Norcantharidin inhibits tumor angiogenesis via blocking VEGFR2/MEK/ERK signaling pathways

Norcantharidin (NCTD), the demethylated form of Cantharidin, a reagent isolated from blister beetles, has been shown to be an anti‐tumor agent capable of inhibiting proliferation as well as inducing apoptosis in many cancer cell lines. However, little is known about the effect of NCTD in tumor angiogenesis. In this study, we demonstrated that NCTD inhibited vascular endothelial growth factor (VEGF)‐induced cell proliferation, migration, invasion, and capillary tube formation of primary human umbilical vein endothelial cells (HUVECs) in a dose‐dependent manner. Furthermore, we showed NCTD inhibited tumor growth and angiogenesis of colon cancer cells (LOVO) in vivo. We then mechanistically described that NCTD specifically abrogated the phosphorylation/activation of vascular endothelial growth factor receptor‐2 (VEGFR2)/MEK/ERK pathway kinases, with little effect on the phosphorylation of p38 MAPK and Akt, and on Cox‐2 expression. In summary, our results indicate that NCTD is a potential inhibitor of tumor angiogenesis by blocking VEGFR2/MEK/ERK signaling.     

Celastrol inhibits the growth of human glioma xenografts in nude mice through suppressing VEGFR expression

Celastrol, a compound purified from Tripterygium wilfordii whose preparations have been used for clinical treatment for rheumatoid arthritis, has been demonstrated to have antiangiogenic activity, and be inhibitory against mice tumor growth by a few recent studies. However, whether its antiangiogenic activity plays a role in the celastrol-mediated suppression of tumor growth and the molecular basis of anti-tumor activity are poorly understood. In this study, we found that celastrol inhibited the growth of human glioma xenografts in mice, which concurred with the suppression of angiogenesis. Interestingly, while celastrol had no effect on either the expression of VEGF or its mRNA levels, celastrol treatment lowered the expression levels of its receptors (VEGFR-1 and VEGFR-2) and their mRNA levels. These findings suggest that celastrol have potential to be used as an antiangiogenesis drug through its role in suppressing VEGF receptors expression that might consequently reduce the signal transduction between VEGF and VEGFR.     

Itraconazole inhibits angiogenesis and tumor growth in non-small cell lung cancer

The antiangiogenic agent bevacizumab has been approved for the treatment of non-small cell lung cancer (NSCLC), although the survival benefit associated with this agent is marginal, and toxicities and cost are substantial. A recent screen for selective inhibitors of endothelial cell proliferation identified the oral antifungal drug itraconazole as a novel agent with potential antiangiogenic activity. In this article, we define and characterize the antiangiogenic and anticancer activities of itraconazole in relevant preclinical models of angiogenesis and lung cancer. Itraconazole consistently showed potent, specific, and dose-dependent inhibition of endothelial cell proliferation, migration, and tube formation in response to both VEGF- and basic fibroblast growth factor-mediated angiogenic stimulation. In vivo, using primary xenograft models of human NSCLC, oral itraconazole showed single-agent growth-inhibitory activity associated with induction of tumor hypoxia-inducible factor 1 alpha expression and marked inhibition of tumor vascularity. Itraconazole significantly enhanced the antitumor efficacy of the chemotherapeutic agent cisplatin in the same model systems. Taken together, these data suggest that itraconazole has potent and selective inhibitory activity against multiple key aspects of tumor-associated angiogenesis in vitro and in vivo, and strongly support clinical translation of its use. Based on these observations, we have initiated a randomized phase II study comparing the efficacy of standard cytotoxic therapy with or without daily oral itraconazole in patients with recurrent metastatic NSCLC.     

二氢杨梅素通过ERK/VEGFA/VEGFR2通路对体内外胃癌血管生成的影响

背景与目的：二氢杨梅素（dihydromyricetin,DHM）通过抑制细胞周期、促进细胞凋亡和抑制新生血管生成等机制发挥抗肿瘤作用。探讨DHM对胃癌的抗癌作用,并研究其可能作用机制。方法：不同剂量DHM作用体外人脐静脉内皮细胞（human umbilical vein endothelial cells, HUVEC）和胃癌细胞系MKN28后,采用细胞计数试剂盒（cell counting kit-8,CCK-8）法筛选无毒剂量的DHM,Transwell小室实验测定细胞侵袭;小管形成实验测定HUVEC体外血管生成能力;采用蛋白质印迹法（Western blot）检测血管内皮生长因子A（vascular endothelial growth factor A, VEGFA）、phospho-血管内皮生长因子受体（vascular endothelial growth factor receptor, VEGFR）2、细胞外信号调节激酶（extracellular signal-regulated kinase,ERK）、phospho-c-Jun氨基端激酶（c-Jun NH2-terminal kinase,JNK）和phospho-p38表达水平;基质胶塞实验测定DHM对体内血管形成的影响;通过皮下肿瘤细胞注射法建立MKN28裸鼠异种移植瘤模型,观察DHM给药对体内胃癌生长的影响,Ki-67增殖指数及CD34、VEGFA在移植瘤组织中的表达通过免疫组织化学法测定。结果：（0.5~2.5 μmol/L）的DHM对HUVEC和MKN28细胞生长无明显抑制作用,该浓度范围的DHM呈浓度依赖性地抑制HUVEC侵袭和小管形成,VEGFA（20 μg/L）可明显逆转DHM对HUVEC侵袭和小管形成的抑制效果;DHM处理可导致MKN28细胞中VEGFA和phospho-ERK表达呈剂量依赖性下降,而对p-p38和p-JNK的表达无明显影响;DHM处理可导致HUVEC中phospho-VEGFR2表达呈剂量依赖性下降;低剂量（30 mg/kg）DHM对裸鼠无明显的不良反应,但可抑制体内血管形成,同时有效延缓体内胃癌生长,体内肿瘤组织中的CD34和VEGFA的表达受到DHM的显著下调作用,但低剂量DHM对Ki-67无明显作用。结论：低剂量的DHM可有效抑制体内外血管生成和体内胃癌生长,该作用至少部分是通过ERK/VEGFA/VEGFR2信号通路来实现的。     

A VEGFR1 antagonistic peptide inhibits tumor growth and metastasis through VEGFR1-PI3K-AKT signaling pathway inhibition

Angiogenesis is central to the growth of cancers and VEGFR-1/Flt-1 plays an important role during the neovascularization under pathological conditions. We previously founded a VEGFR1 antagonistic peptide, F56, by screening the phage peptide library. We showed that DHFR-F56 chimeric protein displayed anti-tumor activity and inhibited angiogenesis, however the anti-tumor activity of monomeric F56 and the mechanism remain unclear. In this study, we found that the F56 didn’t affect VEGF-A induced endothelial cell proliferation, but reduced migration and tube formation of endothelial cells. F56 also inhibited the sprout of rat aortic endothelial cells, the angiogenesis of chicken embryo chorioallantoic membrane as well as the generation of subintestinal vein vessels (SIV) in zebrafish embryos. We found that F56 inhibited VEGF-induced phosphorylation of VEGFR1, as well as the phosphorylation of the downstream of PI3K-AKT axis. However, F56 had no effect on the phosphorylation of VEGFR2. Correlating with these effects, F56 inhibited xenograft growth of HT-29 and MGC-823 cells in BALB/c nude mice, and significantly suppressed the lung metastasis of B16 cells in C57BL/6 mice. Our study demonstrated that monomeric peptide F56 had a significant anti-tumor activity by inhibiting angiogenesis, and laid the foundation for its clinical application.     

Indirubin inhibits tumor growth by antitumor angiogenesis via blocking VEGFR2-mediated JAK/STAT3 signaling in endothelial cell

Tumor angiogenesis is one of the hallmarks of the development in malignant neoplasias and metastasis. Many angiogenesis inhibitors are small molecules from natural products. Indirubin, the active component of a traditional Chinese herbal medicine, Banlangen, has been shown to exhibit antitumor and anti-inflammation effects. But its roles in tumor angiogenesis, the key step involved in tumor growth and metastasis, and the involved molecular mechanism is unknown. Here, we identified that indirubin inhibited prostate tumor growth through inhibiting tumor angiogenesis. Using chick chorioallantoic membrane (CAM) assay and mouse corneal model, we found that indirubin inhibited angiogenesis in vivo. We also showed the inhibition activity of indirubin in endothelial cell migration, tube formation and cell survival in vitro. Furthermore, indirubin suppressed vascular endothelial growth factor receptor 2-mediated Janus kinase (JAK)/STAT3 signaling pathway but had little effects on the activity of extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase in endothelial cell. Our study provided the first evidence for antitumor angiogenesis activity of indirubin and the related molecular mechanism. Our investigations suggested that indirubin was a potential drug candidate for angiogenesis related diseases.     

Fibroblast growth factor signaling in non-small-cell lung cancer

Despite recent progress in the treatment on non-small cell lung cancer (NSCLC), outcomes remain suboptimal. Treatment advances that target the epidermal growth factor receptor (EGFR) and vascular endothelial growth factor (VEGF) signaling pathways highlight the need to understand the multiple convergent growth factor signaling pathways involved in the pathogenesis of NSCLC. Signaling through fibroblast growth factors (FGF), long recognized for its pro-angiogenic activity, has recently emerged as a contributing factor in the pathogenesis and progression of NSCLC through an autocrine signaling loop. In addition, this pathway may function as a mechanism of resistance to anti-EGFR and anti-VEGF treatment. Clinical experience with FGF receptor (FGFR) inhibitors is mounting, and more specific inhibitors of this signaling pathway are in development. This review describes the structure of the FGF signaling pathway, delineates its dual roles in angiogenesis and proliferation in NSCLC, evaluates FGF ligand and receptor expression as prognostic biomarkers in NSCLC, and discusses the development of FGF pathway inhibitors for the treatment of lung malignancies.     

TKI-31 inhibits angiogenesis by combined suppression signaling pathway of VEGFR2 and PDGFRbeta

Tyrosine kinases have been strongly implicated as therapeutic targets that influence the angiogenic process in growing tumors. In this study, we revealed that TKI-31 is a potent broad spectrum tyrosine kinase inhibitor, which inhibits vascular endothelial growth factor receptor 2 (VEGFR2), platelet-derived growth factor receptor beta (PDGFRbeta) and also inhibits kinases of other class, such as c-Kit and c-Src on molecular base, but showed no activity against vascular endothelial growth factor receptor 1 (VEGFR1) and epidermal growth factor receptor (EGFR). TKI-31 inhibits VEGF-induced phosphorylation of VEGFR2 in endothelial cells as well as PDGF(BB)-induced phosphorylation in fibroblast cells, and leading to the inhibition of down-stream signaling triggered by these receptors such as PI3K/Akt/mTOR, MAPK42/44(ERK) and paxillin. TKI-31 also inhibited VEGF-induced endothelial cells proliferation, migration and their differentiation into capillary-like tube formation. Its anti-angiogenic property was further confirmed by the inhibition of neovascularization on CAM, in vivo. These results collectively highlight the therapeutic potential of this compound for the treatment of solid tumors and other diseases where angiogenesis plays an important role.     

VEGFR2 blockade augments the effects of tyrosine kinase inhibitors by inhibiting angiogenesis and oncogenic signaling in oncogene-driven non-small-cell lung cancers

Molecular agents targeting the epidermal growth factor receptor (EGFR)-, anaplastic lymphoma kinase (ALK)- or c-ros oncogene 1 (ROS1) alterations have revolutionized the treatment of oncogene-driven non-small-cell lung cancer (NSCLC). However, the emergence of acquired resistance remains a significant challenge, limiting the wider clinical success of these molecular targeted therapies. In this study, we investigated the efficacy of various molecular targeted agents, including erlotinib, alectinib, and crizotinib, combined with anti-vascular endothelial growth factor receptor (VEGFR) 2 therapy. The combination of VEGFR2 blockade with molecular targeted agents enhanced the anti-tumor effects of these agents in xenograft mouse models of EGFR-, ALK-, or ROS1-altered NSCLC. The numbers of CD31-positive blood vessels were significantly lower in the tumors of mice treated with an anti-VEGFR2 antibody combined with molecular targeted agents compared with in those of mice treated with molecular targeted agents alone, implying the antiangiogenic effects of VEGFR2 blockade. Additionally, the combination therapies exerted more potent antiproliferative effects in vitro in EGFR-, ALK-, or ROS1-altered NSCLC cells, implying that VEGFR2 inhibition also has direct anti-tumor effects on cancer cells. Furthermore, VEGFR2 expression was induced following exposure to molecular targeted agents, implying the importance of VEGFR2 signaling in NSCLC patients undergoing molecular targeted therapy. In conclusion, VEGFR2 inhibition enhanced the anti-tumor effects of molecular targeted agents in various oncogene-driven NSCLC models, not only by inhibiting tumor angiogenesis but also by exerting direct antiproliferative effects on cancer cells. Hence, combination therapy with anti-VEGFR2 antibodies and molecular targeted agents could serve as a promising treatment strategy for oncogene-driven NSCLC.     

MEDI3617, a human anti-angiopoietin 2 monoclonal antibody, inhibits angiogenesis and tumor growth in human tumor xenograft models

Angiopoietin 2 (Ang2) is an important regulator of angiogenesis, blood vessel maturation and integrity of the vascular endothelium. The correlation between the dynamic expression of Ang2 in tumors with regions of high angiogenic activity and a poor prognosis in many tumor types makes Ang2 an ideal drug target. We have generated MEDI3617, a human anti-Ang2 monoclonal antibody that neutralizes Ang2 by preventing its binding to the Tie2 receptor in vitro, and inhibits angiogenesis and tumor growth in vivo. Treatment of mice with MEDI3617 resulted in inhibition of angiogenesis in several mouse models including: FGF2-induced angiogenesis in a basement extract plug model, tumor and retinal angiogenesis. In xenograft tumor models, treatment with MEDI3617 resulted in a reduction in tumor angiogenesis and an increase in tumor hypoxia. The administration of MEDI3617 as a single agent to mice bearing human tumor xenografts resulted in tumor growth inhibition against a broad spectrum of tumor types. Combining MEDI3617 with chemotherapy or bevacizumab resulted in a delay in tumor growth and no body weight loss was observed in the combination groups. These results, combined with pharmacodynamic studies, demonstrate that treatment of tumor-bearing mice with MEDI3617 significantly inhibited tumor growth as a single agent by blocking tumor angiogenesis. Together, these data show that MEDI3617 is a robust antiangiogenic agent and support the clinical evaluation and biomarker development of MEDI3617 in cancer patients.     

Antiapoptotic signaling pathways in non-small-cell lung cancer: biology and therapeutic strategies

One of the hallmarks of lung cancer is the deregulation of apoptotic or programmed cell death mechanisms usually found in normal cells that allow for corrupted cells to undergo cellular suicide. This includes mechanisms that attenuate proapoptotic pathways and/or amplify antiapoptotic pathways. Increasing evidence suggests that lung cancer cells use multiple and perhaps redundant pathways to maintain survival. Increasing knowledge of these pathways offers a better understanding of the biology of lung cancer as well as novel therapeutic strategies that can enhance lung cancer cell death. This review discusses the apoptotic machinery and signal transduction pathways that regulate apoptosis, methods of identifying the presence of activated survival signaling pathways in human lung cancers, and the clinical significance and relevance for therapy for patients with lung cancer.     

Foretinib inhibits angiogenesis,lymphangiogenesis and tumor growth of pancreatic cancer in vivo by decreasing VEGFR-2/3 and TIE-2 signaling

Foretinib, a multiple kinase inhibitor undergoing clinical trials, could suppress the activity of hepatocyte growth factor (HGF) receptor c-MET and vascular endothelial growth factor receptor-2 (VEGFR-2). In addition, Foretinib may inhibit two critical lymphangiogenic signaling receptors VEGFR-3 and TIE-2. However, the effect of Foretinib on lymphatic endothelial cells (LECs) in vitro and lymphangiogenesis in vivo is still unknown. We found Foretinib decreased basal- and HGF-induced c-MET activity at low concentrations. However, Foretinib only reduced the proliferation of pancreatic cancer cells at high concentration reflecting the intrinsic chemoresistance of pancreatic cancer cells. Foretinib inhibited VEGF-A, VEGF-C and Angiopoetin-2 (ANG-2)-stimulated tube formation and sprouting of LECs by reducing VEGFR-2, VEGFR-3 and TIE-2 activation and increased apoptosis of LECs. In xenograft animal study, Foretinib suppressed tumor growth by inhibiting proliferation, angiogenesis and lymphangiogenesis. Additionally, Foretinib inhibited angiogenesis and lymphangiogenesis more significantly and exhibited low detrimental effect in orthotopic animal study. Collectively, we suggested that Foretinib simultaneously inhibits cancer cells and LECs to reduce pancreatic tumor growth in vivo and demonstrated for the first time that Foretinib suppresses angiogenesis and lymphangiogenesis by blocking VEGFR-2/3 and TIE-2 signaling.     

ZD6474 inhibits vascular endothelial growth factor signaling,angiogenesis, and tumor growth following oral administration

ZD6474 [N-(4-bromo-2-fluorophenyl)-6-methoxy-7-[(1-methylpiperidin-4-yl)methoxy]quinazolin-4-amine]is a potent, p.o. active, low molecular weight inhibitor of kinase insert domain-containing receptor [KDR/vascular endothelial growth factor receptor (VEGFR) 2] tyrosine kinase activity (IC(50) = 40 nM). This compound has some additional activity versus the tyrosine kinase activity of fms-like tyrosine kinase 4 (VEGFR3;IC(50) = 110 nM) and epidermal growth factor receptor (EGFR/HER1; IC(50) = 500 nM) and yet demonstrates selectivity against a range of other tyrosine and serine-threonine kinases. The activity of ZD6474 versus KDR tyrosine kinase translates into potent inhibition of vascular endothelial growth factor-A (VEGF)-stimulated endothelial cell (human umbilical vein endothelial cell) proliferation in vitro (IC(50) = 60 nM). Selective inhibition of VEGF signaling has been demonstrated in vivo in a growth factor-induced hypotension model in anesthetized rat: administration of ZD6474 (2.5 mg/kg, i.v.) reversed a hypotensive change induced by VEGF (by 63%) but did not significantly affect that induced by basic fibroblast growth factor. Once-daily oral administration of ZD6474 to growing rats for 14 days produced a dose-dependent increase in the femoro-tibial epiphyseal growth plate zone of hypertrophy, which is consistent with inhibition of VEGF signaling and angiogenesis in vivo. Administration of 50 mg/kg/day ZD6474 (once-daily, p.o.) to athymic mice with intradermally implanted A549 tumor cells also inhibited tumor-induced neovascularization significantly (63% inhibition after 5 days; P < 0.001). Oral administration of ZD6474 to athymic mice bearing established (0.15-0.47 cm(3)), histologically distinct (lung, prostate, breast, ovarian, colon, or vulval) human tumor xenografts or after implantation of aggressive syngeneic rodent tumors (lung, melanoma) in immunocompetent mice, produced a dose-dependent inhibition of tumor growth in all cases. Statistically significant antitumor activity was evident in each model with at least 25 mg/kg ZD6474 once daily (P < 0.05, one-tailed t test). Histological analysis of Calu-6 tumors treated with 50 mg/kg/day ZD6474 for 24 days showed a significant reduction (>70%) in CD31 (endothelial cell) staining in nonnecrotic regions. ZD6474 also restrained growth of much larger (0.9 cm(3) volume) Calu-6 lung tumor xenografts and induced profound regression in established PC-3 prostate tumors of 1.4 cm(3) volume. ZD6474 is currently in Phase I clinical development as a once-daily oral therapy in patients with advanced cancer.     

A notch1 ectodomain construct inhibits endothelial notch signaling, tumor growth, and angiogenesis

Notch signaling is required for vascular development and tumor angiogenesis. Although inhibition of the Notch ligand Delta-like 4 can restrict tumor growth and disrupt neovasculature, the effect of inhibiting Notch receptor function on angiogenesis has yet to be defined. In this study, we generated a soluble form of the Notch1 receptor (Notch1 decoy) and assessed its effect on angiogenesis in vitro and in vivo. Notch1 decoy expression reduced signaling stimulated by the binding of three distinct Notch ligands to Notch1 and inhibited morphogenesis of endothelial cells overexpressing Notch4. Thus, Notch1 decoy functioned as an antagonist of ligand-dependent Notch signaling. In mice, Notch1 decoy also inhibited vascular endothelial growth factor-induced angiogenesis in skin, establishing a role for Notch receptor function in this process. We tested the effects of Notch1 decoy on tumor angiogenesis using two models: mouse mammary Mm5MT cells overexpressing fibroblast growth factor 4 (Mm5MT-FGF4) and NGP human neuroblastoma cells. Exogenously expressed FGF4 induced Notch ligand expression in Mm5MT cells and xenografts. Notch1 decoy expression did not affect tumorigenicity of Mm5MT-FGF4 cells in vitro but restricted Mm5MT-FGF4 xenograft growth in mice while markedly impairing neoangiogenesis. Similarly, Notch1 decoy expression did not affect NGP cells in vitro but disrupted vessels and decreased tumor viability in vivo. These results strongly suggest that Notch receptor signaling is required for tumor neoangiogenesis and provides a new target for tumor therapy.     

Vascular endothelial growth factor, platelet-derived endothelial cell growth factor and angiogenesis in non-small-cell lung cancer

High microvessel density, an indirect measure of angiogenesis, has been shown to correlate with increased tumour size, lymph node involvement and poor prognosis in non-small-cell lung cancer (NSCLC). Tumour cell vascular endothelial growth factor (VEGF) and platelet-derived endothelial cell growth factor (PD-ECGF) expression correlate with angiogenesis and a poor outcome in this disease. In a retrospective study VEGF and PD-ECGF expression and microvessel density were evaluated immunohistochemically in surgically resected specimens (T1-3, N0-2) from 223 patients with operable NSCLC using the VG1, P-GF.44C and JC70 monoclonal antibodies respectively. High VEGF immunoreactivity was seen in 104 (46.6%) and PD-ECGF in 72 (32.3%) cases and both were associated with high vascular grade tumours (P= 0.009 and P= 0.05 respectively). Linear regression analysis revealed a weak positive correlation between VEGF and PD-ECGF expression in cancer cells (r= 0.21; P = 0.002). Co-expression of VEGF and PD-ECGF was not associated with a higher microvessel density than VEGF or PD-ECGF only expressing tumours. Furthermore a proportion of high vascular grade tumours expressed neither growth factor. Univariate analysis revealed tumour size, nodal status, microvessel density and VEGF and PD-ECGF expression as significant prognostic factors. Tumour size (P < 0.02) and microvessel density (P < 0.04) remained significant on multivariate analysis. In conclusion, VEGF and PD-ECGF are important angiogenic growth factors and have prognostic significance in NSCLC. Furthermore the study underlines the prognostic significance of microvessel density in operable NSCLC.     

Gambogic acid inhibits angiogenesis and prostate tumor growth by suppressing vascular endothelial growth factor receptor 2 signaling

Gambogic acid (GA), the main active compound of Gamboge hanburyi, has been previously reported to activate apoptosis in many types of cancer cell lines by targeting transferrin receptor and modulating nuclear factor-kappaB signaling pathway. Whether GA inhibits angiogenesis, which is crucial for cancer and other human diseases, remains unknown. Here, we found that GA significantly inhibited human umbilical vascular endothelial cell (HUVEC) proliferation, migration, invasion, tube formation, and microvessel growth at nanomolar concentration. In a xenograft prostate tumor model, we found that GA effectively inhibited tumor angiogenesis and suppressed tumor growth with low side effects using metronomic chemotherapy with GA. GA was more effective in activating apoptosis and inhibiting proliferation and migration in HUVECs than in human prostate cancer cells (PC3), suggesting GA might be a potential drug candidate in cancer therapy through angioprevention with low chemotoxicity. Furthermore, we showed that GA inhibited the activations of vascular endothelial growth factor receptor 2 and its downstream protein kinases, such as c-Src, focal adhesion kinase, and AKT. Together, these data suggest that GA inhibits angiogenesis and may be a viable drug candidate in antiangiogenesis and anticancer therapies.     

Celecoxib inhibits angiogenesis by inducing endothelial cell apoptosis in human pancreatic tumor xenografts

Previous studies suggest that antagonists of cyclooxygenases 1 and 2 (COX-1, -2) inhibit angiogenesis in tumor xenografts, but the molecular mechanisms involved remain unclear. Here we characterized the effects of non-selective (indomethacin) and selective (NS398, celecoxib) cyclooxygenase inhibitors on parameters of angiogenesis in human pancreatic adenocarcinoma cells. COX-1 expression was constitutive in 9/9 pancreatic cancer cell lines, whereas COX-2 and cytosolic phospholipase A2 (cPLA2) expression were observed in 4/9 cell lines (BxPC3, Capan2, Cfpac1, and L3.6 pl). Production of the COX product, prostaglandin E2, correlated with expression of cPLA2 and COX-2 and was blocked by non-steroidal anti-inflammatory drugs (NSAIDs, indomethacin or NS398). In contrast to the findings of others, neither indomethacin nor NS398 affected tumor cell secretion of angiogenic factors (VEGF, bFGF, IL-8) at concentrations that produced maximal inhibition of PGE2 production, and higher concentrations increased angiogenic factor production. We also studied the effects of celecoxib in orthotopic L3.6 pl xenografts. Immunofluorescence analyses revealed high-level expression of COX-2 in endothelial cells in L3.6 pl xenografts that increased following therapy with celecoxib, whereas the tumor cells expressed uniformly low levels of COX-2. Celecoxib did not decrease tumor-associated VEGF levels in orthotopic human L3.6 pl xenografts, but the drug did decrease tumor microvessel density (MVD) and increase apoptosis in tumor-associated endothelial cells in a dose-dependent fashion. Together, our results demonstrate that the anti-angiogeneic effects of NSAIDs in human pancreatic cancer cells are exerted via direct effects on endothelial cells.