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1 Journal of Pharmacological Sciences 129 (2015) 9e17 HOSTED BY Contents lists available at ScienceDirect Journal of Pharmacological Sciences journal homepage: Full paper CPU-12, a novel synthesized oxazolo[5,4-d]pyrimidine derivative, showed superior anti-angiogenic activity Jiping Liu a, 1, Ya-Hui Deng b, 1, Ling Yang a, Yijuan Chen b, Manzo Lawali a, Li-Ping Sun b, **, Yu Liu a, * a Department of Biochemistry, School of Life Science and Technology, China Pharmaceutical University, Nanjing , PR China b Jiangsu Key Laboratory of Drug Design & Optimization, and Department of Medicinal Chemistry, China Pharmaceutical University, Nanjing , PR China article info abstract Article history: Received 13 February 2015 Received in revised form 2 June 2015 Accepted 3 June 2015 Available online 20 June 2015 Keywords: Angiogenesis Oxazolo[5,4-d]pyrimidine derivatives Vascular endothelial growth factor receptor (VEGFR) Receptor tyrosine kinase (RTK) inhibitor The rat aortic ring assay Angiogenesis is a crucial requirement for malignant tumor growth, progression and metastasis. Tumorderived factors stimulate formation of new blood vessels which actively support tumor growth and spread. Various of drugs have been applied to inhibit tumor angiogenesis. CPU-12, 4-chloro-N-(4-((2-(4- methoxyphenyl)-5-methyloxazolo[5,4-d] pyrimidin-7-yl)amino)phenyl)benzamide, is a novel oxazolo [5,4-d]pyrimidine derivative that showed potent activity in inhibiting VEGF-induced angiogenesis in vitro and ex-vivo. In cell toxicity experiments, CPU-12 significantly inhibited the human umbilical vein endothelial cell (HUVEC) proliferation in a dose-dependent manner with a low IC 50 value at 9.30 ± 1.24 mm. In vitro, CPU-12 remarkably inhibited HUVEC's migration, chemotactic invasion and capillary-like tube formation in a dose-dependent manner. In ex-vivo, CPU-12 effectively inhibited new microvessels sprouting from the rat aortic ring. In addition, the downstream signalings of vascular endothelial growth factor receptor-2 (VEGFR-2), including the phosphorylation of PI3K, ERK1/2 and p38 MAPK, were effectively down-regulated by CPU-12. These evidences suggested that angiogenic response via the induction of VEGFR through distinct signal transduction pathways regulating proliferation, migration and tube formation of endothelial cells was significantly inhibited by the novel small molecule compound CPU-12 in vitro and ex-vivo. In conclusion, CPU-12 showed superior anti-angiogenic activity in vitro Japanese Pharmacological Society. Production and hosting by Elsevier B.V. on behalf of Japanese Pharmacological Society. This is an open access article under the CC BY-NC-ND license ( creativecommons.org/licenses/by-nc-nd/4.0/). 1. Introduction Angiogenesis is a physiological process by which new blood vessels sprouting from pre-existing vessels. Angiogenesis occurs during several physiological and pathological conditions (1). Although angiogenesis is essential for normal physiological processes, it also plays a significant role in the development of diseases, including cancer (2), rheumatoid arthritis (3) and diabetic retinopathy (4). Tumor vessels develop by sprouting from preexisting vessels and supply nutrient for tumor tissue, and hence * Corresponding author. Tel.: þ , þ ** Corresponding author. addresses: chslp@cpu.edu.cn (L.-P. Sun), liuyuyaoda@163.com (Y. Liu). Peer review under responsibility of Japanese Pharmacological Society. 1 These authors contribute to this work equally. most tumor growth and metastasis are angiogenesis-dependent (5). Therefore, inhibition of tumor angiogenesis is one of the promising strategies in the development of novel anticancer therapy. The most vital players for angiogenesis are blood vessel growth-promoting factors and inhibiting factors (6). There are various molecular factors involved in angiogenesis. Among these, members of vascular endothelial growth factor (VEGF) family have a predominant role. VEGF promotes angiogenesis and remodels of the vasculature by activation of vascular endothelial growth factor receptor (VEGFR) and subsequently facilitates tumor growth (7). VEGFR is a key protein tyrosine kinase receptor and locates on the surface of endothelial cells. VEGFR-2 is thought to initiate intracellular signal transduction, regulating endothelial cell proliferation, migration, and angiogenesis. Certain approaches to interfere with VEGF-induced endothelial cell proliferation, are currently in preclinical and clinical development, including neutralizing anti- VEGF monoclonal antibody (8), blocking antibody against VEGF / 2015 Japanese Pharmacological Society. Production and hosting by Elsevier B.V. on behalf of Japanese Pharmacological Society. This is an open access article under the CC BY-NC-ND license (

2 10 J. Liu et al. / Journal of Pharmacological Sciences 129 (2015) 9e17 receptors (9, 10) and potent small molecular inhibitors of the VEGFR-2 tyrosine kinase (11). Researching and developing new inhibitor to block the VEGF/VEGFR signal pathway is meaningful for anti-angiogenesis and antitumor therapy. Pyrimidines possess versatile biological activities and are broadly applied in medical development such as calcium channel modulator, selective 1-adrenoreceptor antagonist, HIV gpl20-cd4 inhibitor, antiviral, anticancer drug with mitotic kinesin inhibition, oral antihypertensive, blood platelet aggregation inhibitor, muscarinic antifungal, and antibacterial drugs (12). Oxazolo[5,4-d] pyrimidine derivatives are associated with diverse biological activities including ricin and shiga toxin inhibition, A2A adenosine receptor antagonism and kinase inhibition (13e15). These pyrimidine and fused heterocyclic pyrimidine derivatives are of great biological interest, especially as antiviral, antitumor, antimicrobial, and anti-inflammatory agents. In our search for potent receptor tyrosine inhibitors, a series of oxazolo[5,4-d]pyrimidine derivatives were synthesized based on a computer-assisted drug design protocol (Ya-Hui Dengz, Ji-Ping Liuz, Yi-Juan Cheng, Yu Liu, Li-Ping Sun, Diarylureas and diarylamides with oxazolo[5,4-d]pyrimidine scaffold as a novel class of antiangiogenesis agents, under submission.). The most promising compound CPU-12 was selected for biological evaluation in a virtual screen. As shown in Fig. 1, the bicyclic oxazolo[5,4-d]pyrimidine moiety served as a mimic of the adenine ring in ATP to afford primary affinity between the compound and target kinase and the amine fragment was intended to exert a hydrophobic interaction with the target. As an elaborately designed compound, CPU-12 may have inhibitory activity against angiogenesis. In this study, CPU-12 was further evaluated for antiproliferative and antiangiogenic activity using various angiogenesis assay models, both in vitro and ex-vivo. CPU-12 reduced human umbilical vein endothelial cells (HUVECs) proliferation and migration and inhibited VEGF-induced tube formation. Meanwhile, CPU-12 was found to significantly inhibit blood capillary-network sprouting in an ex-vivo rat aortic ring assay model in a dose-dependent manner. Moreover, the results of western blot analysis indicated that CPU- 12 efficiently reduced the phosphorylation of PI3K, ERK1/2 and p38 MAPK in a dose-dependent manner, while the expression levels of PI3K, ERK1/2 and p38 MAPK remained unchanged. 2. Materials and methods 2.1. Ethics statement Animal studies were conducted in conformity with the institutional guide for the care and use of laboratory animal. All rat protocols were approved by the Animal Care and Use Committee of China Pharmaceutical University (Nanjing, Jiangsu, China) Chemicals and antibodies DMEM and M199 were purchased from Gibco Life Technologies (Carlsbad, CA, USA). VEGF was expressed and purified from a recombination pichia pastoris. Fetal bovine serum (FBS) was from Wisent biotechnology Co. Ltd (Nanjing, Jiangsu, China). Matrigel was purchased from Corning Life Sciences (Corning, NY, USA). Sources of the antibodies are as follows: PI3K p85/p55 (Ab-467/ 199) antibody, ERK1/2 antibody and p38 MAPK (Ab-180/182) antibody were from Signalway Antibody Inc (College Park, Maryland, USA). PI3K p85/p55 (phospho-tyr467/199) antibody, p44/42 MAPK (Phospho-Thr202/Tyr204) antibody and p38 MAPK (Phospho-Thr180/Tyr182) antibody were obtained from Signalway Antibody Inc (College Park, Maryland, USA). Goat anti-rabbit IgG Secondary Antibody were from Bioworld Technology Inc (Nanjing, Jiangsu, China). The Motic Image System was purchased from Motic China group Co. Ltd (Motic Image Plus 2.0, Xiamen, China). In all experiments, the final DMSO concentration was kept <0.1% throughout the study Cell culture Human Umbilical Vein Endothelial Cell (HUVEC) were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA), and routinely cultured in DMEM containing 10% FBS, 100 mg/ml penicillin and 100 mg/ml streptomycin, maintained in a humidified atmosphere of 95% air, 5% CO 2 at 37 C. Confluent HUVECs within 8 passages were used in the experiments described below Cell proliferation assay Fig. 1. The structure of CPU-12. The logarithm phase cells were seeded onto a 96-well plate ( cells/well) in DMEM containing 10% FBS and incubated for 24 h to produce nearly confluent cell monolayer. Adhered cells were serum-starved and then treated with CPU-12 (0, 1, 10, 20, 50 and 100 mm) in medium supplemented with 20 ng/ml VEGF for 72 h. After incubation, an MTT assay was used to assess the drug effects on cell viability (16) Cell cycle and apoptosis analysis HUVECs were plated in 6-well plates at cells/well and allowed to adhere for 24 h. After serum starvation, HUVECs were activated with 20 ng/ml VEGF for 30 min, and then cells were either incubated in fresh medium alone or supplemented with different concentration of CPU-12 (2.5, 5, and 10 mm) for an additional 48 h. The FITC Annexin V Apoptosis Detection Kit (BD, NJ, USA) was used for the detection of all the stages of apoptosis according to the manufacturer's instructions. Briefly, HUVECs were harvested after trypsin treatment and washed with cold phosphate buffered saline (PBS) and then resuspended in 1 Binding Buffer at a concentration of cells/ml. 100 ml of the solution ( cells) were transferred to a 5 ml culture tube and then 5 ml of Annexin V and 5 ml Propidium Iodide Staining Solution were added to the solution for 15 min at room temperature (25 C) and kept in dark place. Additional 400 ml of 1 Binding Buffer was introduced to each tube and later cell apoptosis were analyzed by flow cytometry (BD LSRII System, BD Biosciences, San Jose, CA). The results were analyzed using FlowJo software (Tree Star, Inc., Asland, USA). For the cell cycle analysis, after compound treatment, cells were harvested after trypsin treatment and washed with phosphate buffered saline (PBS). HUVECs were fixed in precooled 70% ethanol for 12 h, washed, and resuspended in 1 ml PBS; treated with 10 ml RNase A; and stained with 5 ml Propidium Iodide at 0.25 mg/ml for 30 min at room temperature, and kept in dark place. The stained

3 J. Liu et al. / Journal of Pharmacological Sciences 129 (2015) 9e17 11 cells were analyzed by flow cytometry and DNA content was quantified using Modified software (Verity Software House, Inc., Topsham, USA) Endothelial cell migration assay (wound healing assay) HUVEC migration was evaluated using a scratch wound healing assay. HUVECs were cultured in a 6-well plate to form a monolayer. The cell monolayer was wounded with a 1 mm-wide lane per well followed with a gentle wash with PBS; cells were supplied with 2 ml medium plus 1% FBS with or without CPU-12 at various concentrations (0.1, 1 and 10 mm). The extent of wound closure was observed and photographed at 0 h, 24 h and 48 h using an inverted phase-contrast microscope. Images of the denuded endothelial monolayer were captured using a Motic Image System. The effect of CPU-12 on the progression of endothelial cell migration was quantitated by calculating the difference in the denuded area after 48 h. Data were expressed as a percentage of the migration of untreated endothelial cells groups (mean ± SD) Boyden chamber assay of chemotactic endothelial cell invasion The invasion assay measures the ability of cells to attach to the matrixes, invade into and through the matrix, and to migrate toward a chemoattractant (17). The transwell chamber migration system enables to select inhibitors, which may alter the invasive phenotype of the endothelial cells (18). The bottom surface of the entire filter was coated with the diluted Matrigel solution as barriers, leaving the Matrigel layer to air-dry for 30 min. HUVECs were collected and suspended at a final concentration of cells/ml in DMEM medium containing 1% FBS plus various concentrations of CPU-12 (0, 0.1, 1 and 10 mm). Fresh DMEM medium with 10% FBS and 20 ng/ml VEGF were added into the bottom well of the chamber. After 24 h incubation (at 37 C and 5% CO 2 ), HUVECs were fixed and stained with Hematoxylin solution. The noninvading cells were removed from the upper surface of the membrane by scrubbing with a sterile cotton swab. The degree of invaded cells was quantified by manual counting, and five random chosen fields were photographed from each group Tube formation assay When plated on Matrigel, HUVEC spontaneously form capillarylike tube structures in the presence of serum and endothelial cell growth supplement. This method can be used to assess compounds that either inhibit or stimulate angiogenesis (19). Briefly, the precooled 96-well plates were coated with 60 ml thawed Matrigel for 30 min. The logarithm phase cells were harvested and suspended in DMEM medium containing 10% FBS and VEGF (20 ng/ml). HUVECs were seeded on coated plates at cells/well and treated with CPU-12 at various concentrations (0, 2.5, 5, and 10 mm) and further incubated for 8 h. The formed endothelial tubes were photographed and quantified. The degree of tube formation was assessed by manual counting of the number of tubes in five random chosen fields from each well Rat aortic ring assay An additional evidence showing the potential activity of CPU-12 to inhibit angiogenesis is provided by the ex-vivo model of the rat aortic ring assay. The rat aortic ring assay was performed as previously described (20, 21). Young and 6-week-old male SpragueeDawley rats were anesthetized with pentobarbital and then humanely sacrificed. The entire length of the thoracic and abdominal aorta was surgically excised and removed from the sacrificed rat, using sterile dissecting instruments. The excised aorta was transferred into a culture dish containing fresh M199 medium and the surrounding fibroadipose tissues were removed. The proximal and distal 2-mm segments of the aorta were cut away and were sectioned into 1 mm long ring sections after being rinsed in 5e8 washes of M199 medium. Care was ensured for the removal of the remaining blood residues. Then the clotting fibrinogen solution was added into the wells of a 24-well tissue culture plate before these rings were seeded in 24-well plates for clotting. The clotting fibrinogen solution contained M199 medium plus 0.3% fibrinogen and 0.5% 6-aminocaproic acid supplemented with penicillin and streptomycin. After the fibrin clot formation for 30 min, an equal volume of growth media (M199 medium plus 0.5% 6-aminocaproic acid and 10% FBS) containing various concentrations of CPU-12 (0, 2.5, 5 and 10 mm) was added to each well and the whole was incubated at 37 C with 5% CO 2 and the treatment growth media was removed and changed every 2 days for a total of 8 days. The cultures were stained with MTT and the length and density of new growth microvessels quantified and photographed. For each group, five fields were chosen randomly in each well of the replicates, and the inhibition degree of CPU-12 was assessed by manual counting of the number of new microvessels. The use of animals was in accordance with the guidelines established by the National Science Council of The People's Republic of China, with adherence to the ethical guidelines for the care and use of animals Western blot analysis In order to explore the phosphorylation of a key protein in VEGF/VEGFR signal pathway, HUVECs were serum starved overnight and then pre-treated with VEGF (20 ng/ml) for 15 min and treated with or without various concentrations of CPU-12 (2.5, 5, and 10 mm). Cells were washed with cold PBS twice and lysed using RIPA Lysis Buffer (Beyotime Biotechnology, Jiangsu, China) containing protease inhibitors (sodium orthovanadate, sodium fluoride, EDTA, PMSF) and lysates were clarified by centrifugation at 4 C for 10 min at 13,000 g. The concentration of protein in the supernatants was measured by the BCA Assay Kit. Then equal amounts of protein (25 mg) were separated by 10% SDS-PAGE and transferred onto the PVDF membranes (Bio-rad). The blots were incubated for 12 h in 4 C with anti-phospho specific PI3K, ERK1/2, p38 MAPK antibodies or anti-pi3k, ERK1/2, p38 MAPK antibodies, followed by horseradish peroxidase (HRP)-conjugated secondary antibodies (1:5000) for 1 h. Then blots were developed with enhanced chemiluminescence reagent. All blots were stripped and reprobed with polyclonal anti-b-actin antibody to ascertain equal loading of proteins. The densitometric analyses were determined by using ImageJ software (NIH, Bethesda, MD, USA) Statistical analysis The data were presented as the means ± S.D. using GraphPad Prism 5.0 software. All experiments were repeated at least three independent times. The statistical analysis was performed using a two-tailed Student's t-test using Microsoft Excel software (Microsoft Corporation, DC, USA) to calculate differences between groups. A *P value less than 0.05 was considered significant. 3. Result 3.1. CPU-12 inhibits the proliferation of HUVECs Anti-proliferative activity against HUVECs was determined by a normal method i.e. the MTT assay as previously described (16). We

4 12 J. Liu et al. / Journal of Pharmacological Sciences 129 (2015) 9e17 Fig. 2. CPU-12 inhibited cell proliferation of VEGF-stimulated HUVEC in a dosedependent manner. Attached cells were treated with various concentrations (0, 1, 10, 20, 50 and 100 mm) of CPU-12 for 72 h. Cell survival was quantified by MTT assay. Cells receiving 0.1% DMSO and the same concentration of VEGF (20 ng/ml) served as negative control. Data shown were expressed as means ± S.D. The values represented are relative to the negative control. ***P < versus control, n ¼ 5 per group. evaluated the anti-proliferative activity of CPU-12 against HUVECs in vitro. CPU-12 evidently inhibited the proliferation of HUVECs in a dose-dependent manner (Fig. 2). IC 50 values were calculated from a linear regression curve using Graphpad prism software. HUVECs were sensitive to CPU-12 treatment with an IC 50 value at 9.30 ± 1.24 mm CPU-12 has no prominent influence on cell cycle arrest and apoptosis at the tested concentration Cell cycle progression is directly associated with cell proliferation, so we investigated the influence of CPU-12 on the progression of cell cycle. After treatment of HUVECs with or without CPU-12 for 48 h, the percentages of cells in G0/G1, S, and G2/M phages were assessed. In the cell cycle analysis, CPU-12 had no significantly influence on cell populations respectively in the G0/G1, S, and G2/M phages compared with negative control group (Fig. 3A). The results indicated that CPU-12 did not block cell cycle progression. Apoptosis is a normal process which occurs in both physiologic and pathologic conditions of tissue homeostasis. Loss of plasma membrane is one of the earliest certain morphological characteristics of the cell apoptosis (22). Prominent early-stage and latestage apoptotic cells were not observed in the results of flow cytometry (Fig. 3B). These results indicated that the CPU-12- induced inhibition on cell proliferation was not relative to an activity on apoptosis CPU-12 reduces VEGF-stimulated HUVEC migration and chemotactic invasion The migration of HUVECs is a key process of angiogenesis. A wound healing assay was therefore applied to CPU-12 to assess the ability of HUVEC migration inhibition. In the wound healing assay, CPU-12 inhibited cell migration in VEGF stimulated HUVECs in a dose-dependent manner (Fig. 4A). In the negative group, the HUVECs migrated into the denuded area after 24 h and 48 h incubation. Significant inhibition of HUVEC migration into the wounded area was observed in the CPU-12 treated group. Quantitative analysis revealed that treatment with CPU-12 at 0.1 mm, 1 mm, and 10 mm resulted in 83%, 56% and 22% number of migrated cells in contrast with the negative group, respectively. These results demonstrated the potentiality of CPU-12 in inhibiting HUVEC migration in vitro. Fig. 3. CPU-12 can not induce prominent cell cycle arrest and apoptosis at the tested concentration in HUVEC. The VEGF-stimulated HUVECs were treated with various concentrations (2.5, 5, and 10 mm) of CPU-12 for 48 h and analyzed by flow cytometry. (A) No significant cell cycle arrest was observed with the treatment of CPU-12 in HUVEC. HUVECs were treated with various concentrations, while the percentage of cells in G0/G1, S, and G2/M phases were almost the same. (B) No prominent early-stage and late-stage apoptotic cells population appeared in the CPU-12 treatment groups.

5 J. Liu et al. / Journal of Pharmacological Sciences 129 (2015) 9e17 13 Fig. 4. CPU-12 inhibited VEGF-stimulated HUVEC migration and invasion by a scratch wound healing assay and transwell assay. (A) The cell monolayer were wounded (the wound was shown between the two yellow lines in each photo) at 0 h, and then treated with various concentrations (0.1, 1, and 10 mm) of CPU-12 after activated with 20 ng/ml VEGF. Photographs were taken at 0 h, 24 h and 48 h (Magnification: 100.). Quantification of the number of migrated cells (cells between the two yellow lines) was done after 48 h exposure. Data shown were expressed as means ± S.D. The value represented was relative to control (0 mm). **P < 0.01 and ***P < versus control, n ¼ 3 per group. (B) The bottom surface of the entire filter was coated with the diluted Matrigel solution, and HUVECs were treated with various concentrations of CPU-12 and suspended in the upper chamber. Medium in the down chamber contained 20 ng/ml VEGF. After 48 h, the cells on the bottom side of the filter were fixed, stained and visualized using a microscope (Magnification: 100.). Quantitative results of the average number of invasion cells (cells invaded to the lower side of the membrane in the Transwell Chamber) were obtained after 48 h exposure. Data shown were expressed as means ± S.D. The values represented are relative to control (0 mm). *P < 0.05 and **P < 0.01 versus control, n ¼ 3 per group. The penetration of HUVECs into the Matrigel membranes was determined by the transwell chamber assay, as previously described (18). CPU-12 significantly inhibited VEGF-stimulated invasion of HUVECs in a dose-dependent manner (Fig. 4B). In the negative control group, a great quantity of HUVECs invaded from the upper side to the lower side of the membrane in the Transwell Chamber. CPU-12 reduced the VEGF-induced invasion at 74%, 62%, 33% over the control at 0.1 mm, 1 mm, 10 mm respectively. These results demonstrated that CPU-12 could suppress VEGF-induced invasion of HUVECs in vitro CPU-12 reduces VEGF-stimulated capillary tube formation of HUVECs The tube formation assay is based on the differentiation of HUVECs on the Matrigel. HUVEC differentiate and form capillarylike structures on Matrigel in the presence of 10% serum and 20 ng/ml VEGF (23). Under these conditions the formation of tubelike vessels can be used to assess the potential activity of compounds. The scale and density of tube-like structures were effectively reduced by CPU-12 in a dose-dependent manner (Fig. 5). Fig. 5 showed that a reticular and well-defined tube-like structure was formed within 8 h in the negative control group. When HUVECs were exposed to CPU-12, the tube structures were destroyed depending on the compound concentration. At a concentration of 10 mm or higher, the morphogenesis of HUVECs on the Matrigel was evidently inhibited. A partial inhibition of the tube like structure formation which was unable to form meshes was observed when HUVECs were treated with lower concentration (5 mm and 2.5 mm) of CPU-12. Quantitative analysis revealed that treatment with CPU-12 resulted in 31%, 48%, and 68% inhibition of VEGF-induced tube formation at 2.5 mm, 5 mm, and 10 mm, respectively. The interferential tube formation structures and the distinctive morphological cell characteristics resulted are indicative of the potential antiangiogenic activity of CPU CPU-12 suppresses microvessels sprouting from the rat aortic ring The rat aortic ring assay is a valuable method which integrates the advantages of both in vivo and in vitro systems (24). It is a useful assay to test inhibitors in a controlled environment, and it contains the necessary steps involved in angiogenesis (25, 26). In the rat aortic ring assay early formation of new microvessels was first observed on the fourth day in the negative control group. After being cultivated for 8 days, the rat aortic ring was able to generate long and dense new microvessels in the absence of CPU-12. In the treated groups, the formation of microvessels sprouting from the explants of rat aortic ring was inhibited in a dose-dependent manner (Fig. 6). In the group treated with CPU-12 at 2.5 mm, lots

6 14 J. Liu et al. / Journal of Pharmacological Sciences 129 (2015) 9e17 Fig. 5. CPU-12 reduced VEGF-stimulated HUVEC tube formation. The images showed the morphological features of VEGF-stimulated HUVEC on Matrigel in the presence or absence of various concentrations of CPU-12 for 8 h. (A) Negative control (containing 20 ng/ml VEGF), (B) 2.5 mm CPU-12 plus VEGF, (C) 5 mm CPU-12 plus VEGF, (D) 10 mm CPU-12 plus VEGF. (Magnification: 40.). (E) Quantification of the number of tubes after 8 h exposure. Data shown were expressed as means ± S.D. The values represented were relative to the negative control. *P < 0.05 and **P < 0.01 versus control, n ¼ 3 per group. of microvessels still formed, but the area and the length of microvessels were prominently decreased when treated with 5 mm versus the negative group. Almost complete inhibition was observed at the highest concentration of CPU-12 (10 mm). Compared with the negative group, microvessels sprouting from the rat aortic ring were found to be significantly reduced to 82%, 61%, 29% at concentration of 2.5 mm, 5 mm, and 10 mm respectively. These results confirmed that the presence of CPU-12 in the aortic outgrowth has suppressed the process of microvessels sprouting from the aortic wall ex-vivo Influence of CPU-12 on VEGFR-2 related pathways in VEGFstimulated HUVECs To understand the molecular mechanism of action of CPU-12 in VEGF-stimulated HUVECs, we investigated three key proteins expression levels in the VEGFR-2 related downstream signaling, including PI3K, ERK1/2 and p38 MAPK (27). Western blot assays were used for the detection of both phosphorylated and total protein levels of PI3K, ERK1/2 and p38 MAPK, which play significant role in the VEGFR-2 signal pathway. As shown in the results below, the phosphorylation level of these critical proteins was dose-dependently down-regulated in response to the CPU-12 respective treatment groups after the addition of exogenous VEGF into HUVECs (Fig. 7). The phosphorylation of PI3K, ERK1/2 and p38 MAPK activated by VEGF was effectively inhibited by CPU-12, while the total protein levels of PI3K, ERK1/2 and p38 MAPK remained constant. The phosphorylated protein versus total protein values were calculated in order to show the expression level of phosphorylated protein. These results suggest that VEGFR-2 might be a potential target of CPU-12 leading to interference of VEGFstimulated signal transduction.

7 J. Liu et al. / Journal of Pharmacological Sciences 129 (2015) 9e17 15 Fig. 6. CPU-12 suppresses microvessels sprouting from the rat aortic ring. The explants of rat aortic ring were seeded into Matrigel and treated with various concentration of CPU- 12. After further incubation for 8 days, the samples were stained with MTT and visualized using a microscope (Magnification: 40.). (A) Negative control group, (B) 2.5 mm CPU-12 group, (C) 5 mm CPU-12 group, (D) 10 mm CPU-12 group. (E) The number of new microvessels in each treatment group was quantified. Data shown were expressed as means ± S.D. The values represented were relative to the negative control. **P < 0.01 versus control, n ¼ 3 per group. 4. Discussion Angiogenesis is driven by numerous cells including endothelial cells, inflammatory cells and tumor cells by secreting soluble proangiogenic and anti-angiogenic molecules which control neovascularization (28). Angiogenesis plays a major role in the development of pathological diseases, including inflammation, cancer, rheumatic arthritis and arteriosclerosis. Tumors can rarely grow vigorous and metastasize to other organs without the evolvement and promotion of new blood vessels (29). In the process of angiogenesis, many molecules have the capability of inducing the formation of new vessels including angiogenin (30), insulin-like growth factor I (IGF-1) (31), platelet-derived endothelial cell growth factor (PDGF) and vascular endothelial growth factor (VEGF) (32). Among these molecules, VEGF is generally expressed in many different cell types and angiogenesis-related processes as well as diseases. Correspondingly, VEGF receptors are expressed in invading and proliferating endothelial cells and tumor cells. Interfering with the VEGFR-2 signal pathway is promising for antiangiogenesis and antitumor therapy. Oxazolo[5,4-d]pyrimidines have been reported to possess a variety of biological activities including gastric antisecretory activity (33), kinase inhibition, adenosine receptor antagonism and tumor growth inhibition. Interest in this ring system has increased due to recent reports, but the antiangiogenesis activity is barely reported. We try to demonstrate that the newly synthesized compound, CPU- 12, which possesses the oxazolo[5,4-d]pyrimidine structure, has potential in inhibiting angiogenesis. In the inhibition proliferation assay, we found that CPU-12 significantly inhibited HUVEC proliferation in a dose-dependent manner with a relatively low IC 50 value. To evaluate whether the inhibition effect on HUVECs' proliferation by CPU-12 was due to cell

8 16 J. Liu et al. / Journal of Pharmacological Sciences 129 (2015) 9e17 Fig. 7. CPU-12 down-regulate the phosphorylation of PI3K, ERK1/2, and p38 MAPK, and the total steady state remained constant in endothelial cells. Serum-starved HUVECs were activated with VEGF (20 ng/ml) and then treated with or without CPU-12 (0, 2.5, 5, and 10 mm) for 1 h. (A) Protein expression levels of phosphorylated PI3K, ERK1/2, p38 MAPK and total PI3K, ERK1/2, p38 MAPK were analyzed by western blot and normalized with the b-actin. (B), (C) and (D) Representative the densitometric analyses of the expression ratio of p-pi3k vs total PI3K, p-erk1/2 vs total ERK1/2 and p-p38 MAPK vs total p38 MAPK. Data shown were expressed as means ± S.D. The values represented were relative to control (0 mm). *P < 0.05, **P < 0.01 and ***P < versus control, n ¼ 3 per group. cycle arrest or apoptotic activity, cell cycle arrest and apoptosis were assessed by flow cytometry. As the results shown, no prominent early-stage and late-stage apoptotic cells were observed in the dot plot of flow cytometry. It indicated that the CPU-12-induced inhibition proliferative effect was not due to its apoptosis activity. The possible mechanism for CPU-12 to inhibit the HUVEC proliferation is related to the influence of receptor tyrosine kinase, which agrees with the drug design. To determine the antiangiogenic activity of CPU-12, we performed several antiangiogenesis experiments in VEGF-stimulated HUVEC in vitro, including cell migration, invasion and tube formation assay. The results of scratch wound healing assays and transwell assay showed that CPU-12 was capable to inhibit the motility of HUVECs. The tube formation assay is based on the differentiation of HUVECs on a basement membrane matrix. The results showed that CPU-12 disturbed the formation of capillary-like structures. It can be inferred that CPU-12 may affect the differentiation of HUVECs and reduce the tube formation. Several assays are available to assess angiogenesis in vitro and in vivo. Assays in vivo can stimulate the natural process but are often complicated by the recruitment or stimulation of tissue cell which in turn release angiogenic factors that may interfere with angiogenesis (34), while variables of assays in vitro are more likely to be controlled. We use ex-vivo experiments to evaluate the direct effects of compounds on the vascular cells. The rat aortic ring assay, developed by Nicosia et al., can integrate the advantages of both in vitro and in vivo system. It recapitulates the necessary step involved in angiogenesis and can test angiogenic factors or inhibitors in a controlled environment. The results of the rat aortic ring assay showed that the density and length of new microvessels were inhibited by CPU-12. This ex-vivo assay confirmed that CPU- 12 inhibited the nature process of angiogenesis. In our future study, various angiogenesis assay models in vivo will be used for further biological profiling. Vascular endothelial growth factor receptor were formed both homodimers and heterodimers and were accompanied with the autophosphorylation state, by binding with the proper ligand (35). Phosphorylated receptors recruit interacting proteins and induce the activation of signaling pathways that involve a series of second messengers. In the VEGF/VEGFR-2 pathway, the function of ERK1/2 are known to regulate the cell proliferation. While the PI3K and p38 MAPK are associated with cell survival, vascular permeability and cell migration (36e39). Western blot analysis was performed to evaluate the molecular mechanism of CPU-12 against VEGFstimulated angiogenesis in HUVEC. Results indicated that the phosphorylation of PI3K, ERK1/2 and p38 MAPK were effectively inhibited by CPU-12, while the total protein levels of PI3K, ERK1/2 and p38 MAPK remained constant. These decreases of the phosphorylated protein expression level, especially the phosphorylation of PI3K, suggested that CPU-12 might be a potent inhibitor of the VEGFR-2 tyrosine kinase. In conclusion, the oxazolo[5,4-d]pyrimidine derivative, CPU-12 was able to inhibit VEGF-stimulated HUVEC proliferation, migration, invasion and tube formation. We demonstrated that CPU-12 suppressed microvessels sprouting from the rat aortic ring and reduce the phosphorylation of PI3K, ERK1/2 and p38 MAPK

9 J. Liu et al. / Journal of Pharmacological Sciences 129 (2015) 9e17 17 expression level. We demonstrated that oxazolo[5,4-d]pyrimidine derivatives inhibited angiogenesis in vivo and ex-vivo for the first time. These results further suggest that oxazolo[5,4-d]pyrimidine derivatives and similar analogs may be excellent leads in the development of anti-angiogenic and anti-cancer drugs. Conflicts of interest All authors have none to declare. Acknowledgements This work was supported by the National Natural Science Foundation of China (NO , NO ) and Innovation Program for the Postgraduates in Jiangsu Province (NO ). References (1) Wang CH, Duan HJ, He LC. Inhibitory effect of atractylenolide I on angiogenesis in chronic inflammation in vivo and in vitro. Eur J Pharmacol. 2009;612: 143e152. (2) Garcia-Quiroz J, Rivas-Suarez M, Garcia-Becerra R, Barrera D, Martinez-Reza I, Ordaz-Rosado D, et al. 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