cotreatment inhibited B16BL6 pulmonary metastasis formation by 80% while the inhibition induced by individual treatment with pFLAG-AngioK1/3 or pFLAG-Endo was 68% and 71%, respectively. These results provide additional evidence that systemic expression of angiostatin K1-3 and/or endostatin genes is a viable alternative procedure for antiangiogenic cancer therapy.     

Recombinant adeno-associated virus 2-mediated antiangiogenic prevention in a mouse model of intraperitoneal ovarian cancer.

PURPOSE: In the present study, we sought to determine the potential of sustained transgene expression by a single i.m. administration of recombinant adeno-associated virus 2 (rAAV) encoding angiostatin and endostatin in inhibiting i.p. ovarian cancer growth and dissemination in a preclinical mouse model. EXPERIMENTAL DESIGN: Cohorts of female athymic nude mice received either no virus or 1.2 x 10(11) particles of rAAV encoding green fluorescence protein or endostatin plus angiostatin, i.m. Three weeks later, the mice were i.p. injected with 10(6) human epithelial ovarian cancer cell line SKOV3.ip1. As a measure of effectiveness of the therapy, tumor weight, abdominal distension, ascites volume and vascular endothelial growth factor level, and tumor weight were determined. Immunohistochemistry was done to determine tumor cell apoptosis and endothelial cell proliferation following the therapy. Tumor-free survival was recorded as the end point. RESULTS: Results indicated a significant tumor-free survival (P < 0.003) following therapy with rAAV encoding endostatin and angiostatin compared with untreated or rAAV-green fluorescence protein-treated mice. Ascites volume in rAAV endostatin and angiostatin-treated mice was significantly lower than naive mice and contained less hemorrhage and tumor conglomerates. The level of vascular endothelial growth factor in the ascites of antiangiogenic vector treated mice was also significantly less compared with the untreated mice. Immunohistochemical analyses indicated increased tumor cell apoptosis and decreased blood vasculature following rAAV endostatin and angiostatin treatment. CONCLUSION: The results indicate that antiangiogenic genetic prevention from stable systemic levels of angiostatin and endostatin by i.m. administration of rAAV can be used for the treatment of i.p. ovarian cancer growth and dissemination.     

内皮抑素对小鼠肺腺癌LA795生长和转移抑制作用的初步研究

目的：观察人内皮抑互对小鼠肺腺癌ＬＡ７９５生长和转移的抑制作用。方法：对重组人内皮抑素高效表达克隆ｐＣＸ的表达产物进行纯化,得到重组人内皮抑素（ｒｈＥＳ）。用亲和层析及胰弹性蛋白酶消化法从过期人血浆纯化得到人血管抑素（ｈＡＳ）。将接种ＬＡ７９５肺腺癌细胞的Ｔ７３９小鼠随机分成３组,分别给予ｒｈＥＳ,ｈＡＳ或等体积ＰＢＳ皮下注射,１次／日,共１４ｄ。观察３组肿瘤生长情况、肺湿重、肺表面转移结节数、动物生存期,分别进行ｑ检验。结果：ｒｈＥＳ组及ｈＡＳ组肿瘤生长缓慢,８ｄ后肿瘤逐渐回缩;肺湿重、肺表面转移结节数明显减少,动物生存期明显延长。结论：ｒｈＥＳ与ｈＡＳ均可明显抑制ＬＡ７９５所致的小鼠实验性肿瘤的生长与转移,延长动物的生存期。     

Synergy between angiostatin and endostatin: inhibition of ovarian cancer growth

Ovarian cancer is the leading cause of fatality among gynecological malignancies. Ovarian cancer growth is angiogenesis-dependent, and an increased production of angiogenic growth factors such as vascular endothelial growth factor is prognostically significant even during early stages of the disease. Therefore, we investigated whether antiangiogenic treatment can be used to inhibit the growth of ovarian cancer in an experimental model system. Mouse angiostatin (kringle 1-4) and endostatin were expressed in yeast. Purified angiostatin and endostatin were then used to treat established ovarian cancers in athymic mice. These studies showed that both angiostatin and endostatin inhibited tumor growth. However, angiostatin treatment was more effective in inhibiting ovarian cancer growth when compared with endostatin in parallel experiments. Residual tumors obtained from angiostatin- and endostatin-treated animals showed decreased number of blood vessels and, as a consequence, increased apoptosis of tumor cells. Subsequently, the efficacy of a combined treatment with angiostatin and endostatin was investigated. In the presence of both angiostatic proteins, endothelial cell proliferation was synergistically inhibited. Similarly, a combination regimen using equal amounts of angiostatin and endostatin showed more than additive effect in tumor growth inhibition when compared with treatment with individual angiostatic protein. These studies demonstrate synergism between two angiostatic molecules and that antiangiogenic therapy can be used to inhibit ovarian cancer growth.     

IL15 combined with Caspy2 provides enhanced therapeutic efficiency against murine malignant neoplasm growth and metastasis

Interleukin-15 (IL15) is a potential immunotherapeutic treatment for cancer. Caspy2 is an active zebra caspase for inducing apoptosis and immune response in murine tumors. In this study, we aim to evaluate the potential of gene therapy using IL15 and Caspy2 against the murine tumors. Plasmid expressing both Caspy2 and IL15 genes was constructed, encapsulated in DOTAP/cholesterol cationic liposome and injected intratumorally into the mice bearing CT26, B16-F10 and 4T1 carcinoma. We found that coexpression of IL15 and Caspy2 could significant inhibit tumor growth and prolong survival of the mice bearing CT26 or B16F10 tumor. A significant reduction in spontaneous lung metastasis was observed in the 4T1 tumor model. In CT26 model, the mice treated with IL15 and Caspy2 acquired a long-time protective immunity against the parental tumor cell rechallenge. Cytotoxic T lymphocytes and terminal deoxynucleotidyltransferase-mediated nick end labelling assays showed that the combination of capsy2 and IL15 could enhance both the apoptosis and immune response induction, which may account for its extraordinary antitumor effect. Furthermore, we showed that the observed tumor suppression by IL15 and Caspy2 concurred with the Caspy2-mediated downregulation of IL10 and upregulation of interferon-γ and tumor necrosis factor-α. Our results therefore suggested that the combination regimen might be a novel and effective strategy for cancer treatment.     

Effects of sustained antiangiogenic therapy in multistage prostate cancer in TRAMP model

Antiangiogenic therapy is a promising alternative for prostate cancer growth and metastasis and holds great promise as an adjuvant therapy. The present study evaluated the potential of stable expression of angiostatin and endostatin before the onset of neoplasia and during the early and late stages of prostate cancer progression in transgenic adenocarcinoma of mouse prostate (TRAMP) mice. Groups of 5-, 10-, and 18-week-old male TRAMP mice received recombinant adeno-associated virus-6 encoding mouse endostatin plus angiostatin (E+A) by i.m. injection. The effects of therapy were determined by sacrificing groups of treated mice at defined stages of tumor progression and following cohorts of similarly treated mice for long-term survival. Results indicated remarkable survival after recombinant adeno-associated virus-(E+A) therapy only when the treatment was given at an earlier time, before the onset of high-grade neoplasia, compared with treatment given for invasive cancer. Interestingly, early-stage antiangiogenic therapy arrested the progression of moderately differentiated carcinoma to poorly differentiated state and distant metastasis. Immunohistochemical analysis of the prostate from treated mice indicated significantly lower endothelial cell proliferation and increased tumor cell apoptosis. Vascular endothelial growth factor receptor (VEGFR)-2 expression was significantly down-regulated in tumor endothelium after treatment but not VEGFR-1. Analysis of the neuroendocrine marker synaptophysin expression indicated that antiangiogenic therapy given at an early-stage disease reduced neuroendocrine transition of the epithelial tumors. These studies indicate that stable endostatin and angiostatin gene therapy may be more effective for minimally invasive tumors rather than advanced-stage disease.