Endometrial hyperplasia and endometrial carcinoma induce a vasoproliferative response in the chick embryo chorioallantoic membrane

Ten specimens of endometrial adenocarcinoma, of endometrial hyperplasia and uterine prolapse (the latter used as controls), respectively, were grafted onto the chick embryo chorioallantoic membrane (CAM) to investigate their angiogenic activity. The vasoproliferative response was assessed four days after grafting on histologic sections by a planimetric point-count method. Microvessel counts in the CAM area under and around the implants were significantly higher in endometrial adenocarcinoma than in endometrial hyperplasia and in the latter over the controls. These findings show that endometrial hyperplasia and endometrial adenocarcinoma are angiogenic. These results confirm previous observation that angiogenic activity is both a marker of the passage between preneoplastic to neoplastic status and an event that influences tumour progression.     

鸡胚绒毛尿囊膜模型在肿瘤研究领域的进展

鸡胚绒毛尿囊膜(chick embryo chorioallantoie membrane,CAM)是近几年兴起的一种新型的肿瘤模型,与动物肿瘤模型相比具有简便价廉、便于动态观察、敏感性强等优点。利用CAM肿瘤模型可以筛选抗肿瘤药物,对其作用机制如抗血管生成、肿瘤侵袭、转移等方面研究作出一定贡献。但此肿瘤模型的应用目前仍存在一定的不足之处,正确应用该模型将为肿瘤体外实验研究提供有力的说服力。     

A novel 3-d model of chick chorioallantoic membrane for ameliorated studies in angiogenesis

Decisive indulgence of angiogenesis requires a more holistic assessment and several in vivo assays have been developed that permit a more realistic appraisal of the angiogenic response. One of the most popular assays to study angiogenic activity is the chick chorioallantoic membrane (CAM). Although CAM assay is a vital technique used to study normal and putative angiogenesis, a serious drawback in its utilization is the lack of quantitative assessment of vascularization. In this study, we proposed a new 3-D model of the developing CAM for precise quantification of normal vasculature of CAM from Day 4 to Day 13 of incubation. Image probing technique was used to quantify different 3-D parameters of vascular microarchitecture. A significant increase (P < 0.05) in surface roughness (Sa) was observed at Day 5 of incubation, while highly significant increase (P < 0.01) in Sa values was observed at Day 6 of incubation. Maximum increase (P < 0.001) in Sa values was observed from Day 7 to Day 9 of incubation and slight decrease in Sa values was observed in successive days. Similar results were observed for root mean square values (Sq), absolute heights of the surface (Sz), and amount of lowest valleys (Sy) on CAM. A significant increase (P < 0.05) in developed surface area (Sdr) from Day 5 of incubation, reaching to its maximum (P < 0.01) at Day 9 also was noted. Similarly, significant increase (P < 0.05) in fluid core retention (Sci) illustrates presence of copious fluid in blood vessels. Image probing technique offers a useful modality for visualizing 3-D microvascular architecture of CAM to exaggerate the fine details and reveal the hidden information that can be helpful for precise quantification of angiogenesis. This approach can be used to evaluate the angiogenic and antiangiogenic potential of different biological substances and also can be a valuable independent prognostic indicator in a wide variety of human cancers.     

Enhanced photodynamic activity of hypericin by penetration enhancer N-methyl pyrrolidone formulations in the chick chorioallantoic membrane model

Hypericin (HY) was examined for photodynamic therapy (PDT)-induced vascular damage using the chick chorioallantoic membrane (CAM) model. Clinically, plasma protein was used to solubilize HY. Upon binding to albumin, free HY available to be transported through the membrane may be limited. Hence, formulations containing a biocompatible solvent, N-Methyl pyrrolidone (NMP), have the potential to enhance HY delivery into solid tumors. At suitable concentrations, NMP and/or light irradiation did not produce antivascular damage. Hypericin-PDT effects showed to be HY and NMP concentrations-dependent. These findings indicate that NMP is a promising solvent and penetration enhancer for HY-PDT clinical applications.     

Suramin inhibits bFGF-induced endothelial cell proliferation and angiogenesis in the chick chorioallantoic membrane.

The effects of suramin, an inhibitor of growth factor mitogenic activity, were evaluated on basic fibroblast growth factor (bFGF)-induced proliferation of bovine aortic endothelial cells and on angiogenesis in the chorioallantoic membrane (CAM) of chick embryos. The role of bFGF gene expression in endothelial cell growth was also investigated by using an antisense oligodeoxynucleotide to bFGF. The 4-fold increase in [3H]-thymidine uptake in endothelial cells in vitro upon stimulation with 10 ng ml-1 of bFGF was inhibited by suramin 300 micrograms ml-1. bFGF antisense oligomer (10 microM) reduced [3H]-thymidine incorporation in exponentially growing cells by 76%; this effect was reversed by bFGF 10 ng ml-1. In the CAM of chick embryos suramin 50 micrograms was a more potent inhibitor of angiogenesis than the combination of heparin 60 micrograms/hydrocortisone 50 micrograms; the mean value of the area with reduced vascularity was significantly larger in suramin-treated CAMs (2.4 cm2) than in heparin/hydrocortisone (0.6 cm2), while the reduction of vascular density was similar (- 35 and - 29% compared to controls, respectively), In conclusion, the effects of treatments with bFGF and bFGF antisense oligomer demonstrate that bFGF plays a relevant role in endothelial cell proliferation and may be the target of suramin since the drug is able to suppress basal and bFGF-induced endothelial cell growth; in addition to this, suramin is a more potent angiogenesis inhibitor in the CAM than the combination of heparin/hydrocortisone.     

Correlative study of angiogenesis in endometrial cancer assessed by the color Doppler ultrasound and by the chick embryo chorioallantoic membrane

Angiogenesis is required for both tumor growth and progression and the degree of vascularization seems to correlate with prognosis in several human tumors including uterine malignant neoplasms. In this study we have investigated if three Doppler parameters, such as peak systolic velocity (PSV), resistance index (RI) and pulsatily index (PI), measured in patients with endometrial cancer, were correlated to the angiogenic response induced by grafting of bioptic specimens obtained from the same patients onto the chick embryo chorioallantoic membrane (CAM), a useful in vivo model for such an investigation. Results showed that only PSV was directly correlated to the degree of angiogenesis measured by means of the CAM assay. Moreover, these two parameters were also directly correlated to the malignancy grade of the disease.     

Vinblastine inhibits the angiogenic response induced by adrenomedullin in vitro and in vivo

Adrenomedullin (ADM) is protumorigenic by stimulating tumor cell growth and angiogenesis. In this context, ADM is identified as a novel target for antiangiogenic therapy. In this study, we addressed the possibility that vinblastine (VBL), as demonstrated in other experimental conditions, may act as an angiostatic molecule in the angiogenic response induced by ADM in two assays, such as Matrigel tube formation in vitro and angiogenesis in the chick embryo chorioallantoic membrane (CAM) in vivo. When tested on Matrigel, ADM caused a morphogenetic effect. In fact, endothelial cells spread and aligned with each other to form branching anastomosing tubes with multicentric junctions that gave rise to a meshwork of capillary-like structures. When ADM was administered in the presence of VBL, the capillary-like tubes were interrupted, most cells were spherical, either isolated or aggregated in small clumps. In the CAM assay, ADM induced a strong angiogenic response, which was counteracted by the treatment with VBL. Overall, these observations implicate ADM as a promoter of tumor growth and a possible target for anticancer strategies, such as the use of VBL at very low, nontoxic doses. Nevertheless, the antiangiogenic activity of low-dose VBL deserves further investigation, alone or together with other antiangiogenic agents for the treatment of tumors characterized by enhanced angiogenesis.     

Tumors formed by human rhabdomyosarcoma cells in chorioallantoic membrane of embryonated hens' eggs

Cultured human rhabdomyosarcoma (RD) cells were seeded on the chorioallantoic membrane of embryonated hens' eggs at the 10th to 12th day of incubation. When the membranes were harvested after 18 to 19 days' incubation, tumors had formed in eggs seeded with 5 × 10⁵ to 10 × 10⁶ RD cells. The microscopic appearance of the tumors was similar to that of the rhabdomyosarcoma of the patient from whom the cell line was derived. In contrast to cell lines derived from RD cell tumors which formed in a cat or an NIH-Swiss mouse and released the endogenous viruses of the cat and mouse, respectively, none of 36 cell lines derived from 13 tumors formed on the chorioallantoic membrane of hens' eggs released a type-C virus and none contained the avian virus group-specific antigen.     

Muscarinic receptors participation in angiogenic response induced by macrophages from mammary adenocarcinoma-bearing mice

Introduction  

The role of macrophages in tumor progression has generated contradictory evidence. We had previously demonstrated the ability of peritoneal macrophages from LMM3 murine mammary adenocarcinoma-bearing mice (TMps) to increase the angiogenicity of LMM3 tumor cells, mainly through polyamine synthesis. Here we investigate the ability of the parasympathetic nervous system to modulate angiogenesis induced by TMps through the activation of the muscarinic acetylcholine receptor (mAchR).     

环孢霉素A增强羟基喜树碱、三尖杉酯碱对HL-60细胞凋亡的诱导

目的:观察环孢霉素A(CsA)对羟基喜树碱(HCPT)、三尖杉酯碱(HT)诱导白血病细胞凋亡的调节作用.方法:应用细胞形态学检查,DNA凝胶电泳及流式细胞术分析检测.结果:CsA3μg/ml对HL-60细胞生长无明显影响,也没有促进凋亡作用.HL-60细胞加上HCPT0.5μg/ml、HT0.1μg/ml作用4h,细胞凋亡率分别为(35.7±1.3)%和(31.9±0.8)%.先以CsA3μg/ml作用HL-60细胞24h,洗涤,然后再加HCPT0.5μg/ml、HT0.1μg/ml继续作用4h,细胞凋亡率明显增加,达(47.5±0.5)%(P<0.01)和(44.3±0.8)%(P<0.01).CsA+HCPT及CsA+HT组分别比单独HCPT、HT组诱导的DNA梯状条带亮度增强.CsA3μg/ml对HL-60细胞的bcl-2蛋白表达以及周期分布均无影响.结论:CsA能提高HL-60细胞对HCPT或HT诱导凋亡敏感性.提示临床上应用CsA联合HCPT或HT治疗白血病,可能取得较好疗效.     

环孢毒素A增强羟基喜树碱,三尖杉酯碱对HL—60细胞凋亡的诱导

观察环孢霉素A(CsA)对羟基喜树碱 (HCPT)、三尖杉酯碱 (HT)诱导白血病细胞凋亡的调节作用。方法 :应用细胞形态学检查 ,DNA凝胶电泳及流式细胞术分析检测。结果 :CsA3μg/ml对HL 60细胞生长无明显影响 ,也没有促进凋亡作用。HL 60细胞加上HCPT0 5μg/ml、HT0 1μg/ml作用4h ,细胞凋亡率分别为(35 7±1 3) %和(31 9±0 8) %。先以CsA3μg/ml作用HL 60细胞24h ,洗涤 ,然后再加HCPT0 5μg/ml、HT0 1μg/ml继续作用4h ,细胞凋亡率明显增加 ,达(47 5±0 5) % (P<0 01)和(44 3±0 8) % (P<0 01)。CsA +HCPT及CsA +HT组分别比单独HCPT、HT组诱导的DNA梯状条带亮度增强。CsA3μg/ml对HL 60细胞的bcl 2蛋白表达以及周期分布均无影响。结论 :CsA能提高HL 60细胞对HCPT或HT诱导凋亡敏感性。提示临床上应用CsA联合HCPT或HT治疗白血病 ,可能取得较好疗效     

华蟾素联合三氧化二砷抑制鸡胚尿囊膜血管生成的实验研究

目的:观察和比较华蟾素注射液、三氧化二砷注射液单药以及联合用药后对鸡胚尿囊膜(CAM)血管生成的抑制作用。方法:100只七日龄鸡胚随机分为4组(每组25只):生理盐水(NS)对照组、华蟾素组、As2O3组和华蟾素联合As2O3组。CAM上植入甲基纤维素碟,加入各组药物,观察对血管生长的影响。结果:华蟾素、As2O3以及两药联合对鸡胚尿囊膜新生血管均有抑制作用,以两药联合后抑制血管的作用最强。结论:华蟾素、As2O3以及两药联合均具有抗血管生成作用,两药联用后且具有协同作用。     

Inhibition by doxycycline of angiogenesis in the chicken chorioallantoic membrane (CAM)

Doxycycline, a tetracycline derivative, has many properties in addition to its antibiotic activity, including inhibition of matrix metalloproteinases (MMPs) and the ability to chelate divalent cations including Ca²⁺. It has been shown to inhibit endothelial cell growth in vitro, and reduce the development of experimental tumours, especially bone metastasis in a model of breast cancer. We examined the effects of doxycycline on angiogenesis in the chicken chorioallantoic membrane (CAM) model, and showed that doxycycline will cause loss of the chorionic plexus in CAMs when applied at day 8 of incubation, and the duration of this inhibition was dose-dependent. Repeated doses prolonged the inhibition, but following removal of the doxycycline there was rapid recovery of the chorionic plexus. The effects of doxycycline are in part mimicked by the MMP inhibitor 1,10-phenanthroline, and more closely by the Ca²⁺-chelating agent EGTA. Doxycycline was equally effective in causing loss of the chorionic plexus by day 11 in CAMs, a time at which the blood vessels are established. Doxycycline has important potential as an antiangiogenic treatment. It is capable of inhibiting angiogenesis in an in vivo model, including the removal of comparatively mature endothelial cells. The response is sensitive to the dosing regimen and the effect is rapidly reversible.     

E-selectin expression induced by pancreas-carcinoma-derived interleukin-1α results in enhanced adhesion of pancreas-carcinoma cells to endothelial cells

Cellular adhesion of sialyl-Lewis-a(SLe^a)-positive pancreas carcinoma to endothelial cells (EC) is augmented by activation of EC via up-regulated E-selectin expression on EC. Co-cultivation of pancreas-carcinoma cells, PCI-24, with human umbilical-vein endothelial cells (HUVEC) for 5 hr at the PCI-to-HUVEC ratio of 1:10 induced E-selectin expression on the endothelial-cell surface, augmenting SLe^a-positive pancreas-carcinoma cell attachment with HUVEC. Culture supernatants of 6 tested pancreas-carcinoma cell lines contained soluble, E-selectin-inducing factor(s). The E-selectin-inducing effect by the supernatants was blocked by the protein-kinase-C inhibitor, H7. Antibodies against SLe^a and E-selectin but not SLe^x or ICAM-I blocked the increased pancreas-carcinoma-to-endothelial attachment. Paraformaldehyde(PFA)-fixed PCI-24 cells also induced E-selectin on vascular endothelial cells upon direct contact with endothelial cells, indicating the presence of a membrane-bound form. The 6 pancreas-carcinoma lines all produced IL-1α mRNA and protein but not IL-1β or TNF-α protein and/or mRNA Absorption of IL-lα from the supernatants by IL-lα-specific antibody almost completely abolished E-selectin-inducing activity. Anti-IL-lα antibody also abolished the E-selectin-inducing activity of PFA-fixed PCI. IL-1α production by PCI cells was up-regulated by TNF-α. These observations suggest that substance(s) produced by pancreas-carcinoma cells, in this case, IL-1α, may contribute to pancreas-carcinoma-cell colonization in non-inflamed, distant locations in vivo, by activating vascular endothelial cells.     

The antineoplastic ether lipid, s-phosphonate, selectively induces apoptosis in human leukemic cells and exhibits antiangiogenic and apoptotic activity on the chorioallantoic membrane of the chick embryo

Introduction: We investigated the cytotoxic and antiangiogenic activity of the ether lipid, 2′-(trimethylammonio)ethyl 4-(hexadecyloxy)-3(S)-methoxybutane-phosphonate (termed s-phosphonate). Method: Cytotoxicity was determined using an XTT bioassay. Apoptosis was measured by either DNA fragmentation or immunolabelling techniques. Angiogenesis was measured using the in vivo chorioallantoic membrane (CAM) of the chick embryo. Results: s-phosphonate was selectively cytotoxic towards the human leukemic cell lines, HL-60 and AML-14, whereas leukemic K-562 cells and the murine mast cell line, MC-9, were resistant to this agent at concentrations as high as 50 μM. This selectivity resulted from the induction of apoptosis (or programmed cell death) by s-phosphonate in HL-60 and AML-14 cells but not in resistant K-562 or MC-9 cells. S-phosphonate induced localized antiangiogenic effects and membrane thinning in the CAM. This concentration-dependent antiangiogenic effect was associated with apoptosis in the CAM as measured by DNA fragmentation in extracted CAM tissue. The localized areas of membrane thinning and antiangiogenesis on the CAM caused by s-phosphonate were also the only areas of the membrane in which apoptosis occurred. Conclusion: We conclude that s-phosphonate selectively induces apoptosis in human leukemic cells and exhibits antiangiogenic and apoptotic activity on the CAM. Received: 10 March 1996 / Accepted: 14 July 1997     

c-Myc degradation induced by DNA damage results in apoptosis of CHO cells

Although tripchlorolide (TC), a compound purified from a Chinese herb Tripterygium Wilfordii Hook, has been demonstrated to be a potent antitumor agent, its mechanisms of action are unknown. The present study shows that TC induces apoptosis of Chinese Hamster Ovary (CHO) cells. Most strikingly, TC was particularly potent in inducing apoptosis of the UV41 mutant CHO cells, which are deficient in the ERCC4 gene encoding a nucleotide excision repair protein. TC caused a higher level of DNA damage in UV41 cells than those in the wild-type CHO cells or EM9 cells, which are deficient in single-strand break repair. These results provided a critical link between apoptotic hypersensitivity and DNA damage in defective nucleotide excision repair pathway of UV41 cells by TC treatment. Further analysis showed that degradation of the c-Myc protein in TC-treated UV41 cells was much stronger than those in the wild-type CHOAA8 and the EM9. A proteasome inhibitor, MG132, reduced both the degradation of c-Myc and apoptosis in TC-treated UV41 cells. Expression of exogenous c-Myc also inhibited apoptosis of TC-treated UV41 cells. These results indicate that c-Myc degradation induced by DNA damage in the presence of TC contributes to induction of apoptosis of UV41 cells.     

Early-response gene signalling is induced by angiogenic oligosaccharides of hyaluronan in endothelial cells. Inhibition by non-angiogenic,high-molecular-weight hyaluronan

The degradation products of hyaluronan are known to stimulate endothelial-cell proliferation and to promote neovascularization associated with angiogenesis, whilst native high-molecular-weight hyaluronan is inhibitory to these processes. To investigate the cellular signalling pathways coupled to hyaluronan-induced responses in angiogenesis, we have analyzed early-response gene expression in vitro, in cultured bovine aortic endothelial cells. Angiogenic oligosaccharides of hyaluronan induced rapid transient up-regulation of the immediate early genes c-fos, c-jun, jun-B, Krox-20 and Krox-24. In contrast, native hyaluronan when used alone failed to elicit a significant change in expression of any of the genes tested, and when used in combination with angiogenic oligosaccharides of hyaluronan, gave a dose-dependent inhibition of induced gene expression. However, prior addition of angiogenic hyaluronan, as little as one minute before addition of high-molecular-weight hyaluronan, abrogated this inhibition, suggesting that positive or negative responses associated with hyaluronan signalling are integrated at a very early stage following receptor binding. Conversely, prior addition of high-molecular-weight hyaluronan led to an irreversible block in gene expression and proliferative response. These data are consistent with native hyaluronan antagonizing the angiogenic response in part by blocking a signalling cascade at or immediately following ligand-receptor interaction. Finally, we demonstrated that chronic exposure to oligosaccharides of hyaluronan is essential for cell proliferation, indicating that short-term immediate early-gene signalling is insufficient to elicit the proliferation of endothelial cells. Int. J. Cancer 71:251–256, 1997. © 1997 Wiley-Liss, Inc.     

Tumor grafts derived from sarcoma patients retain tumor morphology, viability, and invasion potential and indicate disease outcomes in the chick chorioallantoic membrane model

The chick chorioallantoic membrane (CAM) assay was used to evaluate whether xenotransplanted sarcomas retain the histological characteristics and functional behavior of the original tumors. Metabolically active tumor tissue, identified by dynamic-contrast MRI, from 28 patients with a bone or soft-tissue tumors was applied to the CAM. Angiogenesis and graft and host behaviors were evaluated. The essential features and immunohistochemical characteristics of the original tumors were maintained, illustrating the diversity of sarcomas. Graft viability was inversely related to patient survival, but longer follow-up and more patients are needed to relate tumor graft behavior to natural history. We conclude that the CAM assay is a potential prognostic and predictive preclinical xenograft model for tumors that are difficult to culture in vitro, such as sarcomas; therefore, the use of the CAM assay may facilitate personalized medicine.     

丁酸钠增强U937细胞凋亡敏感性的分子机制研究

目的 探讨丁酸钠（NaBu)对细胞周期检测点效应及对U937细胞凋亡的敏感性。方法 以U937-ASPI3K（ATM阴性）,U937-pZeosv2( )（野生型ATM基因）两种U937的变异细胞系作为细胞模型。用免疫沉淀及激酶活性测定p38MAPK,ERK1的激酶活性。用免疫印迹分析Bad磷酸化灭活。结果 经NaBu预处理的U937-pZeosv2( )细胞经^137Gs照射后。细胞凋亡敏感性呈NaBu剂量依赖性增强,这种增强效应可被p38MAPK阻滞剂OLM阻断,但不能被p34cdc2激酶的特异性抑制剂ALP及CDK2阻滞剂CDK-2-Ⅰ阻断,经NaBu预处理的U937-ASPI3K细胞经^137Cs照射后,细胞凋亡敏感性进一步增强,这种增强效应可被OLM阻断,放射线可显著增强p38MAPK激酶活性,抑制ERK1激酶活性;NaBu预处理与放射线联用后,对p38MAPK激酶活性的增强有极其显著的协同效应,放射线诱导U937-ASPI3K细胞Bad蛋白磷酸化灭活,在NaBu协同作用下,Bad蛋白磷酸化灭活效应进一步增强。结论 NaBu通过p38MAPK激酶活性,增强细胞凋亡敏感性,该效应与ATM基因是否缺失无关,与ATM失活增强的细胞凋亡敏感性各为独立通路。