Preclinical characterization of anlotinib,a highly potent and selective vascular endothelial growth factor receptor‐2 inhibitor

Abrogating tumor angiogenesis by inhibiting vascular endothelial growth factor receptor‐2 (VEGFR2) has been established as a therapeutic strategy for treating cancer. However, because of their low selectivity, most small molecule inhibitors of VEGFR2 tyrosine kinase show unexpected adverse effects and limited anticancer efficacy. In the present study, we detailed the pharmacological properties of anlotinib, a highly potent and selective VEGFR2 inhibitor, in preclinical models. Anlotinib occupied the ATP‐binding pocket of VEGFR2 tyrosine kinase and showed high selectivity and inhibitory potency (IC₅₀ <1 nmol/L) for VEGFR2 relative to other tyrosine kinases. Concordant with this activity, anlotinib inhibited VEGF‐induced signaling and cell proliferation in HUVEC with picomolar IC₅₀ values. However, micromolar concentrations of anlotinib were required to inhibit tumor cell proliferation directly in vitro. Anlotinib significantly inhibited HUVEC migration and tube formation; it also inhibited microvessel growth from explants of rat aorta in vitro and decreased vascular density in tumor tissue in vivo. Compared with the well‐known tyrosine kinase inhibitor sunitinib, once‐daily oral dose of anlotinib showed broader and stronger in vivo antitumor efficacy and, in some models, caused tumor regression in nude mice. Collectively, these results indicate that anlotinib is a well‐tolerated, orally active VEGFR2 inhibitor that targets angiogenesis in tumor growth, and support ongoing clinical evaluation of anlotinib for a variety of malignancies.     

AMG 706, an oral, multikinase inhibitor that selectively targets vascular endothelial growth factor, platelet-derived growth factor, and kit receptors, potently inhibits angiogenesis and induces regression in tumor xenografts

The growth of solid tumors is dependent on the continued stimulation of endothelial cell proliferation and migration resulting in angiogenesis. The angiogenic process is controlled by a variety of factors of which the vascular endothelial growth factor (VEGF) pathway and its receptors play a pivotal role. Small-molecule inhibitors of VEGF receptors (VEGFR) have been shown to inhibit angiogenesis and tumor growth in preclinical models and in clinical trials. A novel nicotinamide, AMG 706, was identified as a potent, orally bioavailable inhibitor of the VEGFR1/Flt1, VEGFR2/kinase domain receptor/Flk-1, VEGFR3/Flt4, platelet-derived growth factor receptor, and Kit receptors in preclinical models. AMG 706 inhibited human endothelial cell proliferation induced by VEGF, but not by basic fibroblast growth factor in vitro, as well as vascular permeability induced by VEGF in mice. Oral administration of AMG 706 potently inhibited VEGF-induced angiogenesis in the rat corneal model and induced regression of established A431 xenografts. AMG 706 was well tolerated and had no significant effects on body weight or on the general health of the animals. Histologic analysis of tumor xenografts from AMG 706-treated animals revealed an increase in endothelial apoptosis and a reduction in blood vessel area that preceded an increase in tumor cell apoptosis. In summary, AMG 706 is an orally bioavailable, well-tolerated multikinase inhibitor that is presently under clinical investigation for the treatment of human malignancies.     

Selection of high affinity human neutralizing antibodies to VEGFR2 from a large antibody phage display library for antiangiogenesis therapy.

Compelling evidence suggests that vascular endothelial growth factor (VEGF) and its receptors play an important role in angiogenesis associated with tumor growth and metastasis. VEGF exerts its biologic activities through 2 transmembrane tyrosine kinase receptors: the fms-like tyrosine kinase receptor (Flt-1, or VEGFR1) and kinase insert domain-containing receptor (KDR or VEGFR2). We have previously produced a panel of antibodies directed against KDR from mice immunized with the recombinant form receptor. These antibodies efficiently neutralized VEGF-induced KDR activation and mitogenesis of human umbilical vascular endothelial cells (HUVEC). Murine antibodies, however, may not be suitable candidates for human therapy because of their propensity to elicit human anti-mouse antibody response. Here we isolated several high-affinity human Fab antibody fragments directed against KDR from an antibody phage display library constructed from the pooled B lymphocytes of nonimmunized healthy human donors. These human Fab fragments bind specifically to KDR with nanomolar affinity and block KDR/VEGF interaction with IC(50) of approximately 2-20 nM. Further, they effectively inhibit VEGF-stimulated mitogenesis of HUVEC and migration of human leukemia cells. Epitope mapping studies demonstrated that all neutralizing human antibodies bound the epitope(s) located within the first 3 N-terminal immunoglobulin-like domains of KDR, the same region that encompasses the binding site of VEGF. Our results suggest that these human anti-KDR antibodies may have potential application in the treatment of cancer and other diseases in which pathologic angiogenesis occurs.     

Tumor angiogenesis suppression by alpha-eleostearic acid, a linolenic acid isomer with a conjugated triene system, via peroxisome proliferator-activated receptor gamma

We have shown previously that alpha-eleostearic acid (ESA), a linolenic acid isomer with a conjugated triene system, suppresses tumor growth in vivo. In our earlier study, blood vessels were observed at the tumor surface in control mice, whereas in ESA-treated mice no such vessels were observed and the inner part of the tumor was discolored. These observations suggested that ESA might suppress cancer cell growth through malnutrition via a suppressive effect on tumor angiogenesis. In the current study, the antiangiogenic effects of ESA were investigated in vivo and in vitro. Tumor cell-induced vessel formation was clearly suppressed in mice orally administered ESA at doses of 50 and 100 mg/kg/day in a dose-dependent manner. ESA also inhibited the formation of capillary-like networks by human umbilical vein endothelial cells (HUVEC) and moderately inhibited HUVEC proliferation and migration in a dose-dependent manner. The mechanism by which ESA inhibited angiogenesis was through suppression of the expression of vascular endothelial growth factor receptors 1 and 2, activation of peroxisome proliferator-activated receptor gamma (PPARgamma) and induction of apoptosis in HUVEC. We thus demonstrated that, like troglitazone, ESA is a PPARgamma ligand and that it activates PPARgamma, induces apoptosis in HUVEC and inhibits angiogenesis. Our findings suggest that ESA has potential use as a therapeutic dietary supplement and medicine for minimizing tumor angiogenesis.     

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。     

Antiangiogenic mechanisms of PJ-8, a novel inhibitor of vascular endothelial growth factor receptor signaling

Angiogenesis occurs not only during tissue growth and development but also during wound healing and tumor progression. Angiogenesis is a balanced process controlled by proangiogenic and antiangiogenic molecules. As a critical factor in the induction of angiogenesis, vascular endothelial growth factor (VEGF) has become an attractive target for antiangiogenic and cancer therapeutic agents. In an effort to develop novel inhibitors to block VEGF signaling, we selected Pj-8, a benzimidazole derivative, and investigated its inhibitory mechanisms in human umbilical vascular endothelial cells (HUVECs). Pj-8 concentration-dependently inhibited VEGF-induced proliferation, migration and tube formation of HUVECs. Pj-8 also suppressed VEGF-induced microvessel sprouting from aortic rings ex vivo and suppressed neovascularization of implanted matrigel plugs in vivo. Pj-8 inhibited VEGF-induced phosphorylation of VEGF receptor (VEGFR) 2 and the downstream protein kinases, including Akt, focal adhesion kinase, extracellular signal-regulated kinases and Src. Results from in vitro kinase assay further demonstrated that Pj-8 suppressed the kinase activity of 3-phosphoinositide-dependent kinase 1 (PDK1). Using xenograft tumor angiogenesis model, Pj-8 markedly eliminated tumor-associated angiogenesis. Taken together, our findings suggest that Pj-8 inhibits VEGF and tumor cells MDA-MB-231-induced angiogenesis, and it may be a potential drug candidate in anticancer therapy. Downregulation of VEGFR2-mediated signaling may contribute to its antiangiogenic actions.     

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.     

Administration of VEGF receptor tyrosine kinase inhibitor increases VEGF production causing angiogenesis in human small-cell lung cancer xenografts

Angiogenesis is mediated mainly by vascular endothelial growth factor (VEGF), and VEGF causes rapid growth in cancers, including human small-cell lung cancer (SCLC). The anti-angiogenic strategy of treating cancer using VEGF receptor (VEGFR) inhibition is currently of great interest. We tested the effects of the VEGFR2 tyrosine kinase inhibitor (TKI) vandetanib on the proliferation of two kinds of SCLC cell lines: SBC-1 cells, with detectable VEGFR2 expression and MS-1-L cells, without detectable VEGFR2 expression. To evaluate the anti-tumor and anti-angiogenic effects of vandetanib in vivo, we grafted SBC-1 and MS-1-L cells into mice. After a 3-week treatment, we measured the tumor size and histologically evaluated necrosis and apoptosis using H&E and TUNEL staining, respectively. The microvessels in the xenografts were also quantified by immunostaining of CD31. Vandetanib did not affect the proliferation of SBC-1 cells, but stimulated the growth of MS-1-L cells. In the SCLC xenograft model, vandetanib inhibited growth and tumor angiogenesis in a dose-dependent manner in SBC-1 xenografts. Vandetanib inhibited the growth of MS-1-L xenografts at a low dose (<12.5 mg/kg/day), but it did not affect tumor size or change microvessel counts at a higher dose. Interestingly, secretion of VEGF increased significantly in the MS-1-L cell line in the presence of a high dose of vandetanib in vitro. The effects of vandetanib on tumor angiogenesis were different in SBC-1 and MS-1-L cell lines. Production of angiogenic factors such as VEGF by the tumor potentially stimulates tumor angiogenesis and results in the acquisition of resistance to VEGFR TKI.     

FTY720 enhances TRAIL-mediated apoptosis by up-regulating DR5 and down-regulating Mcl-1 in cancer cells

FTY720, Fingolimod, is a functional antagonist to the sphingosine-1-phoaphate (S1P) receptor and an inhibitor of sphingosine kinase 1. Here, we showed that a combination of FTY720 and TRAIL induced apoptosis in human renal, breast, and colon carcinoma cells. Most importantly, this combination had no effect on normal cells. Furthermore, the combined treatment with FTY720 and TRAIL reduced tumor growth in xenograft models. FTY720 up-regulated death receptor (DR)5 at post-translational level. Knockdown of DR5 markedly blocked apoptosis induced by the combined treatment. FTY720 also inhibited Mcl-1 expression at the post-translational level. Over-expression of Mcl-1 blocked apoptosis induced by FTY720 and TRAIL. Interestingly, phospho-FTY720 and inhibitors of sphingosine kinase failed to enhance TRAIL-induced apoptosis. Thus, FTY720 enables TRAIL-induced apoptosis through up-regulation of DR5 and down-regulation of Mcl-1 in human cancer cells.     

YM-359445, an orally bioavailable vascular endothelial growth factor receptor-2 tyrosine kinase inhibitor, has highly potent antitumor activity against established tumors.

PURPOSE: The vascular endothelial growth factor receptor-2 (VEGFR2) tyrosine kinase has been implicated in the pathologic angiogenesis associated with tumor growth. YM-359445 was a (3Z)-3-quinolin-2(1H)-ylidene-1,3-dihydro-2H-indol-2-one derivative found while screening based on the inhibition of VEGFR2 tyrosine kinase. The aim of this study was to analyze the efficacy of this compound both in vitro and in vivo. EXPERIMENTAL DESIGN: We tested the effects of YM-359445 on VEGFR2 tyrosine kinase activity, cell proliferation, and angiogenesis. The antitumor activity of YM-359445 was also tested in nude mice bearing various established tumors and compared with other VEGFR2 tyrosine kinase inhibitors (ZD6474, CP-547632, CGP79787, SU11248, and AZD2171), a cytotoxic agent (paclitaxel), and an epidermal growth factor receptor tyrosine kinase inhibitor (gefitinib). RESULTS: The IC50 of YM-359445 for VEGFR2 tyrosine kinase was 0.0085 micromol/L. In human vascular endothelial cells, the compound inhibited VEGF-dependent proliferation, VEGFR2 autophosphorylation, and sprout formation at concentrations of 0.001 to 0.003 micromol/L. These concentrations had no direct cytotoxic effect on cancer cells. In mice bearing various established tumors, including paclitaxel-resistant tumors, once daily oral administration of YM-359445 at doses of 0.5 to 4 mg/kg not only inhibited tumor growth but also reduced its vasculature. YM-359445 had greater antitumor activity than other VEGFR2 tyrosine kinase inhibitors. Moreover, in human lung cancer A549 xenografts, YM-359445 markedly regressed the tumors (73%) at a dose of 4 mg/kg, whereas gefitinib caused no regression even at 100 mg/kg. CONCLUSION: Our results show that YM-359445 is more potent than orally bioavailable VEGFR2 tyrosine kinase inhibitors, which leads to great expectations for clinical applicability.     

Augmentation of radiation response with the vascular targeting agent ZD6126

PURPOSE: To examine the antivascular and antitumor activity of the vascular targeting agent ZD6126 in combination with radiation in lung and head-and-neck (H and N) cancer models. The overall hypothesis was that simultaneous targeting of tumor cells (radiation) and tumor vasculature (ZD6126) might enhance tumor cell killing. METHODS AND MATERIALS: A series of in vitro studies using human umbilical vein endothelial cells (HUVEC) and in vivo studies in athymic mice bearing human lung (H226) and H and N (squamous cell carcinoma [SCC]1, SCC6) tumor xenografts treated with ZD6126 and/or radiation were performed. RESULTS: ZD6126 inhibited the capillary-like network formation in HUVEC. Treatment of HUVEC with ZD6126 resulted in cell cycle arrest in G2/M, with decrease of cells in S phase and proliferation inhibition in a dose-dependent manner. ZD6126 augmented the cell-killing effect of radiation and radiation-induced apoptosis in HUVEC. The combination of ZD6126 and radiation further decreased tumor vascularization in an in vivo Matrigel angiogenesis assay. In tumor xenografts, ZD6126 enhanced the antitumor activity of radiation, resulting in tumor growth delay. CONCLUSIONS: These preclinical studies suggest that ZD6126 can augment the radiation response of proliferating endothelial H and N and lung cancer cells. These results complement recent reports suggesting the potential value of combining radiation with vascular targeting/antiangiogenic agents.     

Epoxyquinol B shows antiangiogenic and antitumor effects by inhibiting VEGFR2, EGFR, FGFR, and PDGFR

Angiogenesis is the development of new blood vessels to provide oxygen and nutrients and is indispensable for solid tumor growth. Therefore, the inhibition of angiogenesis is an important modality for cancer chemotherapy. Here we report the antiangiogenic mechanism and antitumor effects of epoxyquinol B (EPQB), which was isolated from fungal metabolites. Short-term treatment of EPQB resulted in the reduction of tumor growth and the number of blood vessels directed to the tumor in a murine xenografts model. Furthermore, EPQB inhibited vascular endothelial growth factor (VEGF)-induced migration and tube formation in human umbilical vein endothelial cells (HUVECs) without cytotoxicity. VEGF-stimulated phosphorylation of VEGF receptor 2 (VEGFR2), phospholipase Cgamma-1 (PLCgamma1), and p44/42 MAP kinases (ERK) was inhibited by EPQB in a dose-dependent manner, and in vitro assay using kinase domain of VEGFR2 showed that EPQB covalently bound and inhibited the VEGFR2 kinase. Its binding site on VEGFR2 was different from SU5614, a well-known VEGFR2 kinase inhibitor. Interestingly, EPQB inhibited growth factor-induced activation of not only VEGFR2 but also epidermal growth factor receptor (EGFR), fibroblast growth factor receptor (FGFR), and platelet-derived growth factor receptor (PDGFR), suggesting that EPQB is a novel multiple kinase inhibitor. These findings suggest that EPQB would be a good lead compound for the development of potent antiangiogenic and antitumor drugs.     

Mechanism of inhibition of tumor angiogenesis by β-hydroxyisovalerylshikonin

Shikonin and β-hydroxyisovalerylshikonin (β-HIVS) from Lithospermum erythrorhizon inhibit angiogenesis via inhibition of vascular endothelial growth factor receptors (VEGFR) in an adenosine triphosphate-non-competitive manner, although the underlying molecular mechanism has not been fully understood. In the present study, we found that β-HIVS inhibited angiogenesis within chicken chorioallantoic membrane approximately threefold more efficiently than shikonin. β-HIVS also significantly inhibited angiogenesis in two other assays, induced either by Lewis lung carcinoma cells implanted in mouse dorsal skin or by VEGF in s.c. implanted Matrigel plugs and metastasis of Lewis lung carcinoma cells to lung. Therefore, using β-HIVS as a bioprobe, we investigated the molecular mechanism of shikonin's anti-angiogenic actions. β-HIVS inhibited the phosphorylation and expression of VEGFR2 and Tie2 without affecting VEGFR1 and fibroblast growth factor receptor 1 levels. β-HIVS suppressed the phosphorylation but not the expression of extracellular signal-regulated kinase, and an Sp1-dependent transactivation of the VEGFR2 and Tie2 promoters, thereby suppressing the proliferation of vascular endothelial and progenitor cells. This was mimicked by an Sp1 inhibitor mithramycin A and partially rescued by Sp1 overexpression. These results implicate potential use of shikonin and β-HIVS as leading compounds for clinical application in the future by virtue of their unique properties including: (i) inhibition of VEGFR2 and Tie2 phosphorylation in an adenosine triphosphate-non-competitive manner; (ii) simultaneous inhibition of the phosphorylation and expression of VEGFR2 and Tie2; and (iii) bifunctional inhibition of the growth in endothelial cells and vascular remodeling. ( Cancer Sci 2009; 100: 269–277)     

Rationale for antiangiogenic cancer therapy with vaccination using epitope peptides derived from human vascular endothelial growth factor receptor 2

Angiogenesis is a critical mechanism for tumor progression. Multiple studies have suggested that tumor growth can be suppressed if tumor angiogenesis can be inhibited using various types of antiangiogenic agents. Recent studies in mouse systems have shown that tumor angiogenesis can also be inhibited if cellular immune response could be induced against vascular endothelial growth factor receptor 2 (VEGFR2), which is one of the key factors in tumor angiogenesis. In this study, we examined the possibility of developing this novel immunotherapy in clinical setting. We first identified the epitope peptides of VEGFR2 and showed that stimulation using these peptides induces CTLs with potent cytotoxicity in the HLA class I-restricted fashion against not only peptide-pulsed target cells but also endothelial cells endogenously expressing VEGFR2. In A2/Kb transgenic mice that express alpha1 and alpha2 domains of human HLA-A*0201, vaccination using these epitope peptides in vivo was associated with significant suppression of the tumor growth and prolongation of the animal survival without fatal adverse effects. In antiangiogenesis assay, tumor-induced angiogenesis was significantly suppressed with the vaccination using these epitope peptides. Furthermore, CTLs specific to the epitope peptides were successfully induced in cancer patients, and the specificities of the CTLs were confirmed using functional and HLA-tetramer analysis. These results in vitro and in vivo strongly suggest that the epitope peptides derived from VEGFR2 could be used as the agents for antiangiogenic immunotherapy against cancer in clinical settings.     

Bortezomib potentially inhibits cellular growth of vascular endothelial cells through suppression of G2/M transition

Bortezomib, a selective 26S proteasome inhibitor, has shown clinical benefits against refractory multiple myeloma. The indirect anti‐angiogenic activity of bortezomib has been widely recognized; however, the growth‐inhibitory mechanism of bortezomib on vascular endothelial cells remains unclear, especially on the cell cycle. Here, we showed that bortezomib (2 nM of the IC₅₀ value) potently inhibited the cellular growth of human umbilical vascular endothelial cells (HUVECs) via a vascular endothelial growth factor receptor (VEGFR)‐independent mechanism resulting in the induction of apoptosis. Bortezomib significantly increased the vascular permeability of HUVECs, whereas a VEGFR‐2 tyrosine kinase inhibitor decreased it. Interestingly, a cell cycle analysis using flow cytometry, the immunostaining of phospho‐histone H3, and Giemsa staining revealed that bortezomib suppressed the G2/M transition of HUVECs, whereas the mitotic inhibitor paclitaxel induced M‐phase accumulation. A further analysis of cell cycle‐related proteins revealed that bortezomib increased the expression levels of cyclin B1, the cdc2/cyclin B complex, and the phosphorylation of all T14, Y15, and T161 residues on cdc2. Bortezomib also increased the ubiquitination of cyclin B1 and wee1, but inhibited the kinase activity of the cdc2/cyclin B complex. These protein modifications support the concept that bortezomib suppresses the G2/M transition, rather than causing M‐phase arrest. In conclusion, we demonstrated that bortezomib potently inhibits cell growth by suppressing the G2/M transition, modifying G2/M‐phase‐related cycle regulators, and increasing the vascular permeability of vascular endothelial cells. Our findings reveal a cell cycle‐related mode of action and strongly suggest that bortezomib exerts an additional unique vascular disrupting effect as a vascular targeting drug. (Cancer Sci 2010)     

The tyrosine kinase inhibitor cediranib blocks ligand-induced vascular endothelial growth factor receptor-3 activity and lymphangiogenesis

Solid tumors express a range of factors required to sustain their growth and promote their dissemination. Among these are vascular endothelial growth factor-A (VEGF-A), the key angiogenic stimulant, and VEGF-C, a primary mediator of lymphangiogenesis. Small molecule tyrosine kinase inhibitors offer the potential to inhibit more than one kinase and impede tumor growth by multiple mechanisms. However, their potency toward individual targets can vary. Cediranib (RECENTIN; AZD2171) is an inhibitor of VEGF signaling that has been shown in experimental models to prevent VEGF-A-induced angiogenesis and primary tumor growth, yet the effects of cediranib on VEGF receptor (VEGFR)-3-mediated endothelial cell function and lymphangiogenesis are unknown. To better understand the activity of cediranib against VEGFR-3 and its associated signaling events compared with its activity against VEGFR-2, we used the receptor-specific ligands VEGF-E and VEGF-C156S. In human endothelial cells, cediranib inhibited VEGF-E-induced phosphorylation of VEGFR-2 and VEGF-C156S-induced phosphorylation of VEGFR-3 at concentrations of 相似文献   

The multi-targeted kinase inhibitor SU5416 inhibits small cell lung cancer growth and angiogenesis, in part by blocking Kit-mediated VEGF expression

SU5416 is a multi-targeted kinase inhibitor that potentially has the ability to directly block tumor growth by inhibiting Kit signaling, as well as blocking angiogenesis by inhibiting vascular endothelial growth factor receptor (VEGFR) signaling. Previous work has demonstrated that SU5416 efficiently blocks Kit-mediated growth of small cell lung cancer (SCLC) in vitro. To determine the drug's effect on in vivo growth of SCLC, we studied its activity, alone and in combination with carboplatin, in chemotherapy-resistant H526, and chemotherapy-sensitive H209 murine xenograft models. SU5416 efficiently inhibited Kit activity in vivo when administered on a twice-weekly schedule. When administered over a 3-week period to animals bearing established tumors, it inhibited growth by at least 70%. It was at least as effective as carboplatin in suppressing growth of H526 xenografts. However, the combination with carboplatin was not superior to the most active single agent in either xenograft model at the doses and schedule utilized. SU5416 clearly inhibited growth in part by inhibiting angiogenesis, with microvessel density dropping by approximately 50% in treated xenografts. In addition to the recognized mechanism of inhibition of VEGFR, we uncovered a novel mechanism of angiogenesis suppression by demonstrating reduced VEGF expression in SU5416-treated xenografts. In vitro, stem cell factor treatment of the H526 cell line enhanced expression of VEGF, which was efficiently blocked with SU5416. Thus, we have demonstrated that SU5416 can inhibit SCLC growth by directly inhibiting tumor cell proliferation and by inhibiting angiogenesis, in part by inhibiting Kit-mediated VEGF expression. These data suggest that kinase inhibitors that target both Kit and VEGFR could play an important role in the treatment of SCLC, as well as other malignancies that express Kit.     

Soluble Eph A receptors inhibit tumor angiogenesis and progression in vivo

The Eph family of receptor tyrosine kinases and their ligands, known as ephrins, play a crucial role in vascular development during embryogenesis. The function of these molecules in adult angiogenesis has not been well characterized. Here, we report that blocking Eph A class receptor activation inhibits angiogenesis in two independent tumor types, the RIP-Tag transgenic model of angiogenesis-dependent pancreatic islet cell carcinoma and the 4T1 model of metastatic mammary adenocarcinoma. Ephrin-A1 ligand was expressed in both tumor and endothelial cells, and EphA2 receptor was localized primarily in tumor-associated vascular endothelial cells. Soluble EphA2-Fc or EphA3-Fc receptors inhibited tumor angiogenesis in cutaneous window assays, and tumor growth in vivo. EphA2-Fc or EphA3-Fc treatment resulted in decreased tumor vascular density, tumor volume, and cell proliferation, but increased cell apoptosis. However, EphA2-Fc had no direct effect on tumor cell growth or apoptosis in culture, yet inhibited migration of endothelial cells in response to tumor cells, suggesting that the soluble receptor inhibited blood vessel recruitment by the tumor. These data provide the first functional evidence for Eph A class receptor regulation of pathogenic angiogenesis induced by tumors and support the function of A class Eph receptors in tumor progression.     

Regulatory effects of PTEN gene transfection on vascular endothelial growth factor (VEGF) in leukemia cells北大核心

Objective: To investigate the effects of wild type tumor-suppressing gene PTEN (phosphatase and tensin hemology deleted on chromosome ten gene) on vascular endothelial growth factor (VEGF) and VEGF receptor 1 (VEGFR1) in leukemia cells and elucidate the mechanism. Methods: The recombinant adenovirus containing wild type PTEN and green fluorescent protein (GFP) (Ad-PTEN-GFP) or empty vector (Ad-GFP) was transfected into K562 leukemia cells. PTEN, VEGF, and VEGFR1 mRNA expression levels were detected using quantitative PCR and the protein expression levels were detected using Western blotting. Transwell invasion test was used to examine the invasion ability of the leukemia cells. Human umbilical vein endothelial cells ECV304 were also transfected with or without PTEN gene. Then the proliferation and apoptosis were measured using MTT assay and flow cytometry (FCM), respectively. Chick chorioallantoic membrane (CAM) test was used to determine the effect of PTEN gene transfection on angiogenesis. Results: The expressions of VEGF and VEGFR1 were significantly inhibited after transfection of Ad-PTEN-GFP compared with the control group transfected with empty Ad-GFP. The inhibitory effects were in a dose-dependent manner. The invasion capability of K562 cells was markedly weakened after transfection of Ad-PTEN-GFP. PTEN gene transfection inhibited the proliferation and induced apoptosis of vascular endothelial ECV304 cells. The cells were arrested at S phase. CAM test showed that PTEN gene inhibited the angiogenesis in vivo. Conclusion: Tumor suppressor gene PTEN inhibited the growth of endothelial cells and the invasion of leukemia cells. The action mechanism may be related with down-regulation VEGF expression and angiogenesis of leukemia cells.