Antiangiogenic effect by SU5416 is partly attributable to inhibition of Flt-1 receptor signaling

Interaction between vascular endothelial growth factor (VEGF) and its cognate receptors, KDR/Flk-1 and Flt-1, of vascular endothelial cells is expected to induce an angiogenesis "switch" in tumors and other angiogenesis-associated diseases. SU5416, a selective inhibitor of the KDR/Flk-1 tyrosine kinase, is known to be a potent inhibitor of tumor angiogenesis. In this study, we first observed that SU5416 inhibited Flt-1 tyrosine kinase activity at similar doses, in addition to inhibiting KDR/Flk-1 tyrosine kinase activity in response to VEGF. SU5416 inhibited cell migration of human vascular endothelial cells expressing both Flt-1 and KDR in response to VEGF and also inhibited the cell migration in response to placenta growth factor (PIGF), a specific ligand for Flt-1. Chemotaxis of monocytes expressing only Flt-1 was also inhibited by SU5416 in a dose-dependent manner. Moreover, SU5416 was found to inhibit tyrosine kinase of Flt-1 in response to PIGF in vitro. And angiogenesis induced by PIGF in a Matrigel plug assay was inhibited by administration of SU5416. The antiangiogenic effects by this VEGF receptor-targeting compound appeared to be mediated through interference not only with KDR/Flk-1 but also with Flt-1. Cell migration of vascular endothelial cells and monocytic cells through Flt-1, thus, might play a key role in VEGF-induced tumor angiogenesis in concert with KDR/Flk-1.     

Contribution of individual targets to the antitumor efficacy of the multitargeted receptor tyrosine kinase inhibitor SU11248

Recent achievements in the development of multitargeted molecular inhibitors necessitate a better understanding of the contribution of activity against individual targets to their efficacy. SU11248, a small-molecule inhibitor targeting class III/V receptor tyrosine kinases, including the platelet-derived growth factor (PDGF) and vascular endothelial growth factor (VEGF) receptors, KIT and FLT3, exhibits direct effects on cancer cells as well as antiangiogenic activity. Here, we investigated the contributions of inhibiting individual SU11248 target receptors to its overall antitumor efficacy in tumor models representing diverse signaling paradigms. Consistent with previous results, SU11248 was highly efficacious (frequently cytoreductive) in all models tested. To elucidate the specific contributions of inhibition of PDGF and VEGF receptors to the in vivo efficacy of SU11248, we employed two selective inhibitors, SU10944 (VEGF receptor inhibitor) and Gleevec (PDGF receptor inhibitor). SU10944 alone induced a tumor growth delay in all models evaluated, consistent with a primarily antiangiogenic mode of action. In contrast, Gleevec resulted in modest growth inhibition in tumor models in which the cancer cells expressed its targets (PDGFRbeta and KIT), but was not efficacious against tumors not driven by these target receptor tyrosine kinases. Strikingly, in all but one tumor model evaluated, the antitumor efficacy of SU10944 combined with Gleevec was similar to that of single-agent SU11248, and was greatly superior to that of each compound alone, indicating that the antitumor potency of SU11248 in these models stems from combined inhibition of both PDGF and VEGF receptors. The one exception was a model driven by an activated mutant of FLT3, in which the activity of SU11248, which targets FLT3, was greater than that of SU10944 plus Gleevec. Moreover, SU10944 combined with Gleevec inhibited tumor neoangiogenesis to an extent comparable to that of SU11248. Thus, the potent efficacy of SU11248 in models representing diverse signaling paradigms results from simultaneous inhibition of individual target receptors expressed both in cancer cells and in the tumor neovasculature, supporting the hypothesis that multitargeted inhibitors have the cumulative antitumor efficacy of combined single-target inhibitors.     

血管内皮生长因子对神经细胞的保护作用及机制

目的 探讨VEGF对体外培养的神经元保护作用及机制。方法 体外培养大鼠皮质神经元细胞，建立神经细胞缺氧缺糖/再灌注损伤模型(OGSD)，在加入VEGF基础上，加入VEGFR-2/Flk-1受体抑制剂SU1498；检测以下指标：①观察细胞形态学变化②细胞活力：MTT法③线粒体跨膜电位④凋亡相关蛋白：Bc1-2和Bax表达。结果 ①OGSD组、VEGF164+SU1498组细胞损害重，VEGF组细胞损害轻(p<0.05)；②OGSD组、VEGF164+SU1498组细胞活力下降明显(p<0.05)，而VEGF组与正常组无明显区别；③OGSD组、VEGF164+SU1498组线粒体跨膜电位不稳定(p<0.05)，VEGF能稳定线粒体跨膜电位；④OGSD组、VEGF164+SU1498组Bcl- 2、Bax蛋白表达均增多，VEGF组Bcl- 2蛋白表达增多最明显(p<0.01)，而Bax蛋白表达增多最不明显(p<0.01)。结论 VEGF对神经元起直接的保护作用，并通过与VEGFR-2受体结合减少了缺血、缺氧下培养的体外神经元细胞的凋亡。     

SU11248 inhibits KIT and platelet-derived growth factor receptor beta in preclinical models of human small cell lung cancer

The purpose of this study was to evaluate the activity of the indolinone kinase inhibitor SU11248 against the receptor tyrosine kinase KIT in vitro and in vivo, examine the role of KIT in small cell lung cancer (SCLC), and anticipate clinical utility of SU11248 in SCLC. SU11248 is an oral, multitargeted tyrosine kinase inhibitor with direct antitumor and antiangiogenic activity through targeting platelet-derived growth factor receptor (PDGFR), vascular endothelial growth factor receptor, KIT, and FLT3 receptors. Treatment of the KIT-expressing SCLC-derived NCI-H526 cell line in vitro with SU11248 resulted in dose-dependent inhibition of stem cell factor-stimulated KIT phosphotyrosine levels and proliferation. The biological significance of KIT inhibition was evaluated in vivo by treating mice bearing s.c. NCI-H526 tumors with SU11248 or another structurally unrelated KIT inhibitor, STI571 (Gleevec), which is also known to inhibit Bcr-Abl and PDGFRbeta. SU11248 treatment resulted in significant tumor growth inhibition, whereas inhibition from STI571 treatment was less dramatic. Both compounds reduced phospho-KIT levels in NCI-H526 tumors, with a greater reduction by SU11248, correlating with efficacy. Likewise, phospho-PDGFRbeta levels contributed by tumor stroma and with known involvement in angiogenesis were strongly inhibited by SU11248 and less so by STI571. Because platinum-based chemotherapy is part of the standard of care for SCLC, SU11248 was combined with cisplatin, and significant tumor growth delay was measured compared with either agent alone. These results expand the profile of SU11248 as a KIT signaling inhibitor and suggest that SU11248 may have clinical potential in the treatment of SCLC via direct antitumor activity mediated via KIT as well as tumor angiogenesis via vascular endothelial growth factor receptor FLK1/KDR and PDGFRbeta.     

Antiangiogenic drugs for chemotherapy of bladder tumours

BACKGROUND: Bladder cancers have different angiogenic pathways distinguishing not only papillary from solid tumours, but even papillary superficial from papillary invasive ones, thus representing selective targets for antiangiogenic drugs. METHODS: The bacterial wall component tecogalan, inhibiting basic fibroblast growth factor (bFGF), the fumagillin derivative TNP-470, inhibiting vascular endothelial growth factor (VEGF), the distamycin A derivative PNU153429, and the tetracycline minocycline were administered to nude mice injected with the human bladder cancer cell lines 639V, causing bFGF-expressing papillary superficial tumours, or T24, causing VEGF-expressing papillary invasive tumours. RESULTS: Tecogalan had no effect even on 639V tumour growth, where bFGF was unaffected. TNP-470 only had an effect on T24 tumours, delaying tumour appearance and growth and lowering VEGF; these effects were augmented by adding minocycline. PNU153429 had no effect on 639V tumours, and a slight effect on T24 tumours. CONCLUSION: TNP-470 may represent a selective drug for the treatment of VEGF-expressing invasive papillary bladder tumours.     

Gene therapy for malignant liver disease

For most patients with advanced or multifocal hepatocellular carcinoma (HCC) or with metastatic malignant liver disease treatment options are limited, resulting in a poor prognosis. Novel therapeutic strategies such as gene therapy are therefore urgently required. Gene therapeutic approaches use gene delivery systems (vectors) to introduce DNA constructs as therapeutic agents into living cells. Antitumour strategies include the reintroduction of tumour suppressor genes into tumour cells, the expression of foreign enzymes to render tumours susceptible to treatment with chemotherapeutic agents and the enhancement of tumour immunogenicity by expressing immunomodulatory genes or by genetic vaccination with tumour antigens. Furthermore, gene therapy may be also used for anti-angiogenesis to reduce tumour growth and metastatic potential. Other novel approaches aim at the development of genetically altered replication competent viruses, which selectively replicate in tumour cells inducing cell lysis. Although most clinical trials of antitumour gene therapy so far have failed to induce strong therapeutic effects, further improvement of antitumour gene therapy may finally result in potent clinical treatment options for patients with malignant liver tumours.     

Benefits of targeting both pericytes and endothelial cells in the tumor vasculature with kinase inhibitors

Functions of receptor tyrosine kinases implicated in angiogenesis were pharmacologically impaired in a mouse model of pancreatic islet cancer. An inhibitor targeting VEGFRs in endothelial cells (SU5416) is effective against early-stage angiogenic lesions, but not large, well-vascularized tumors. In contrast, a kinase inhibitor incorporating selectivity for PDGFRs (SU6668) is shown to block further growth of end-stage tumors, eliciting detachment of pericytes and disruption of tumor vascularity. Importantly, PDGFRs were expressed only in perivascular cells of this tumor type, suggesting that PDGFR(+) pericytes in tumors present a complimentary target to endothelial cells for efficacious antiangiogenic therapy. Therapeutic regimes combining the two kinase inhibitors (SU5416 and SU6668) were more efficacious against all stages of islet carcinogenesis than either single agent. Combination of the VEGFR inhibitor with another distinctive kinase inhibitor targeting PDGFR activity (Gleevec) was also able to regress late-stage tumors. Thus, combinatorial targeting of receptor tyrosine kinases shows promise for treating multiple stages in tumorigenesis, most notably the often-intractable late-stage solid tumor.     

Preclinical evaluation of the tyrosine kinase inhibitor SU11248 as a single agent and in combination with "standard of care" therapeutic agents for the treatment of breast cancer

SU11248 is an oral multitargeted tyrosine kinase inhibitor with antitumor and antiangiogenic activities through targeting platelet-derived growth factor receptor, vascular endothelial growth factor receptor, KIT, and FLT3, the first three of which are expressed in human breast cancer and/or its supporting tissues. The purpose of the present studies was to demonstrate the potent anticancer activity of SU11248 alone or in combination with conventional cytotoxic agents against several distinct preclinical models of breast cancer. SU11248 was administered as a monotherapy to (1) mouse mammary tumor virus-v-Ha-ras mice and 7,12-dimethylbenz(a)anthracene-treated rats bearing mammary tumors and (2) mice bearing human breast cancer xenografts of s.c. MX-1 tumors and osseous metastasis of a MDA-MB-435-derived cell line (435/HAL-Luc). SU11248 was also administered in combination with docetaxel both in xenograft models and in combination with 5-fluorouracil and doxorubicin in the MX-1 model. SU11248 treatment potently regressed growth of mammary cancers in mouse mammary tumor virus-v-Ha-ras transgenic mice (82% regression) and 7,12-dimethylbenz(a)anthracene-induced mammary tumors in rats (99% regression at the highest dose; P < 0.05 for both). This agent also inhibited MX-1 tumor growth by 52%, with markedly enhanced anticancer effects when administered in combination with docetaxel, 5-fluorouracil, or doxorubicin compared with either agent alone (P < 0.05). SU11248 treatment in combination with docetaxel effectively prolonged survival of mice, with 435/HAL-Luc cancer xenografts established in bone compared with either agent alone (P < 0.05). These results demonstrate that SU11248 is effective in preclinical breast cancer models and suggest that it may be useful in the treatment of breast cancer in the clinic.     

Activated VEGFR2/KDR pathway in tumour cells and tumour associated vessels of colorectal cancer

BACKGROUND: Vascular endothelial cell growth factor (VEGF) acts by phosphorylating specific tyrosine kinase receptors on endothelial cell membrane promoting angiogenesis. The study of the activation status of VEGF receptors in human malignancies has recently become feasible by means of specific monoclonal antibodies recognising the phosphorylated form of these receptors. MATERIALS AND METHODS: In the current study, we investigate the expression of the phosphorylated VEGFR2/KDR receptor in normal colon and colorectal adenocarcinomas in parallel with histopathological parameters, prognosis and the expression of the 'hypoxia inducible factor' HIF1alpha. RESULTS: pVEGFR2/KDR was weakly expressed in the normal colon, but it was expressed strongly in the cytoplasm and nuclei of cancer cells and in the tumour associated vasculature, mainly at the invading tumour edge. pVEGFR2/KDR expression in cancer cells was significantly associated with a tumour diameter > 6 cm (P = 0.04), poor histological differentiation (P = 0.004) and with high CEF1alpha expression (P = 0.05). High pVEGFR2/KDR expressing vascular density was significantly related with a high VEGF and HIF1alpha expression in cancer cells (P = 0.02 and 0.03, respectively). This was also related significantly to high pVEGFR2/KDR expression in cancer cells. In multivariate analysis, the most significant predictors for death were lympho-vascular invasion (P < 0.001) followed by VEGF (P = 0.014), node status (P = 0.015), standard vascular density (P = 0.022) and necrosis (P = 0.032). CONCLUSIONS: pVEGFR2 receptors are largely expressed in colon cancer cells and intratumoural vasculature. As VEGF targeting agents enter the clinical practice, the role of monoclonal antibodies recognising the phosphorylated form of VEGF receptors as predictors of response to targeted therapies should be sought in clinicopathological trials.     

The vascular endothelial growth factor (VEGF)/VEGF receptor system and its role under physiological and pathological conditions

The VEGF (vascular endothelial growth factor) family and its receptors are essential regulators of angiogenesis and vascular permeability. Currently, the VEGF family consists of VEGF-A, PlGF (placenta growth factor), VEGF-B, VEGF-C, VEGF-D, VEGF-E and snake venom VEGF. VEGF-A has at least nine subtypes due to the alternative splicing of a single gene. Although the VEGF165 isoform plays a central role in vascular development, recent studies have demonstrated that each VEGF isoform plays distinct roles in vascular patterning and arterial development. VEGF-A binds to and activates two tyrosine kinase receptors, VEGFR (VEGF receptor)-1 and VEGFR-2. VEGFR-2 mediates most of the endothelial growth and survival signals, but VEGFR-1-mediated signalling plays important roles in pathological conditions such as cancer, ischaemia and inflammation. In solid tumours, VEGF-A and its receptor are involved in carcinogenesis, invasion and distant metastasis as well as tumour angiogenesis. VEGF-A also has a neuroprotective effect on hypoxic motor neurons, and is a modifier of ALS (amyotrophic lateral sclerosis). Recent progress in the molecular and biological understanding of the VEGF/VEGFR system provides us with novel and promising therapeutic strategies and target proteins for overcoming a variety of diseases.     

Inhibition of VEGF receptors causes lung cell apoptosis and emphysema

Pulmonary emphysema, a significant global health problem, is characterized by a loss of alveolar structures. Because VEGF is a trophic factor required for the survival of endothelial cells and is abundantly expressed in the lung, we hypothesized that chronic blockade of VEGF receptors could induce alveolar cell apoptosis and emphysema. Chronic treatment of rats with the VEGF receptor blocker SU5416 led to enlargement of the air spaces, indicative of emphysema. The VEGF receptor inhibitor SU5416 induced alveolar septal cell apoptosis but did not inhibit lung cell proliferation. Viewed by angiography, SU5416-treated rat lungs showed a pruning of the pulmonary arterial tree, although we observed no lung infiltration by inflammatory cells or fibrosis. SU5416 treatment led to a decrease in lung expression of VEGF receptor 2 (VEGFR-2), phosphorylated VEGFR-2, and Akt-1 in the complex with VEGFR-2. Treatment with the caspase inhibitor Z-Asp-CH(2)-DCB prevented SU5416-induced septal cell apoptosis and emphysema development. These findings suggest that VEGF receptor signaling is required for maintenance of the alveolar structures and, further, that alveolar septal cell apoptosis contributes to the pathogenesis of emphysema.     

Angiogenesis in cancer. Basic mechanisms and therapeutic advances

Etiological concepts on cancer development, malignant growth and tumour propagation have undergone a revolutionary development during recent years: Among other aspects, the discovery of angiogenesis - the growth of new blood vessels from pre-existing vasculature - as a key element in the pathogenesis of malignancy has opened an abundance of biologic insights and subsequent therapeutic options, which have led to improved prognosis in many cancers including those originating from colon, lung, breast and kidney. Thereby, targeting the major pro-angiogenic stimulus vascular endothelial growth factor (VEGF) became the focus for therapeutic interventions. However, the use of VEGF-targeting drugs has been shown to be of limited efficacy, which might lie in the fact that tumor angiogenesis is mediated by a variety of different subcellular systems. This review focuses on the basic mechanisms involved in angiogenesis, which potentially represent novel targets for pharmacological agents in the treatment of malignancies.     

VEGF as an inhibitor of tumor vessel maturation: implications for cancer therapy

Tumor angiogenesis is an important component of cancer biology driven in part by the thesis that inhibition of tumor vessel growth would be expected to starve and thereby disrupt tumor growth. A significant portion of research on tumor angiogenesis has focused on VEGF and its blockade with the expectation that dramatic results would be demonstrated in cancer patients as previously documented in animal models. However, to date, anti-VEGF (bevacizumab, Avastin) therapy alone has demonstrated little if any antitumor activity or survival benefit in humans. Interestingly, bevacizumab in combination with chemotherapeutic agents appears to result in a modest survival benefit in patients with various malignancies. This has prompted the re-evaluation of the biological effects resulting from VEGF blockade. Recent reports indicate that inhibition of VEGF and its receptor can have dramatic and unexpected effects on mural and perivascular cells in the tumor microenvironment, leading to vascular smooth muscle cell/pericyte activation and vessel normalization/maturation. Specifically, when VEGF levels in tumors are high, recent studies suggest that this leads to reduced responsiveness of the mural cell to the related growth factor, platelet-derived growth factor. This raises the possibility that in addition to the demonstrated anti-angiogenic effect of VEGF neutralization, mural cell recruitment and thus vascular maturation might be among the most critical activities of anti-VEGF agents. This review seeks to explore how VEGF blockade impacts tumor vascular maturation and considers its implications for cancer therapy.     

Manipulating the angiotensin system--new approaches to the treatment of solid tumours

Angiotensin II (Ang II), a main effector peptide in the renin-angiotensin system (RAS), plays a fundamental role as a vasoconstrictor in controlling cardiovascular function and renal homeostasis. Ang II also acts as a growth promoter or angiogenic factor via type 1 angiotensin II receptors (AT1Rs) in certain tumour cell lines. Recent studies have shown the activation of the local RAS in various tumour tissues, including the abundant generation of Ang II by angiotensin-converting enzyme (ACE) and the upregulation of AT1R expression. Thus, considerable attention has been paid to the role of the RAS in cancer and its blockade as a new approach to the treatment of cancer. There is increasing evidence that the Ang II-AT1R system is involved in tumour growth, angiogenesis and metastasis in experimental models, suggesting the therapeutic potential of an ACE inhibitor and AT1R blocker, both of which have been used as antihypertensive drugs. In addition, specific Ang II-degrading enzymes are expressed in tumours and play a regulatory role in cell proliferation and invasion. This review focuses on the role of the Ang II-AT1R system in solid tumours, particularly in the progression of gynaecological cancer, and presents the clinical potential of manipulating the angiotensin system as a novel and promising strategy for cancer treatment.     

Protein tyrosine kinase inhibitors as novel therapeutic agents.

Protein tyrosine kinases (PTKs) play a key role in normal cell and tissue development. Enhanced PTK activity is intimately correlated with proliferative diseases, such as cancers, leukemias, psoriasis, and restenosis. This realization prompted us to systematically synthesize tyrosine phosphorylation inhibitors (tyrphostins) as potential drugs. Over the years, we have demonstrated the ability to synthesize selective tyrphostins aimed at different receptor, as well as at nonreceptor, tyrosine kinases. Some of these tyrphostins have shown efficacy in vivo as antileukemic agents and antirestenosis agents. AG 490, a Jak-2 inhibitor, is potent against recurrent pre-B acute lymphoblastic leukemia. AG 1295, a selective platelet-derived growth factor receptor kinase inhibitor, inhibits 50% of balloon injury-induced stenosis in the phemoral arteries of pigs. AG 1517 (SU 5271), a potent epiderminal growth factor receptor kinase inhibitor, is currently in clinical trials for psoriasis. Similarly, SU 5416, a potent kinase inhibitor of the vascular endothelial growth factor receptor/kinase domain receptor/Flk-1, is currently in clinical trials as an anticancer agent by virtue of its strong anti-angiogenic activity. These findings demonstrate that the identification of PTKs that play a key role in a defined disease state can lead to a selective drug. Tyrphostins also show efficacy in vivo in inflammatory diseases such as sepsis, cirrhosis, and experimental autoimmune encephalitis.     

A selective and oral small molecule inhibitor of vascular epithelial growth factor receptor (VEGFR)-2 and VEGFR-1 inhibits neovascularization and vascular permeability

Vascular endothelial growth factor (VEGF) is a key driver of the neovascularization and vascular permeability that leads to the loss of visual acuity in diabetic retinopathy and neovascular age-related macular degeneration. Our aim was to identify an orally active, selective small molecule kinase inhibitor of vascular endothelial growth factor receptor (VEGFR)-2 with activity against both VEGF-induced angiogenesis and vascular permeability. We used a biochemical assay to identify 3-[5-methyl-2- (2-oxo-1,2-dihydro-indol-3-ylidenemethyl)-1H-pyrrol-3-yl]-proprionic acid (SU10944), a pyrrole indolinone, which is a potent ATP-competitive inhibitor of VEGFR-2 (Ki of 21 +/- 5 nM). In cellular assays, SU10944 inhibited VEGF-induced receptor autophosphorylation (IC50 of 227 +/- 80 nM) as well as downstream signaling (IC50 of 102 +/- 27 nM). In biochemical assays, SU10944 exhibits potent inhibitory activity against VEGFR-1; weak activity against other related subgroup members, including stem cell factor receptor (SCFR), platelet-derived growth factor receptor beta (PDGFRbeta), and fibroblast growth factor receptor-1 (FGFR-1); and no detectable activity against other protein tyrosine kinases such as epidermal growth factor receptor (EGFR), Src, and hepatocyte growth factor receptor. In cellular assays, the selectivity for SU10944 to inhibit VEGFR is maintained compared with other tyrosine kinases (IC50 for SCFR of 1.6 +/- 0.3 microM, for PDGFRbeta of 30.6 +/- 13.3 microM, for FGFR-1 of >50 microM, and for EGFR of >50 microM). Upon oral administration, SU10944 gave a clear dose response in the corneal micropocket model with an ED50 value for inhibition of neovascularization of approximately 30 mg/kg and a maximum inhibition of 95% at 300 mg/kg. Similarly, upon oral administration in the Miles assay, SU10944 potently inhibited VEGF-induced vascular permeability. Our data indicate that small molecule inhibitors of VEGFR signaling have the potential to ameliorate VEGF-induced neovascularization as well as vascular permeability.     

Hair depigmentation is a biological readout for pharmacological inhibition of KIT in mice and humans

Deregulated activation of the KIT receptor tyrosine kinase has been implicated in several human cancers and in inflammation, making it an attractive target for therapeutic intervention. Conversely, deficiencies in KIT signaling have been implicated in human and animal hair pigmentation disorders, reflecting a role for KIT in the development and function of melanocytes. The goal of this study was to explore the potential utility of hair depigmentation as a biological readout for systemic inhibition of KIT by SU11248 5-[5-fluoro-2-oxo-1,2-dihydroindol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3-carboxylic acid (2-diethylaminoethyl)amide), an oral multitargeted tyrosine kinase inhibitor with antitumor and antiangiogenic activity through targeting platelet-derived growth factor receptors, vascular endothelial growth factor receptors, KIT, and FLT3. Oral SU11248 treatment induced dose-dependent depigmentation of newly regrown hair in depilated C57BL/6 mice. Similar effects were seen after administration of a KIT-neutralizing antibody. SU11248-induced hair depigmentation was reversible with cessation of treatment. Histological and immunohistochemical evaluation of mouse skin samples supported these observations and revealed that SU11248 has no effect on levels of KIT-positive melanocytes associated with hair follicles, indicating that the inhibitory effect is at the level of melanocyte function rather than their development/survival. Similar hair depigmentation has been noted in several cancer patients receiving SU11248 in phase I trials. Strikingly, patient scalp hair exhibits bands of depigmentation and pigmentation that correspond, respectively, to periods of treatment and dosing rest periods. These data demonstrate that hair pigmentation can serve as a dose-dependent, dynamic, biological readout for KIT inhibition in mice, and, apparently, in humans.     

Detecting vascular changes in tumour xenografts using micro-ultrasound and micro-ct following treatment with VEGFR-2 blocking antibodies

Blockade of vascular endothelial growth factor (VEGF) binding to its receptors on endothelial cells has been shown preclinically to induce tumour growth inhibition. Using ultrasound biomicroscopy (UBM) or micro-ultrasound imaging and micro-computed tomography (micro-CT) analysis, we have examined the effects of DC101, a highly specific vascular endothelial growth factor receptor-2 (VEGFR-2)-targeting antibody, in inducing growth inhibition and functional vascular changes in established melanoma (MeWo) xenografts in mice. Postprocessing of UBM imaging loops for speckle variance was introduced to estimate the level of functional blood flow in tumours. Perfused tumour area visualized by speckle variance revealed decreased blood flow within 48 h after DC101 injection (control versus DC101: 1.90 +/- 0.25% versus 1.01 +/- 0.11%, p < 0.01) and following a 3-wk DC101 therapy (control versus DC101: 0.76 +/- 0.14% versus 0.45 +/- 0.05%, p = 0.04), suggesting that VEGFR-2 blockade mediates both early and long-term effects on tumour blood flow. The growth of xenografts was significantly inhibited after treating with DC101 for 3 wk compared with controls. In addition to UBM, we examined the tumour vasculature in three-dimension (3D) using contrast-enhanced Micro-CT imaging, which displayed a reduction in the number of tumour vessels following extended VEGFR-2 blockade (vascular density of control versus DC101: 48.4 +/- 5.4% versus 20.6 +/- 1.8%). Lastly, decreased microvessel density (MVD) was noted in DC101-treated xenografts (3 wk) by performing immunohistochemical staining of endothelial marker CD34. Our study investigates tumour response to DC101 using complementing micro-ultrasound and micro-CT imaging tools.     

Induction of vascular endothelial growth factor secretion by childhood acute lymphoblastic leukemia cells via the FLT-3 signaling pathway

Human leukemia cells secrete VEGF, which can act in a paracrine manner within the bone marrow microenvironment to promote leukemia cell survival and proliferation. The FLT-3 receptor tyrosine kinase plays an essential role in regulating normal hematopoiesis, but its constitutive activation via mutation in acute leukemias is generally associated with poor outcome. The aim of this study was to investigate interactions between the FLT-3 and VEGF signaling pathways in acute leukemia using cell lines and ex vivo cultures of pediatric acute lymphoblastic leukemia cells following expansion of direct patient explants in immunodeficient mice. Different xenograft lines exhibited variable cell surface FLT-3 expression, as well as basal and FLT-3 ligand-induced VEGF secretion, whereas the MV4;11 cell line, which expresses constitutively active FLT-3, secreted high levels of VEGF. The FLT-3 inhibitor, SU11657, significantly reduced VEGF secretion in three of six xenograft lines and MV4;11 cells, in conjunction with inhibition of FLT-3 tyrosine phosphorylation. Moreover, exposure of xenograft cells to the FLT-3-blocking antibody, D43, also reduced VEGF secretion to basal levels and decreased FLT-3 tyrosine phosphorylation. In terms of downstream signaling, SU11657 and D43 both caused dephosphorylation of extracellular signal-regulated kinase 1/2, with no changes in AKT or STAT5 phosphorylation. Finally, partial knockdown of FLT-3 expression by short interfering RNA also resulted in inhibition of VEGF secretion. These results indicate that FLT-3 signaling plays a central role in the regulation of VEGF secretion and that inhibition of the FLT-3/VEGF pathway may disrupt paracrine signaling between leukemia cells and the bone marrow microenvironment.     

Development of novel angiogenesis inhibitors targeting VEGF (vascular endothelial growth factor) for cancer chemotherapy

Recent progress in cancer biology has revealed that angiogenesis is a promising target for new anticancer drugs. Angiogenesis is tightly regulated by the balance between stimulatory and inhibitory angiogenic factors, and the imbalance of these regulators causes dysfunction of angiogenesis. Vascular endothelial growth factor (VEGF) is one of the best characterized pro-angiogenic factors, and multiple strategies have been studied to inhibit the pathway; i. e. production and secretion of VEGF receptor, VEGF binding to its receptor, tyrosine kinase activity of VEGF, and signaling pathway downstream induced by VEGF. In this article, the summary of function of VEGF family as well as recent promising drugs under clinical trials including bevacizumab (Avastin), a humanized monoclonal antibody developed against VEGF, and several small molecule inhibitors targeting VEGF function are described.