SCIENCE CHINA Life Sciences, Volume 60, Issue 2: 202-214(2017) https://doi.org/10.1007/s11427-016-0369-6

A novel synthetic small molecule YF-452 inhibits tumor growth through antiangiogenesis by suppressing VEGF receptor 2 signaling

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  • ReceivedNov 30, 2016
  • AcceptedDec 26, 2016


Tumor angiogenesis is characterized by abnormal vessel morphology, endowing tumor with highly hypoxia and unresponsive toward treatment. To date, mounting angiogenic factors have been discovered as therapeutic targets in antiangiogenic drug development. Among them, vascular endothelial growth factor receptor 2 (VEGFR2) inhibitors exerts potent antiangiogenic activity in tumor therapy. Therefore, it may provide a valid strategy for cancer treatment through targeting the tumor angiogenesis via VEGFR2 pathway. In this study, we established a high-profile compounds library and certificated a novel compound named N-(N-pyrrolidylacetyl)-9-(4-bromobenzyl)-1,3,4,9-tetrahydro-β-carboline (YF-452), which remarkably inhibited the migration, invasion and tube-like structure formation of human umbilical vein endothelial cells (HUVECs) with little toxicity invitro. Rat thoracic aorta ring assay indicated that YF-452 significantly blocked the formation of microvascular exvivo. In addition, YF-452 inhibited angiogenesis in chick chorioallantoic membrane (CAM) and mouse corneal micropocket assays. Moreover, YF-452 remarkably suppressed tumor growth in xenografts mice model. Furthermore, investigation of molecular mechanism revealed that YF-452 inhibited VEGF-induced phosphorylation of VEGFR2 kinase and the downstream protein kinases including extracellular signal regulated kinase (ERK), focal adhesion kinase (FAK) and Src. These results indicate that YF-452 inhibits angiogenesis and may be a potential antiangiogenic drug candidate for cancer therapy.

Funded by

Major State Basic Research Development Program of China(2015CB910400)

National Natural Science Foundation of China(81272463,81472788,81330049,81673304)

The Science and Technology Commission of Shanghai Municipality(15431902200)


This work was supported by Major State Basic Research Development Program of China (2015CB910400), National Natural Science Foundation of China (81272463, 81472788, 81330049, 81673304), and The Science and Technology Commission of Shanghai Municipality (15431902200).

Interest statement

The author(s) declare that they have no conflict of interest. All procedures performed in studies involving animals were in accordance with the ethical standards of the animal investigation committee of the Institute of Biomedical Sciences, East China Normal University.



Figure S1 Migration inhibitory rate of selected nine compounds on HUVECs.

Figure S2 YF-452 concentration and time dependently suppressed HUVECs migration.

Figure S3 Vandetanib and sunitinib concentration dependently suppressed HUVECs invision.

Figure S4 Compared with vandetanib and sunitinib, YF-452 showed a better selectivity on HUVEC than PC-3 cells in MTS assay.

Figure S5 The synthesis route of small molecule compounds YF-452.

The supporting information is available online at life.scichina.com and www.springerlink.com. The supporting materials are published as submitted, without typesetting or editing. The responsibility for scientific accuracy and content remains entirely with the authors.


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  • Figure 1

    (Color online) Inhibitory effect on HUVECs migration, invasion and tube formation and toxicity evaluation of YF-452 in vitro. A, Chemical structure of YF-452. B, YF-452 inhibited HUVECs migration in wound healing assay. The cells were pretreated with mitomycin C to inhibit proliferation before inducing migration. Confluent HUVEC monolayers were scratch wounded and the cells were treated with indicated concentrations of YF-452 (0, 2.5, 5, 10, 20 μmol L−1), then photographed at different times (6 or 12 h), and the migrated cells were quantified by manual counting. Technical replicates n=3. C, YF-452 inhibited HUVECs invasion in transwell assay. HUVECs were seeded in the top chamber and treated with different concentrations of YF-452. After 12 h, the cells that invaded through the membrane were stained and quantified. Technical replicates n=3. D, YF-452 inhibited capillary-like tube formation of HUVECs. After treated with indicated concentrations of YF-452 for 10 h, capillary-like structure in each group was quantified by IPP 6.0 software. The percentage of inhibition was expressed using control as 100%. Technical replicates n=3. E, HUVECs YF-452 treated for 12 h were assayed for apoptosis by flow cytometry using an annexin V-FITC/PI apoptosis detection kit. F, HUVECs were plated on 0.1% gelatin-coated 96 well plates and then treated with the indicated concentrations of YF-452. Cell viability at different time (6, 12, 24, 48 h) was determined by MTS assay. Technical replicates n=3. Error bars, SD. **, P<0.01; ***, P<0.001.

  • Figure 2

    YF-452 inhibits angiogenesis ex vivo and in vivo. A, Representative images (left panel) and the optical density (right panel) of microvessels sprouting from rat thoracic aorta rings in the absence or presence of YF-452. The microvessel density of the untreated group was considered as 100%. Technical replicates n=3. B, YF-452 inhibited the formation of new blood vessels branches in CAM assay. Arrows pointed the new microvessels. Statistics indicated the percentage of inhibition and expressed using untreated group as 100%. Technical replicates n=8. C, YF-452 inhibited VEGF-induced angiogenesis in the mouse corneal micropocket assay. Red arrows pointed the micropellets and yellow arrows pointed VEGF-induced neovasculature. The statistical results showed the clock number, new vessel length and vessel area in each group. Images of both CAM and mouse corneal micropocket assays were obtained with OLYMPUS stereomicroscope. Technical replicates n=8. Error bars, SD. **, P<0.01; ***, P<0.001.

  • Figure 3

    (Color online) Antitumor effect and toxicity evaluation of YF-452 in vivo. PC-3 tumor-bearing nude mice were treated as described with YF-452 at 20, 40 mg kg−1 or with vehicle. A, Compared with vehicle group, YF-452-treated groups showed significant inhibition of tumor growth. Technical replicates n=8. B, The treatment with YF-452 resulted in significant inhibition of tumor volume versus vehicle control. Technical replicates n=8. C, YF-452 did not cause obvious change of body weights. D, YF-452 did not cause obvious pathologic abnormalities in normal tissues. HE staining of paraffin-embedded sections of the hearts, livers, spleens, lungs and kidneys. Error bars, SD. **, P<0.01; ***, P<0.001.

  • Figure 4

    YF-452 inhibits tumor angiogenesis and induces necrosis as well as apoptosis in xenograft mouse model. A, YF-452 inhibited angiogenesis in tumor. B, CD31 staining was performed to indicate the blood vessels in tumor tissues. YF-452 apparently decreased CD31 fluorescent signal (green) in treated groups verse vehicle group. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (blue). Graph depicting CD31 immunofluorescence signal strengths in tumor tissues. C, Effect of YF-452 on the structural changes of tumors. Histopathological changes were observed under OLYMPUS stereomicroscope. Statistics indicated the percentage of necrotic areas per field (asterisks showed the necrotic tissue). D, Apoptosis was measured by cleaved caspase-3 staining in tumor sections. The treatment with YF-452 resulted in remarkably increased apoptosis versus vehicle control. Arrows indicated cleaved caspase-3 positive apoptotic cells. Statistics were calculated by cleaved caspase-3 positive cells/the total number of cells per field. Three random tumors were selected in three groups. Error bars, SD. *, P<0.05; **, P<0.001; ***, P<0.001.

  • Figure 5

    YF-452 inhibits the phosphorylation of VEGFR2 and its downstream signaling mediators. A, YF-452 suppressed the phosphorylation of VEGFR2 induced by VEGF in HUVECs. YF-452 also suppressed VEGFR2-mediated protein kinase activation of FAK, Src and ERK. B, To rule out YF-452 affecting tumor cells directly, expression of VEGFR2 and p-VEGFR2 were detected in HUVECs and PC-3 cells. Compared with HUVECs, a significantly lower expression of VEGFR2 in PC-3 cells was not sufficient to activate the phosphorylation of VEGFR2 and various downstream signaling molecules responsible for cell proliferation. C, The sensitivity of COS-7 cells to YF-452 was significantly increased after overexpression of VEGFR2. Error bars, SD. ***, P<0.001.

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