VEGF-stimulated HUVEC Research Articles

Therapeutic potential of elema-1,3,7(11),8-tetraen-8,12-lactam from Curcuma wenyujin on diabetic retinopathy via anti-inflammatory and anti-angiogenic pathways

Ethnopharmacological relevanceIn traditional Chinese medicine, the causes of diabetic retinopathy (DR) are blood stasis and heat. Curcuma wenyujin Y. H. Chen & C. Ling and its extracts have the effects of promoting blood circulation to remove blood stasis, clearing the heart, and cooling the blood, and have been used in the treatment of DR. Elema-1,3,7 (11),8-tetraen-8,12-lactam (Ele), an N-containing sesquiterpene isolated from this plant. However, the anti-inflammatory and anti-angiogenic effects of Ele and its therapeutic potential in DR are still unknown. Aim of the studyTo evaluate the anti-inflammatory and anti-angiogenic effects of Ele and its therapeutic potential in DR. Materials and methodsIn vitro, anti-inflammatory and anti-angiogenic effects were assessed using TNF-α or VEGF-stimulated HUVECs. Protein expression was analyzed using Western blotting. ICAM-1 and TNF-α mRNA expressions were analyzed using real-time quantitative RT-PCR. The therapeutic potential in DR was assessed using both animal models of STZ-induced diabetes and oxygen-induced retinopathy. The retinal vascular permeability was measured using Evans blue, and the quantitation of retinal leukostasis using FITC-coupled Con A. The retinal neovascular tufts were analyzed using fluorescein angiography and counting pre-retinal vascular lumens. ResultsEle inhibited NF-κB pathway, and ICAM-1, TNF-α mRNA expression in TNF-α- stimulated HUVECs. It also inhibits the multistep process of angiogenesis by inhibiting the phosphorylation of VEGFR2 and its downstream signaling kinases Src, Erk1/2, Akt, and mTOR in VEGF-stimulated HUVECs. Intravitreal injection of Ele can significantly reduce retinal microvascular leakage, leukostasis, and expression of ICAM-1, TNF-α in diabetic rats and inhibits oxygen-induced retinal neovascularization and VEGFR2 phosphorylation in OIR mice. ConclusionsEle has anti-inflammatory and anti-angiogenic effects through inhibiting NF-κB and VEGFR2 signaling pathways, and it may be a potential drug candidate for DR.

Dual blockade of MET and VEGFR2 signaling pathways as a potential therapeutic maneuver for peritoneal carcinomatosis in scirrhous gastric cancer

Scirrhous gastric cancer frequently develops into peritoneal carcinomatosis with malignant ascites, leading to an extremely poor prognosis. We had demonstrated that paracrine hepatocyte growth factor (HGF)-induced MET activation promotes peritoneal carcinomatosis with ascites formation. The vascular endothelial growth factor (VEGF) receptor (VEGFR)/VEGF axis facilitates tumor progression and formation of malignant ascites. This study investigated the role of MET and VEGFR2 in the development of peritoneal carcinomatosis with malignant ascites. Cabozantinib is a dual inhibitor of MET and VEGFR2. We examined the effects of cabozantinib on MET- and VEGFR2-mediated progression of peritoneal carcinomatosis in human scirrhous gastric cancer in vitro and in vivo. Cabozantinib inhibited HGF-stimulated proliferation of scirrhous cancer cell lines NUGC4 and GCIY, with a high potential to generate peritoneal carcinomatosis with ascites fluid, as well as the constitutive proliferation of MKN45 cells with MET amplification. Cabozantinib also inhibited the phosphorylation of both MET and VEGFR2 in scirrhous cancer cells and HGF- or VEGF-stimulated HUVECs. It effectively reduced ascitic fluid and prolonged the survival of NUGC4-inoculated nude mice. In clinical specimens, malignant ascites fluid from patients with peritoneal carcinomatosis contained high levels of HGF and VEGF. Our results strongly suggest that MET- and VEGFR2-mediated signaling pathways play pivotal roles in the pathogenesis of peritoneal carcinomatosis in scirrhous gastric cancer. Thus, the dual blockade of MET and VEGFR2 signaling may be a potential therapeutic maneuver for peritoneal carcinomatosis in scirrhous gastric cancer.

P727Metabolic and functional differences between male and female endothelial cells from umbilical cords (HUVEC) of twin pairs

Abstract Background Personalised Medicine is one of the hallmarks of future medicine. Sex and gender differences exist in the incidence, clinical manifestation and outcome of cardiovascular diseases. Gonadal hormones are thought to account for most of these sex differences. However, besides hormones, sexual dimorphisms at the cellular level may also contribute to physiological and pathophysiological cardiovascular differences between women and men. Purpose To analyse intrinsic sex differences at the cellular level, we aimed to elucidate sex-specific differences in endothelial cell migration and energy metabolism under pro-migratory conditions in male and female HUVECs. To reduce biological variability, we used HUVECS obtained from umbilical cords from twin pairs of the opposite sex. These cells are exposed in utero to the same maternal environment, and therefore represent a valuable tool to study intrinsic sex-specific differences at the cellular level. Methods Vascular endothelial growth factor (VEGF)-stimulated migration was determined with IBIDI migration chambers. Sex-specific levels of proteins were studied using proteome profiling. Cellular metabolism was measured by Seahorse and levels of intracellular metabolites were analysed using GC-MS based technology. Results Female cells showed significantly higher VEGF-induced cell migration than male HUVECs. Proteomic profiling revealed a sex-specific response to VEGF treatment. Mitochondrial respiration rate was higher in VEGF-stimulated male HUVECs compared to female cells. Whereas mean glycolytic rates did not significantly differ between sexes, the ratio of glycolysis/mitochondrial respiration after VEGF stimulation was higher in female than in male HUVECs. Female cells had higher intracellular ATP levels after serum starvation and treatment with VEGF. Under both conditions, female cells showed altered levels of metabolite pools compared to male HUVECs. Conclusions Higher intracellular ATP and metabolite levels in female cells after serum starvation and VEGF may contribute to the observed functional sexual dimorphisms, and may also point to an increased stress tolerance of female cells. The results of our study provide a strong argument to discriminate between male and female cells in in vitro experiments. Acknowledgement/Funding The work was supported by the DZHK (German Centre for Cardiovascular Research) and by the BMBF (German Ministry of Education and Research).

Hydrangenol suppresses VEGF-stimulated angiogenesis by targeting p27KIP1-dependent G1-cell cycle arrest, VEGFR-2-mediated signaling, and MMP-2 expression

ABSTRACTWe previously reported that hydrangenol has potent antitumor activity against human bladder cancer EJ cells. Here, we investigated the antiangiogenic activity of hydrangenol using in vitro and ex vivo models. Treatment with hydrangenol significantly inhibited the proliferation of vascular endothelial growth factor (VEGF)-induced HUVECs in a concentration-dependent manner (EC50 = 10 μM). Flow cytometry analysis revealed that hydrangenol suppressed the VEGF-induced inhibition of G1-cell cycle phase and also decreased cyclin D1, cyclin E, CDK2, and CDK4 levels. Hydrangenol-mediated arrest in the G1-cell cycle phase was associated with p27KIP1 level, but not p21WAF1 or p53 level. Hydrangenol also significantly inhibited VEGFR-2-mediated signaling pathways including ERK1/2, AKT, and endothelial nitric oxide synthase. Interestingly, immunoprecipitation assay demonstrated that the inhibition of VEGFR-2 activation was independent of VEGF binding, thereby suggesting an allosteric regulation of hydrangenol against VEGFR-2. Additionally, hydrangenol inhibited migration, invasion, and capillary-like tubular formation in VEGF-stimulated HUVECs. Zymography and immunoblot analyses revealed that these inhibitory activities were partially owing to the VEGF-induced inhibition of matrix metalloproteinase-2 activity. Finally, VEGF-mediated microvessel sprouting was inhibited in the presence of hydrangenol in ex vivo aortic ring assay. Taken together, hydrangenol possesses a potent antiangiogenesis potential; thus we believe that hydrangenol may be developed as a therapeutic reagent to treat angiogenesis-mediated diseases.

Abstract 3782: Development of a dual inhibitor of pan-RAF and VEGFR2 for the treatment of metastatic colorectal cancer with mutant K-RAS

Abstract Signal transduction in the RAS-RAF-MEK-ERK (MAPK) pathway plays a key role in cell survival, growth and proliferation. The pathway is controlled by extracellular signals through receptor tyrosine kinases (RTK) and is activated by oncogenic mutations in many types of cancer. K-RAS mutation has been reported in 40% of colorectal cancer patients. These patients have acquired resistance to EGFR tyrosine kinase inhibitors. Therefore, there is a need for the development of new therapeutic approaches for the treatment of these cancers. The B-RAF selective inhibitors have been approved for the treatment of human melanoma with a B-RAF V600E mutation. However, B-RAF specific inhibitor activates C-RAF in RAS mutant cells by inducing RAF dimer formation and paradoxical activation of C-RAF. These observations require the development of pan-RAF inhibitors without inducing paradoxical activation. In addition, as VEGFR2 represents one crucial promoter of tumor angiogenesis in colorectal cancer, the inhibition of VEGFR2 has been expected to be synergistic for the treatment of colorectal cancer. Therefore, we tried to find dual inhibitors of pan-RAF and VEGFR2 using structure based molecular modeling and identified a selective inhibitor, C1005, which binds to the DFG-out inactive conformation of B-RAF. In addition, C1005 potentially inhibited B-RAF, C-RAF, B-RAF V600E and VEGFR2 with IC50 value of 2.7 nM, 0.7 nM, 5.2 nM and 11nM, respectively. C1005 completely inhibited the phosphorylation of MEK-ERK without paradoxical activation in K-RAS mutant cells compared with Vemurafenib and Regorafenib. We examined the effects of C1005 on cell proliferation of colorectal cancer cells with K-RAS or B-RAF mutation. C1005 displayed potent anti-proliferative activities not only in K-RAS mutant colorectal cancer cells but also in B-RAF mutant colorectal cancer cells. C1005 effectively blocked cell proliferation of HCT-116, LS-513 and WiDr at EC50 of 145 nM, 79 nM and 636 nM, respectively. In addition, growth inhibition of VEGF-stimulated HUVEC indicates that C1005 is a potent inhibitor of the angiogenesis. To study in vivo effect, we tested C1005 in colorectal cancer xenografts with mutant K-RAS. Oral treatment of animals bearing xenograft tumors by 30 and 60 mg/kg, twice daily (BID) of C1005, induced a dose-dependent tumor growth inhibition. We observed 77% and 89% reduction of tumor volume, respectively. In this study, we discovered an orally active dual inhibitor of pan-RAF and VEGFR2. C1005 has been shown to suppress mutant K-RAS colorectal cancer cells without activating the MAPK pathway. Our findings suggest that C1005 could be an excellent preclinical candidate to treat mutant K-RAS driven colorectal cancer. Citation Format: Sungpyo Hong, Young-Il Choi, Jihye Choi, Wonyoung Kim, Ho-Seok Kwon, Yong Bin Park, Min-Hyo Ki, Hee Jong Shin, Michael Lee, Soon Kil Ahn. Development of a dual inhibitor of pan-RAF and VEGFR2 for the treatment of metastatic colorectal cancer with mutant K-RAS. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3782.

Esculetin Inhibits VEGF-Induced Angiogenesis Both In Vitro and In Vivo.

Esculetin is known to inhibit tumor growth, but its effect in angiogenesis has not been studied. Here, we report the efficacy of esculetin on VEGF-induced angiogenesis. Esculetin treatment inhibited VEGF-induced proliferation and DNA synthesis of HUVECs with no cell toxicity. G1-phase cell-cycle arrest was associated with a decreased expression of cyclins and CDKs via the binding of p27KIP1. Esculetin down-regulated the MMP-2 expression in VEGF-stimulated HUVECs, which suppressed colony tube formation and migration. Esculetin reduced the phosphorylation of VEGFR-2 and the downstream signaling of VEGFR-2, including ERK1/2 and eNOS/Akt pathways. Esculetin suppressed microvessel outgrowth from an aortic ring ex vivo model treated with VEGF, and blocked the VEGF-induced formation of new blood vessels and hemoglobin content in an in vivo Matrigel plug model. Collectively, VEGF-stimulated responses in angiogenesis were inhibited in vitro and in vivo, providing a theoretical basis for effective use against anti-angiogenic therapies.

Abstract 770: E7090: A potent and selective FGFR inhibitor with activity in multiple FGFR-driven cancer models with distinct mechanisms of activation

Abstract The fibroblast growth factor (FGF) signaling pathway comprises 18 ligands and 4 FGF receptor subtypes, FGFR1, 2, 3 and 4, which are known as receptor-type tyrosine kinases corresponding to those ligands. Upon ligand binding, FGFRs activate an array of downstream signaling pathways, such as the mitogen activated protein kinase (MAPK) and the phosphoinositide-3-kinase (PI3K)/Akt pathways. Genetic abnormalities (gene fusion, mutation and amplification) of FGFRs are known to cause constitutive activation of their signaling pathways, which play an important role in proliferation, survival and migration of cancer cells, tumor angiogenesis, drug resistance, etc. These abnormalities have also been reported to be involved in different cancer types including lung, breast, endometrial, gastric, and bladder cancer so far. Therefore, FGFRs are considered as one of potential targets for cancer therapy. E7090 is an orally available, selective and potent inhibitor of FGFR1, 2 and 3 tyrosine kinase activities. E7090 displayed inhibition of FGFR1, 2, and 3 kinase activities with the IC50 values of approximately 1 nmol/L. E7090 also inhibited the growth of SNU-16, human gastric cancer cell line harboring FGFR2 amplification with an IC50 value of 3 nmol/L. This activity was about 60-fold stronger than that against VEGF-stimulated HUVEC growth. Kinase profiling assay consisting of 93 kinases including non-receptor and serine/threonine kinases, also demonstrated that E7090 inhibited limited kinases including FGFR-1, -2 and -3. In addition, E7090 inhibited proliferation of human cancer cell lines harboring various types of FGFRs gene abnormalities such as amplification, mutation, or translocation in vitro, which are confirmed by the inhibition of FGFR signaling. E7090 also showed significant antitumor activities on various human xenografts harboring FGFRs gene abnormalities in a dose dependent manner and demonstrated tumor shrinkage at several doses in some models. Furthermore, pharmacodynamics analysis revealed that E7090 inhibited phosphorylation of FGFRs in SNU-16 xenograft tumors in a dose-dependent manner. Overall, the in vitro and in vivo studies confirm that E7090 is a potent and selective FGFRs inhibitor, showing promising antitumor activities with wider therapeutic windows in preclinical cancer models harboring FGFRs gene abnormalities. E7090 has been advanced through preclinical development and we disclose here the first details of its preclinical profile. Citation Format: Saori Watanabe Miyano, Yuji Yamamoto, Kotaro Kodama, Setsuo Funasaka, Satoshi Nagao, Naoko Hata Sugi, Hiroko Kuramochi, Katsuyuki Ishikawa, Kiyoshi Okamoto, Yukinori Minoshima, Takayuki Nakagawa, Yusuke Nakatani, Yuki Karoji, Isao Ohashi, Yoshinobu Yamane, Keigo Tanaka, Toshimi Okada, Tomohiro Matsushima, Junji Matsui, Masao Iwata, Akihiko Tsuruoka, Toshimitsu Uenaka. E7090: A potent and selective FGFR inhibitor with activity in multiple FGFR-driven cancer models with distinct mechanisms of activation. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 770. doi:10.1158/1538-7445.AM2015-770

THE PLASMA MEMBRANE CALCIUM ATPASE 4 SPECIFIC INHIBITOR AURINTRICARBOXYLIC ACID ENHANCES VEGF-INDUCED ANGIOGENESIS

Angiogenesis, the formation of new capillaries from pre-existing ones, is a biological process essential for successful embryonic development, organ growth and tissue repair. Excessive or insufficient growth of blood vessels, due to de-regulation of this process, plays a major role in the pathophysiology of several human diseases. The pro-angiogenic factor Vascular Endothelial Growth Factor (VEGF) has been identified as a crucial regulator of both physiological and pathological angiogenesis. VEGF activates the calcineurin/Nuclear Factor of Activated T-cells (NFAT) signalling pathway which is a critical mediator of angiogenesis. Our group has recently identified a novel role for the plasma membrane calcium ATPase 4 (PMCA4) as a negative regulator of VEGF-dependent angiogenesis. The small molecule Aurintricarboxylic acid (ATA) has been characterised as a selective inhibitor of PMCA4. We hypothesise that ATA-mediated inhibition of PMCA4 in endothelial cells will enhance VEGF-driven angiogenesis. Consistent with this hypothesis, we demonstrate in this work that inhibition of PMCA4 by treatment with ATA increases calcineurin/NFAT activity and the expression of the NFAT-dependent, pro-angiogenic protein RCAN1.4 in VEGF-stimulated HUVEC. Moreover, ATA treatment significantly enhanced endothelial cell migration and tubular morphogenesis in response to VEGF-stimulation. Interestingly, incubation of HUVEC with ATA had no effect on the cell viability or Erk1/2 phosphorylation (activation) status of VEGF-stimulated cells. Our results show a novel role for the PMCA4 specific inhibitor ATA as a stimulator of VEGF-induced angiogenesis. Thus, ATA might lead to the design of new therapeutic strategies to improve blood vessel formation in diseases associated with insufficient angiogenesis.

Abstract 1755: CT053PTSA, a novel c-MET and VEGFR2 inhibitor, potently suppresses angiogenesis and tumor growth

Abstract Background: In many human cancers, c-Met is activated via receptor overexpression, amplification, mutation and/or a ligand-dependent autocrine/paracrine loop. c-MET-driven tumor growth and metastasis may be further stimulated by hypoxic conditions in the tumor microenvironment, which are often exacerbated by the VEGF-pathway inhibition. Therefore, blockades of c-Met and VEGFR2 simultaneously may yield synergistic inhibition on tumor growth. Methods and Results: CT053PTSA is a potent, orally bioavailable small molecule tyrosine kinase inhibitor (TKI) against c-Met, VEGFR2 as well as Axl, Mer, and FLT3, all of which have been implicated in tumor pathogenesis. In cell-based assays, CT053PTSA showed IC50s of 6.7, 1.9 and < 1.0 nM against c-Met, VEGFR-2 and Axl. In cell-based functional assays, CT053PTSA inhibited HGF and VEGF-stimulated HUVEC proliferation and microvascular angiogenesis in rat aortic rings with IC50 values of 8.6 and 6.3 nM, respectively. When single dosed orally (3mg/kg) to U87MG tumor-bearing nude mice, CT053PTSA potently inhibited the phosphorylation of c-Met and its downstream signaling kinases AKT and ERK1/2 for up to 6 hours in tumor tissues. The antitumor efficacy of CT053PTSA was evaluated in human tumor subcutaneous xenograft models in athymic mice, which included Caki-1 (kidney carcinoma), U-87MG (glioblastoma), NCI-H441 (lung adenocarcinoma), MDA-MB-231 (breast adenocarcinoma), SMMC-7721 (hepatoma), 5637 (bladder carcinoma) and SK-OV-3 (ovarian carcinoma). The ED50s of CT053PTSA to inhibit tumor growth in these studies ranged from < 1mg/kg (NCI-H441) to ∼ 6 mg/kg (SMMC-7721). In orthotopic U87MG human glioblastoma xenograft model, CT053PTSA prolonged the median survival time (MST) and yielded significant increase in life-span value (ILS = 32%, p = 0.003) at an oral dose of 20 mg/kg/day (dosed 21 days) versus the vehicle-treated group. Conclusions: CT053PTSA displayed excellent oral bioavailability (> 60% across animal species) and desirable drug exposures in mice, rats, dogs and monkeys. Based on its potent antitumor activity, favorable PK profile and toxicological data, CT053PTSA was granted to enter clinical trials in China and is currently under phase I evaluation as a potential treatment for human malignancies. Citation Format: Ning Xi, Yingjun Zhang, Zhaohe Wang, Yanjun Wu, Tingjin Wang. CT053PTSA, a novel c-MET and VEGFR2 inhibitor, potently suppresses angiogenesis and tumor growth. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1755. doi:10.1158/1538-7445.AM2014-1755

Abstract 5083: Dual targeting of the type 1 insulin-like growth factor receptor and its ligands as an effective anti-angiogenic strategy.

Abstract Background. Several childhood sarcomas, exhibit autocrine or paracrine growth through secretion of insulin-like growth factors 1 and 2 (IGF-1, IGF-2) and signaling through the Type 1 receptor (IGF-1R). In some childhood sarcoma xenografts IGF-1R antibodies dramatically decrease VEGF transcription and have potential anti-angiogenic activity. However, tumor-derived IGF-2 results in intrinsic resistance to IGF-1R-targeted antibodies, and continued tumor angiogenesis. We evaluated the anti-angiogenic activity of a ligand-binding antibody (MEDI-573) alone or in combination with IGF-1 receptor-binding antibodies as a potential strategy for effectively suppressing angiogenesis in pediatric sarcomas. Methods. IGF-stimulated signaling was monitored by increased Akt phosphorylation in sarcoma and human vascular endothelial (HUVEC) cells. Effects on angiogenesis were determined in vitro by capillary tube formation in HUVECs and in vivo using a VEGF-stimulated Matrigel assay followed by assay of hemoglobin and CD34 immunostaining. Results. The IGF-ligand binding antibody MEDI-573 suppressed Akt phosphorylation induced by exogenous IGF-1 and IGF-2 in sarcoma cells. Receptor binding antibodies, MAB391 and CP1-B02, suppressed IGF-1-stimulated phosphorylation of Akt, but not that induced by IGF-2. Both receptor-binding antibodies suppressed VEGF-induced HUVEC tube formation in vitro, but this was abrogated by exogenous IGF-2. MEDI-573 inhibited VEGF-stimulated HUVEC proliferation and tube formation in vitro, but did not inhibit angiogenesis in vivo, probably because MEDI-573 binds murine IGF-1 with low affinity. However, in vitro the anti-angiogenic activity of MEDI-573 was also circumvented by human recombinant IGF-1. The combination of receptor-binding and ligand-binding antibodies completely suppressed VEGF-stimulated proliferation of vascular endothelial cells in the presence of IGF-1 and IGF-2, and prevented ligand-induced phosphorylation of IGF-1R/IR receptors. Further, combining receptor-binding and ligand-binding antibodies completely suppressed VEGF-driven angiogenesis in a mouse model where Matrigel plugs containing both IGF-1 and IGF-2 were incorporated with VEGF. Conclusions. Our previous study showed that IGF-2 produced by tumor cells in the microenvironment could circumvent the anti-angiogenic effects of IGF-1 receptor binding antibodies. In contrast, IGF-1 was able to overcome the anti-angiogenic effect of the IGF-ligand binding antibody, MEDI-573. Our current study demonstrates that combining receptor and ligand binding antibodies effectively suppresses VEGF-stimulated angiogenesis in vitro and in a mouse model. Studies are ongoing to evaluate this therapeutic strategy using pediatric sarcoma xenograft models with characterized IGF expression. (Supported by grants CA77776, CA23099 and MedImmune). Citation Format: Hemant K. Bid, Cheryl A. London, Jin Gao, Haihong Zhong, Robert H. Hollingsworth, Peter J. Houghton. Dual targeting of the type 1 insulin-like growth factor receptor and its ligands as an effective anti-angiogenic strategy. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5083. doi:10.1158/1538-7445.AM2013-5083

Abstract 3277: Insulin-like growth factor-2 (IGF-2) circumvents the antiangiogenic activity of SCH717454, a human antibody that blocks ligand binding to the Type 1 insulin-like growth factor receptor (IGF-1R)

Abstract Background. Deregulated insulin-like growth factor signaling through IGF-1R is common to many human cancers, including childhood solid tumors, and has become an important target for therapeutics development. We reported that there was a rapid decrease in tumor-associated VEGF in childhood sarcoma xenografts responding to an IGF-1R-targeted antibody (Kurmasheva RT Cancer Res, 2010). Subsequently the IGF-1R targeted antibody SCH717454 (SCH) was shown to reduce blood vessel formation in tumor xenografts (Wang Y Mol Cancer Ther. 2010). However, relatively few xenograft tumors respond to treatment (Kolb EA Ped Blood &Cancer, 2009). Here we examine a possible contributing factor to resistance to IGF-1R blockade. Methods. IGF-1R activity was measured by phosphorylation of IGF-1R, insulin receptor (IN-R), and Akt subsequent to addition of ligand (IGF-1, IGF-2). Effects of SCH on angiogenesis in vitro were determined by HUVEC proliferation and tube formation assays. Angiogenesis in vivo was determined using Matrigel plugs infused with PBS (control), VEGF or VEGF+IGF-2 and assay of hemoglobin and CD34 immunostaining. Results. SCH rapidly blocked IGF-1 but not IGF-2 stimulated phosphorylation of Akt in all sarcoma cell lines. Immunoprecipitation using anti-IGF-1R and anti-IN-R antibodies revealed that in the presence of SCH, IGF-2 partially signaled through IGF-1R but significantly induced phosphorylation of IN-R. SCH completely blocked VEGF-stimulated proliferation and tube formation of HUVECs indicating that IGF-1R signaling is essential to these processes. Exogenous IGF-2, but not IGF-1, circumvented SCH inhibition of VEGF- stimulated proliferation and tube formation. In mice SCH (20 mg/kg) dramatically inhibited angiogenesis in VEGF-infused Matrigel plugs, demonstrating anti-angiogenic activity. However, mice lack circulating IGF-2, and many childhood sarcomas secrete IGF-2. To test whether IGF-2 could circumvent the antiangiogenic activity of SCH in vivo, Matrigel plugs infused with VEGF or VEGF+IGF-2 were implanted. SCH suppressed angiogenesis into VEGF-infused Matrigel plugs, but had no inhibitory activity when plugs contained both VEGF+IGF-2. Similarly, in vitro co-culture of HUVECs with IGF-2-secreting tumor cells (Rh30) in transwell plates completely abrogated SCH inhibition of VEGF-stimulated HUVEC proliferation/tube formation. Conclusions. These results support the anti-angiogenic activity of SCH. Both in vitro and in vivo IGF-2 circumvented the effects of SCH and other IGF-1R-targeted antibodies examined in clinical development. Many childhood cancers secrete IGF-2, suggesting that tumor-derived IGF-2 in the microenvironment maintains angiogenesis in the presence of antibodies such as SCH. (Supported by NCI awards CA77776, CA23099 and from Schering-Plough). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3277. doi:10.1158/1538-7445.AM2011-3277

Synthesis and antitumor activity of 5-[1-(3-(dimethylamino)propyl)-5-halogenated-2-oxoindolin-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3-carboxamides

We report herein the design and synthesis of novel 1-[3-(dimethylamino)propyl]indolin-2-one derivatives based on the structural features of Sunitinib, a known multitargeted receptor tyrosine kinase inhibitor, and TMP-20, a previously discovered compound with good antitumor activity in our lab. These newly synthesized derivatives were evaluated for in vitro activity against five human cancer cell lines and VEGF/bFGF-stimulated HUVECs. Results revealed that all of the target compounds 1a-p show potent antitumor activity, compounds 1e-h (IC(50)'s: 0.45-5.08 μM) are more active than Sunitinib (IC(50)'s: 1.35-6.61 μM), and the most active compound 1 h (IC(50): 0.47-3.11 μM) is 2.1-4.6-fold more potent than Sunitinib against all five cancer cell lines. In addition, like Sunitinib, 1a-p have higher selectivity on VEGF-stimulated HUVEC other than bFGF-stimulated HUVEC.

Decursin and decursinol inhibit VEGF-induced angiogenesis by blocking the activation of extracellular signal-regulated kinase and c-Jun N-terminal kinase

The root of Angelica gigas Nakai contains two major coumarins, which have been previously identified as decursin and decursinol. Decursin has been demonstrated to exhibit potent anti-cancer activity both in vitro and in vivo. In this study, we found that decursin and decursinol at non-cytotoxic doses inhibited the VEGF-induced proliferation, migration, and capillary-tube formation of HUVECs. Moreover, decursin and decursinol suppressed microvessel formation on chorioallantoic membranes in fertilized eggs and into mouse Matrigel plugs. The oral administration of decursin and decursinol also reduced VEGF-induced angiogenesis in Matrigel. Furthermore, decursin and decursinol reduced the phosphorylation of ERK and JNK, but not p38 MAPK, in VEGF-stimulated HUVECs. Taken together, our results reveal that decursin and decursinol inhibit VEGF-induced angiogenesis by reducing the activation of ERK and JNK in HUVECs, and possess potent in vivo anti-angiogenic activity, coupled with the advantage of oral dosing. Thus, these compounds may have the potential for the treatment of cancers dependent on VEGF-induced vascularization.

YM-201627: An orally active antitumor agent with selective inhibition of vascular endothelial cell proliferation

We developed an oral administration-compatible, small molecular weight antitumor agent, YM-201627 by screening for the inhibition of the proliferation of VEGF-stimulated HUVECs. YM-201627 selectively inhibited the proliferation of various endothelial cell lines induced by VEGF, bFGF, and FBS (at IC 50 s of 0.0039–0.12 μM), that would not be expected to have any direct antiproliferative effect on other cell types. YM-201627 inhibited angiogenesis in vitro at a concentration of 0.01 μM. In the in vivo studies, it inhibited microvessel formation induced by human melanoma A375 cells suspended in Matrigel (86% with twice-daily doses of 30 mg/kg). Moreover, once-daily oral dosing of YM-201627 to mice bearing A375 xenografts elicited significant antitumor activity (73% with daily doses of 10 mg/kg). These results suggest that YM-201627 is a selective growth inhibitor of endothelial cells, which may be useful for treatment of solid tumors.

A Phase II Study of ZD6474, a Vascular Endothelial Growth Factor Receptor (VEGFR) and Epidermal Growth Factor Receptor (EGFR) Tyrosine Kinase Inhibitor (TKI) in Patients with Relapsed Multiple Myeloma (MM).

ZD6474 targets angiogenesis by inhibiting VEGFR TK and also affects EGFR signaling by inhibiting EGFR TK activity. This oral agent showed dose-related tumor growth inhibition in a broad range of in vivo xenograft models. In Phase I studies, drug- and dose-related adverse events included rash, diarrhea, hypertension, and asymptomatic QT/QTc prolongation. Four patients with NSCLC included in the phase I studies exhibited partial responses. Because MM is a disease in which angiogenesis is postulated to be a relevant target for therapy, we conducted a phase II trial of ZD6474 100 mg p.o. daily in patients with MM who had relapsed following either high-dose chemotherapy and stem cell transplantation, or following non-high-dose chemotherapy. Although a higher dose was recommended for solid tumour trials, a dose of 100 mg p.o. daily produced plasma levels in phase I in excess of the IC50 for in vitro inhibition of VEGF-stimulated HUVEC proliferation and seemed well tolerated. For the study reported here, patients all had a measurable M protein in the serum or urine and were required to have an ECOG PS of 0, 1 or 2. Patients were excluded if they had any of: AGC <1.0x109/L or platelet count <50x109/L; bilirubin, AST and/or ALT >1.5xUNL; creatinine >2xUNL; K+ <4.0mmol/L; abnormal Ca2+ or Mg2+ levels, prior thalidomide treatment or pre-existing cardiac dysfunction. The primary efficacy endpoint was objective response as assessed by reduction in M protein. Following written, informed consent, 18 patients were entered into the study from five centers (9 male/9 female; mean age 64 yrs [35– 81]). Fourteen patients had relapsed following high-dose chemotherapy with stem cell transplant. One patient was ineligible due to elevated baseline calcium and one was not evaluable (off study due to non drug-related infection with < 14 days of therapy) . Patients were treated for 3.0– 29.4 weeks (mean 9.8 weeks). Overall, ZD6474 was well tolerated with only three patients missing doses of ZD6474. The most common drug-related adverse events were nausea, vomiting, fatigue, rash, pruritis, headache, diarrhea, dizziness, and sensory neuropathy, all of which were Grade I – II in severity. There were no drug-related serious adverse events. Laboratory adverse events were infrequent: one patient had Grade III anemia, and there were no Grade III changes in biochemistry. No significant QTc interval changes were seen. Fifteen patients had plasma samples obtained for at least 4 weeks after starting treatment. Trough levels of ZD6474 achieved or exceeded the IC50 for inhibition of VEGF-stimulated HUVEC proliferation in 13 of the 15 patients by day 28. There were no responses in M protein levels. In conclusion, ZD6474 was well tolerated at a dose of 100 mg per day and achieved plasma levels predicted to inhibit VEGF signaling. However, this was not reflected in clinical benefit since none of the patients had a reduction in M protein. ZD6474 development in solid tumors is continuing in a series of Phase II trials.

Antiangiogenic and antitumor effects of a protein kinase Cbeta inhibitor in human breast cancer and ovarian cancer xenografts.

In cell culture, the compound 317615 2HCl, a potent inhibitor of VEGF-stimulated HUVEC proliferation, was not very effective against MX-1 breast cancer cells (IC50= 8.1 microM) or SKOV-3 ovarian carcinoma cells (IC50 = 9.5 microM). Exposure to combinations of paclitaxel or carboplatin and 317615 x 2HCl with MX-1 cells in culture resulted in cell survival that reflected primarily additivity of the two agents. Exposure of SKOV-3 cells to paclitaxel or carboplatin along with 317615 2HCl resulted in cell survivals that reflected additivity of 317615 x 2HCl with paclitaxel and greater-than-additive cytotoxicity with carboplatin. Administration of 317615 x 2HCI orally twice daily to nude mice bearing subcutaneous MX-1 tumors or SKOV-3 tumors resulted in a decreased number of intratumoral vessels as determined by CD31 and CD105 staining with decreases of 35% and 43% in MX-1 tumors and 60% and 75% in SKOV-3 tumors, respectively. 317615 x 2HCl was an active antitumor agent against the MX-1 xenograft and increased the tumor growth delay produced by paclitaxel by 1.7-fold and the tumor growth delay produced by carboplatin by 3.8-fold. Administration of 317615 x 2HCl also increased the tumor growth delay produced by fractionated radiation therapy in the MX-1 tumor. Treatment with 317615 x 2HCl alone increased the lifespan of animals bearing intraperitoneal SKOV-3 xenografts by 1.9 fold compared with untreated control animals. The combination of paclitaxel and 317615 x 2HCl resulted in 100% 120-day survival of SKOV-3 bearing animals. Administration of 317615 x 2HCl along with carboplatin to animals bearing the SKOV-3 tumor produced a 1.8-fold increase in lifespan compared with carboplatin alone. 317615 x 2HCl is a promising new antiangiogenic agent that is in early phase clinical testing.

Antiangiogenic effects of a protein kinase Cbeta-selective small molecule.

Protein kinase C frequently plays a central role in the intracellular signal transduction of growth factors and cytokines. The acyclic bisindolylmaleimide 317615 x 2HCl was identified as a potent selective inhibitor of protein kinase Cbeta. The compound 317615 x 2HCl was tested in culture and in vivo in the rat corneal micropocket and in the SW2 small-cell lung carcinoma human tumor xenograft. In cell culture, 317615 x 2HCl was a more potent inhibitor of VEGF-stimulated HUVEC proliferation (IC50 150 nM, 72 h) than of human SW2 small-cell lung carcinoma cell proliferation (IC50 3.5 microM, 72 h). When administered orally twice daily for 10 days, the compound 317615 x 2HCl markedly decreased the neoangiogenesis induced by VEGF or bFGF in the rat corneal micropocket assay. To assess antitumor efficacy, 317615 x 2HCl was administered orally twice daily to nude mice bearing SW2 xenograft tumors on days 14 through 30 after tumor implantation. The number of countable intratumoral vessels was decreased in a dose-dependent manner reaching as low as one-quarter the number in the control tumors. The decrease in intratumoral vessels was paralleled by increases in tumor growth delay. Treatment of the tumor-bearing animals with paclitaxel or carboplatin followed by treatment with 317615 x 2HCl resulted in a 2.5- to 3.0-fold increase in tumor growth delay compared with the standard chemotherapeutic agents alone. 317615 x 2HCl represents a new approach to antiangiogenic therapy in cancer-blocking multiple growth factor signaling pathways in endothelial cells with a single agent. 317615 x HCl is in early clinical testing.

Antiangiogenic and antitumor effects of a protein kinase Cbeta inhibitor in murine lewis lung carcinoma and human Calu-6 non-small-cell lung carcinoma xenografts.

In cell culture, the compound 317615.2HCl, a potent inhibitor of VEGF-stimulated HUVEC proliferation, was not very effective against Calu-6 non-small-cell lung carcinoma cells (IC50 26 microM). Exposure to combinations of paclitaxel or carboplatin and 317615.2HCl with Calu-6 cells in culture resulted in cell survival that reflected less-than-additivity to additivity of the two agents. Administration of 317615.2HCl orally twice daily to nude mice bearing subcutaneous Calu-6 tumors resulted in a decreased number of intratumoral vessels as determined by CD31 and CD105 staining to 50% of the number in control tumors. 317615.2HCl showed antitumor activity against the Lewis lung carcinoma and increased the tumor growth delay produced by paclitaxel by 5-fold, that produced by gemcitabine by 2-fold and that produced by carboplatin by 1.7-fold. There was a decrease in the number of lung metastases in the Lewis lung carcinoma that paralleled the increased response of the primary tumor with each treatment combination. Administration of 317615.2HCl also increased the tumor growth delay produced by fractionated radiation therapy in the Lewis lung tumor. Treatment with 317615.2HCl was an effective therapy in the Calu-6 non-small-cell lung carcinoma xenograft when the compound was administered early (days 4-18) or later (days 14-30). Combination treatment regimens in which 317615.2HCl was administered along with or sequentially with paclitaxel or carboplatin were much more effective than the chemotherapeutic agents administered alone. 317615.2HCl is in early clinical testing.