抗肿瘤血管生成抑制剂TNP-470的研究进展

肿瘤是一种血管生成依赖性疾病,肿瘤的生成、转移均与血管生成密切相关.烟曲霉素合成类似物TNP-470能有效抑制肿瘤血管生成,破坏血管内皮细胞增殖,从而抑制肿瘤的生成与扩散.当前TNP-470正处于治疗各种肿瘤的临床试验中.本文就近几年来,对TNP-470的研究进展作一综述.     

Preparation of poly-lactic acid microspheres containing the angiogenesis inhibitor TNP-470 with medium-chain triglyceride and the in vitro evaluation of release profiles.

TNP-470 (AGM-1470, 6-0-(N-chloroacetylcarbamoyl)-fumagillol), a derivative of fumagillin, is a promising angiogenesis inhibitor. However, as TNP-470 is very unstable in in vitro and in vivo, it has been difficult to verify its pharmacological efficacy in the clinical medicine. The preparation of a drug delivery system (DDS) in a microsphere form was studied for the stable inclusion and controlled release of TNP-470. Medium-chain triglyceride (MCTG) as an effective stabilizer and poly-lactic acid (PLA) as a biodegradable carrier were used for this purpose. The release of TNP-470 from the MCTG containing DDS continued for approximately 2 weeks, while the release of TNP-470 from the one without MCTG stopped after only 5 days. It was proved that TNP-470 could be released much more stable for much longer period from the MCTG containing DDS compared to the one without DDS.     

Optimal formulation of an angiogenesis inhibitor,TNP-470, for arterial injection determined by in vitro drug release and stability,and in vivo antitumor activity

TNP-470 (6-O-(N-chloroacetylcarbamoyl)fumagillol, AGM-1470) is an angiogenesis inhibitor, a new type of anticancer drug which prevents tumor neovascularization, thereby blocking the nutrient supply to tumors. In this study, we sought the optimal formulation of TNP-470 for arterial injection in order to achieve strong anticancer activity due to the tumor-selective targeting of the drug, by investigating in vitro release and stability and in vivo rabbit VX-2 antitumor activity. We found that a medium-chain triglyceride (MCT) solution containing TNP-470 facilitated the 2-week sustained release of TNP-470 in vitro and fairly good long-term stability of the agent, although it was very labile in aqueous solution. In a rabbit VX-2 tumor model, 3 weeks after inoculation on the inner side of the leg, the antitumor activities of various formulations of TNP-470 were evaluated by administration into the femoral artery feeding the tumor. Compared with Lipiodol solution or PLGA microspheres containing TNP-470, the MCT solution containing TNP-470 exerted stronger and more persistent antitumor activity accompanied by tumor regression for 3 weeks subsequently. The release sustainability of TNP-470 in the in vitro release test was suggested to be an important factor in the antitumor activity of each formulation. From these results, we conclude that the MCT solution is the most promising formulation of TNP-470 as an arterial injection for treatment of cancers.     

Inhibition of angiogenesis and tumor growth by beta and gamma-secretase inhibitors

The involvement of beta-secretase and gamma-secretase in producing the beta-amyloid component of senile plaques found in the brain of Alzheimer's patients has fueled a major research effort to design selective inhibitors of these proteases. Interestingly, gamma-secretase cleaves several proteins including Notch, E-cadherin, CD44 and ErbB-4 (erythroblastic leukemia viral oncogene homolog 4), which are important modulators of angiogenesis. The beta-amyloid precursor protein, which is cleaved by beta-secretase and gamma-secretase to produce beta-amyloid, is highly expressed in the endothelium of neoforming vessels suggesting that it might play a role during angiogenesis. These data prompted us to explore the effects of beta and gamma-secretase inhibitors of different structures on angiogenesis and tumor growth. Both the gamma and beta-secretase inhibitors tested reduce endothelial cell proliferation without inducing cellular toxicity, suppress the formation of capillary structures in vitro and oppose the sprouting of microvessel outgrowths in the rat aortic ring model of angiogenesis. Moreover, they potently inhibit the growth and vascularization of human glioblastoma and human lung adenocarcinoma tumors xenotransplanted into nude mice. Altogether these data suggest that the gamma and beta-secretases play an essential role during angiogenesis and that inhibitors of the beta and gamma-secretases may constitute new classes of anti-angiogenic and anti-tumoral compounds.     

梓醇通过调控肿瘤血管生成及能量代谢抑制肝癌生长

目的探讨梓醇对HepG2肝癌细胞增殖、侵袭、生长、血管生成和能量代谢的影响。方法细胞增殖实验研究梓醇对HepG2细胞体外增殖的影响;LDH试剂盒检测梓醇对HepG2细胞的毒性作用;构建人肝癌HepG2裸小鼠细胞移植瘤模型,研究梓醇对体内肝癌生长的影响;Transwell实验研究梓醇对HepG2细胞体外侵袭的影响;小管形成实验检测梓醇对HepG2细胞诱导的血管生成的影响;免疫组化研究梓醇对移植瘤中能量代谢关键指标单羧酸转运蛋白-1 (monocarboxylate transporter-1,MCT-1)、单羧酸转运蛋白-4(monocarboxylate transporter-4,MCT-4)和葡萄糖转运蛋白1(Glucose transporter 1,GLUT1)蛋白表达的影响。结果在一定浓度范围内,梓醇能够抑制HepG2细胞的体外增殖及侵袭,且具有一定的浓度依赖性;LDH检测显示其在50μmol·L~(-1)时对HepG2细胞表现出毒性作用;与对照组相比,梓醇能够显著抑制HepG2裸小鼠移植瘤的体内生长;HepG2细胞培养上清液能够显著刺激HUVEC细胞的小管形成,而梓醇能够在体外抑制HepG2细胞诱导的小管形成;梓醇能够抑制HepG2裸小鼠移植瘤组织中MCT-1,MCT4和GLUT1蛋白的表达。结论梓醇具有抑制肝癌细胞增殖、侵袭及生长的能力,其机制可能与抑制肝癌的血管生成及肿瘤能量代谢密切相关。     

Plasminogen activator inhibitor-1 in tumor growth,angiogenesis and vascular remodeling

Plasminogen activator inhibitor-1 (PAI-1) is the principal inhibitor of urokinase type plasminogen activator (uPA) and tissue-type plasminogen activator (tPA), and as such is thought to play an important role in the regulation of extracellular matrix remodeling. In blood, PAI-1 is bound to the adhesion protein vitronectin and is associated with vitronectin in fibrin clots and the provisional matrix. Elevated levels of PAI-1 are associated with atherosclerosis and an increased thrombotic tendency, while PAI-1 deficiency leads to increased fibrinolysis and bleeding. PAI-1 is also elevated in many solid tumors and is associated with a poor prognosis in cancer. PAI-1 has been shown to be a potent regulator of both vascular cell migration in vitro and of angiogenesis and tumor growth in vivo. PAI-1 can both promote and inhibit tumor growth and angiogenesis. Low concentrations of PAI-1 can stimulate tumor angiogenesis while treatment of animals with high doses of PAI-1 inhibits angiogenesis and tumor growth. Hence, PAI-1 appears to have a multifunctional role in regulating the migratory and fibrinolytic activity of vascular cells, and this, in turn, may help to explain the many varied actions of PAI-1.     

Enhanced suppression of tumor growth by concomitant treatment of human lung cancer cells with suberoylanilide hydroxamic acid and arsenic trioxide

The efficacy of arsenic trioxide (ATO) against acute promyelocytic leukemia (APL) and relapsed APL has been well documented. ATO may cause DNA damage by generating reactive oxygen intermediates. Suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor, modulates gene and protein expression via histone-dependent or -independent pathways that may result in chromatin decondensation, cell cycle arrest, differentiation, and apoptosis. We investigated whether ATO and SAHA act synergistically to enhance the death of cancer cells. Our current findings showed that combined treatment with ATO and SAHA resulted in enhanced suppression of non-small-cell lung carcinoma in vitro in H1299 cells and in vivo in a xenograft mouse model. Flow cytometric analysis of annexin V+ cells showed that apoptotic cell death was significantly enhanced after combined treatment with ATO and SAHA. At the doses used, ATO did not interfere with cell cycle progression, but SAHA induced p21 expression and led to G1 arrest. A Comet assay demonstrated that ATO, but not SAHA, induced DNA strand breaks in H1299 cells; however, co-treatment with SAHA significantly increased ATO-induced DNA damage. Moreover, SAHA enhanced acetylation of histone H3 and sensitized genomic DNA to DNase I digestion. Our results suggest that SAHA may cause chromatin relaxation and increase cellular susceptibility to ATO-induced DNA damage. Combined administration of SAHA and ATO may be an effective approach to the treatment of lung cancer.     

Oxidative stress, endothelial function and angiogenesis induced by cell therapy and gene therapy

Recently, clinical studies have shown that novel therapies, including cell implantation and transfer of gene encoding for angiogenic growth factors, are effective in patients with critical limb ischemia who have no other treatment option. This concept is called therapeutic angiogenesis. Cell therapy involves implantation of bone-marrow or peripheral mononuclear cells and endothelial progenitor cells (CD34(+) cells) in the gastrocnemius of the ischemic leg. Gene therapy involves delivery of vascular endothelial growth factor and fibroblast growth factor using a plasmid or adenoviral vector. Critical limb ischemia is associated with endothelial dysfunction as well as excess oxidative stress. A balance of oxidative stress and nitric oxide play an important role in the development of atherosclerosis in patients with peripheral arterial diseases. It has been reported that both cell therapy and gene therapy improve endothelial function in the resistant artery of an ischemic limb. Cell therapy or gene therapy in combination with pharmacological therapy as an antioxidant could be useful for restoration of endothelial function and prevention of development of atherosclerosis in patients with critical limb ischemia. In this review, we discuss the relationships between oxidative stress, endothelial function, and angiogenesis and the mechanism by which therapeutic angiogenesis improves endothelial function.     

ADH1, an N-cadherin inhibitor, evaluated in preclinical models of angiogenesis and androgen-independent prostate cancer

The conversion from E-cadherin to N-cadherin has been observed in several human cancer types, including prostate cancer, with more homogenous expression of N-cadherin detected in high-grade prostate tumors. N-cadherin, in vitro, has been shown to promote cell mobility, migration and invasion of several cancer cell lines, indicating the possibility of N-cadherin as a molecular target of cancer therapy. Herein, we examined the potential of an N-cadherin inhibitor, ADH1, in reducing tumor angiogenesis ex vivo and delaying tumor progression in vivo. Our data demonstrate that ADH1, at the dosages evaluated, does not display either antiangiogenic activity in a rat aortic ring assay or antitumor potential in a PC3 subcutaneous xenograft tumor model. We detected cytotoxic activity in human umbilical vein endothelial cells, PC3, and Tsu-Pr1 cells, when ADH1 exposure was evaluated at 500 micromol/l or above.     

Discovery of PG545: a highly potent and simultaneous inhibitor of angiogenesis, tumor growth, and metastasis

Increasing the aglycone lipophilicity of a series of polysulfated oligosaccharide glycoside heparan sulfate (HS) mimetics via attachment of a steroid or long chain alkyl group resulted in compounds with significantly improved in vitro and ex vivo antiangiogenic activity. The compounds potently inhibited heparanase and HS-binding angiogenic growth factors and displayed improved antitumor and antimetastatic activity in vivo compared with the earlier series. Preliminary pharmacokinetic analyses also revealed significant increases in half-life following iv dosing, ultimately supporting less frequent dosing regimens in preclinical tumor models compared with other HS mimetics. The compounds also displayed only mild anticoagulant activity, a common side effect usually associated with HS mimetics. These efforts led to the identification of 3β-cholestanyl 2,3,4,6-tetra-O-sulfo-α-d-glucopyranosyl-(1→4)-2,3,6-tri-O-sulfo-α-d-glucopyranosyl-(1→4)-2,3,6-tri-O-sulfo-α-d-glucopyranosyl-(1→4)-2,3,6-tri-O-sulfo-β-d-glucopyranoside, tridecasodium salt (PG545, 18) as a clinical candidate. Compound 18 was recently evaluated in a phase I clinical trial in cancer patients.     

Effects of Cordyceps militaris extract on angiogenesis and tumor growth

INTRODUCTIONAngiogenesis is the process of new blood vesselformation from existing blood vessels and a natural re-sponse of tissues to ischemia[1]. The steps of angio-genesis involve proteolytic degradation of extracelluarmatrix, endothelial cell-matrix adhesion, migration,proliferation, and differentiation[2]. This biological re-action is often associated with some diseases, such assolid tumor[3], diabetic retinopathy[4], and rheumatoidarthritis[5].Tumor formation requires the developm…     

Applicability of anti-neovascular therapy to drug-resistant tumor: suppression of drug-resistant P388 tumor growth with neovessel-targeted liposomal adriamycin

Anti-neovascular therapy, one of the effective anti-angiogenic chemotherapy, damages new blood vessels by cytotoxic agents delivered to angiogenic endothelial cells and results in indirect eradication of tumor cells. We previously reported that liposomes-modified with a pentapeptide, Ala-Pro-Arg-Pro-Gly (APRPG-Lip) homing to angiogenic site, highly accumulated in tumor tissue, and APRPG-Lip encapsulating adriamycin (APRPG-LipADM) effectively suppressed tumor growth in tumor-bearing mice. In the present study, we examined the topological distribution of fluorescence-labeled APRPG-LipADM as well as TUNEL-stained cells in an actual tumor specimen obtained from Colon 26 NL-17 carcinoma-bearing mice. The fluorescence-labeled APRPG-Lip dominantly localized to vessel-like structure: a part of which was also stained with anti-CD31 antibody. Furthermore, TUNEL-stained cells were co-localized to the same structure. These data indicated that APRPG-LipADM bound to angiogenic endothelial cells and induced apoptosis of them. We also investigated the applicability of anti-neovascular therapy using APRPG-LipADM to ADM-resistant P388 solid tumor. As a result, APRPG-LipADM significantly suppressed tumor growth in mice bearing the ADM-resistant tumor. These data suggest that APRPG-LipADM is applicable to various kinds of tumor including drug-resistant tumor since it targets angiogenic endothelial cells instead of tumor cells, and eradicates tumor cells through damaging the neovessels.     

Altered tumor angiogenesis and metastasis of B16 melanoma in transgenic mice overexpressing tissue inhibitor of metalloproteinases-1

Tissue inhibitor of metalloproteinases-1 (TIMP-1) has emerged as a multifunctional protein that plays contrasting roles during angiogenesis and cancer spread. We have investigated the growth, vascularization and metastasis of B16 melanoma cells in a transgenic mouse model with elevated TIMP-1 levels in the systemic circulation. Transgenic C57BL/6j-CBA mice overexpressing human TIMP-1 in the liver under the control of the mouse albumin promoter/enhancer were employed. An early subcutaneous growth advantage and an increased tumor angiogenic response were observed in transgenic animals with respect to wild-type hybrid mice. On the contrary, there was a dramatic decrease in the lung colonizing ability of B16 melanoma cells in TIMP-1 transgenic mice. No significant effect on metastasis formation was observed in another transgenic mouse model with increased TIMP-1 expression in lungs but low plasma levels, where the transgene was placed under the control of the murine mammary tumor virus promoter. These results support the notion that TIMP-1 displays paradoxical effects on tumor progression and suggest that circulating TIMP-1 is efficient in suppressing lung colonization of melanoma cells.     

Artemisinin inhibits tumor lymphangiogenesis by suppression of vascular endothelial growth factor C

We have previously reported that dihydroartemisinin is found to have a potent ability in influencing lymphatic endothelial cell migration and tube formation. In this study, we investigated the effect of artemisinin on tumor growth, lymphangiogenesis, metastasis and survival in mouse Lewis lung carcinoma (LLC) models. We found that orally administered artemisinin inhibited lymph node and lung metastasis and prolonged survival without retarding tumor growth. Consistent with the decrease in lymph node metastasis, tumor lymphangiogenesis and expression of vascular endothelial growth factor C (VEGF-C) was significantly decreased in artemisinin-treated mice, as compared to control mice. Furthermore, IL-1beta-induced p38 mitogen-activated protein kinase (MAPK) activation and upregulation of VEGF-C mRNA and protein in LLC cells was also suppressed by artemisinin or by the p38 MAPK inhibitor SB-203580, suggesting that p38 MAPK could serve as a mediator of proinflammatory cytokine-induced VEGF-C expression. These data indicate that artemisinin may be useful for the prevention of lymph node metastasis by downregulating VEGF-C and reducing tumor lymphangiogenesis.     

Increased expression of CYP4Z1 promotes tumor angiogenesis and growth in human breast cancer

Cytochrome P450 (CYP) 4Z1, a novel CYP4 family member, is over-expressed in human mammary carcinoma and associated with high-grade tumors and poor prognosis. However, the precise role of CYP4Z1 in tumor progression is unknown. Here, we demonstrate that CYP4Z1 overexpression promotes tumor angiogenesis and growth in breast cancer. Stable expression of CYP4Z1 in T47D and BT-474 human breast cancer cells significantly increased mRNA expression and production of vascular endothelial growth factor (VEGF)-A, and decreased mRNA levels and secretion of tissue inhibitor of metalloproteinase-2 (TIMP-2), without affecting cell proliferation and anchorage-independent cell growth in vitro. Notably, the conditioned medium from CYP4Z1-expressing cells enhanced proliferation, migration and tube formation of human umbilical vein endothelial cells, and promoted angiogenesis in the zebrafish embryo and chorioallantoic membrane of the chick embryo. In addition, there were lower levels of myristic acid and lauric acid, and higher contents of 20-hydroxyeicosatetraenoic acid (20-HETE) in CYP4Z1-expressing T47D cells compared with vector control. CYP4Z1 overexpression significantly increased tumor weight and microvessel density by 2.6-fold and 1.9-fold in human tumor xenograft models, respectively. Moreover, CYP4Z1 transfection increased the phosphorylation of ERK1/2 and PI3K/Akt, while PI3K or ERK inhibitors and siRNA silencing reversed CYP4Z1-mediated changes in VEGF-A and TIMP-2 expression. Conversely, HET0016, an inhibitor of the CYP4 family, potently inhibited the tumor-induced angiogenesis with associated changes in the intracellular levels of myristic acid, lauric acid and 20-HETE. Collectively, these data suggest that increased CYP4Z1 expression promotes tumor angiogenesis and growth in breast cancer partly via PI3K/Akt and ERK1/2 activation.     

Gene delivery and gene therapy of prostate cancer

Surgery, radiation or hormonal therapy are not adequate to control prostate cancer. Clearly, other novel treatment approaches, such as gene therapy, for advanced/recurrent disease are desperately needed to achieve long-term local control and particularly to develop effective systemic therapy for metastatic prostate cancer. In the last decade, significant progress in gene therapy for the treatment of localised prostate cancer has been demonstrated. A broad range of different gene therapy approaches, including cytolytic, immunological and corrective gene therapy, have been successfully applied for prostate cancer treatment in animal models, with translation into early clinical trials. In addition, a wide variety of viral and nonbiological gene delivery systems are available for basic and clinical research. Gene therapy approaches that have been developed for the treatment of prostate cancer are summarised.     

新型酪氨酸激酶抑制剂BSSD-1对斑马鱼血管生成及大肠癌HT29裸鼠肿瘤的抑制作用

目的探讨化合物BSSD-1对斑马鱼新生血管的影响以及对大肠癌HT29裸鼠肿瘤的抑制作用。方法采用24hpf(hours post fertilization)健康TG(VEGFR2:GFP)系血管荧光转基因斑马鱼作为实验动物模型,加入不同剂量BSSD-1与胚胎共同孵育24 h,在荧光显微镜下观察背部节间血管(intersegmental vessels,ISV)生成情况,并计数评价抑制程度;进而建立人大肠癌HT29细胞裸小鼠模型,观察BSSD-1不同剂量作用下对裸鼠肿瘤的抑制作用,根据瘤体积及瘤重,计算相对肿瘤增殖率和肿瘤生长抑制率。结果化合物BSSD-1在0.25～25.0 mg·L-1范围抑制斑马鱼背部节间血管生成具有剂量相关性,12.5 mg·L-1抑制率可达98.8%;裸鼠肿瘤抑制试验中,大、中、小剂量组肿瘤生长抑制率试验结果分别为31.1%、40.0%和40.0%,统计学处理3组差异均有显著性。结论 BSSD-1可以抑制荷瘤裸鼠移植瘤的生长,该作用与抑制肿瘤新生血管生成有关。     

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。