Eriocalyxin B, a natural diterpenoid, inhibited VEGF-induced angiogenesis and diminished angiogenesis-dependent breast tumor growth by suppressing VEGFR-2 signaling

Abstract

Eriocalyxin B (EriB), a natural ent-kaurane diterpenoid isolated from the plant Isodon eriocalyx var. laxiflora, has emerged as a promising anticancer agent. The effects of EriB on angiogenesis were explored in the present study. Here we demonstrated that the subintestinal vein formation was significantly inhibited by EriB treatment (10, 15 μM) in zebrafish embryos, which was resulted from the alteration of various angiogenic genes as shown in transcriptome profiling. In human umbilical vein endothelial cells, EriB treatment (50, 100 nM) could significantly block vascular endothelial growth factors (VEGF)-induced cell proliferation, tube formation, cell migration and cell invasion. Furthermore, EriB also caused G1 phase cell cycle arrest which was correlated with the down-regulation of the cyclin D1 and CDK4 leading to the inhibition of phosphorylated retinoblastoma protein expression. Investigation of the signal transduction revealed that EriB inhibited VEGF-induced phosphorylation of VEGF receptor-2 via the interaction with the ATP-binding sites according to the molecular docking simulations. The suppression of VEGFR-2 downstream signal transduction cascades was also observed. EriB was showed to inhibit new blood vessel formation in Matrigel plug model and mouse 4T1 breast tumor model. EriB (5 mg/kg/day) treatment was able to decrease tumor vascularization and suppress tumor growth and angiogenesis. Taken together, our findings suggested that EriB is a novel inhibitor of angiogenesis through modulating VEGFR-2 signaling pathway, which could be developed as a promising anti-angiogenic agent for treatment of angiogenesis-related human diseases, such as cancer.

Keywords: Eriocalyxin B; angiogenesis; breast cancer; vascular endothelial growth factor (VEGF); vascular endothelial growth factor receptor 2 (VEGFR-2).

Figure 1. The inhibitory effects of EriB on the formation of subintestinal vessel (SIV) of Tg(fli1:EGFP) zebrafish embryos

A. Chemical structure of EriB. B. The SIVs of zebrafish embryos treated with vehicle control (0.1% DMSO) developed into a smooth basket-like structure. Embryos treated with EriB (5, 10, 15 μM) for 72 h leads to an inhibition of SIV formation (magnification, ×100). C. The average length of SIVs was measured as described in materials and methods in total 80 zebrafish embryos. Each values was presented as means + SEM (n=80), **p < 0.01, ***p < 0.001 compared with control group (one-way ANOVA).

Figure 2. EriB exerted anti-angiogenic effect via the modulations of angiogenic genes expression and interaction with the ATP-binding sites of VEGFR-2

A. Heat map of 72 angiogenic genes (Supporting Information Table S1) expressions in zebrafish embryos after treatment with control or EriB (10 and 15 μM) for 72 h determined by transcriptome analysis (p < 0.05). The up-regulated mRNA expression in treated group with respect to control was represented by red colour and down-regulated mRNA expression was presented as green colour. The scale of color intensity was positively correlated to the fold change. B. Real-time PCR validation of selected gene expressions (Fold change >2). The specific genes name: anxa6, annexin A6; cdkn1a, cyclin-dependent kinase inhibitor 1A; Cebpg, CCAAT/enhancer binding protein (C/EBP), gamma; gdf5, growth differentiation factor 5; igfbp6b, insulin-like growth factor binding protein 6b; kdr, kinase insert domain receptor; lgals3bpb, galactoside-binding, soluble, 3 binding protein b; loxl1, lysyl oxidase-like 1; pcdh10a, protocadherin 10a; serpinf1, serpin peptidase inhibitor, clade F (alpha-2 antiplasmin, pigment epithelium derived factor), member 1; sesn2, sestrin 2; sfrp2, secreted frizzled-related protein 2; spon2b, extracellular matrix protein; txn, thioredoxin. C. The interactions of EriB to the amino acid residues in the ATP-binding site of VEGFR-2. EriB could stably bind to the ATP-binding pocket near the hinge region, with the interaction with residues Cys 919, Phe 1045, Val 848, Glu 917, Phe 918, Leu 1035, Leu 840.

Figure 3. EriB inhibited VEGF-induced cell viability, cell proliferation and also suppressed in vitro angiogenesis in HUVECs

A. Different concentrations of EriB (25, 50 and 100 nM) were added to HUVECs in the absence or presence of VEGF (10 ng/mL) for 48 h, then the cell viability were assessed by MTT assay. B. BrdU incorporation assay was performed to detect the effect of EriB on cell proliferation when treated with EriB for 48 h in the absence or in the presence of VEGF. C. EriB inhibited the VEGF-induced tube formation after incubation for 8 h and tube structures of HUVECs were photographed (magnification, ×40). D. EriB suppressed the VEGF-induced cell migration after the incubation for 16 h and the wounded area of each well was captured at 0 h and 16 h of incubation (magnification, ×40). E. EriB attenuated VEGF-induced cell invasion after 8 h incubation, the migrated cells on the lower side of membranes were stained and counted (magnification, ×100). Each value was presented as means + SD (n=3). * p < 0.05, ** p < 0.05 compared with control, ^# p < 0.05, ^## p < 0.01, ^### p < 0.001 compared with VEGF alone group (one-way ANOVA).

Figure 4. EriB caused G1 arrest via the modulation of p21-cyclin D1/CDK4-pRb pathway

A. Representative histograms of the cell cycle obtained after flow cytometry analysis when HUVECs were treated with EriB (25, 50 and 100 nM) in the presence of VEGF (10 ng/mL) for 24 h. B. Quantification of the cell populations in each phase of the cell cycle was presented as means + SD (n=3). ** p < 0.01 compared with control, ^# p < 0.05 compared with VEGF group (one-way ANOVA). C. HUVECs were incubated with EriB and VEGF (10 ng/ml) for 16 h. Then the cells were harvested and western blotting was performed. D. The histograms showed quantified results of protein levels, which were adjusted with corresponding GAPDH protein level. Each value was expressed as fold of control mean + S.D. (n=3). * p < 0.05 compare with control, ^# p <0.05 compared with VEGF group (one-way ANOVA).

Figure 5. EriB inhibited the activation of VEGFR-2 induced by VEGF and suppressed VEGFR-2-mediated downstream signaling pathway

A. HUVECs were pretreated with various concentrations (25, 50 and 100 nM) of EriB for 24 h before exposure to VEGF (10 ng/mL) for 30 min. Then the cells were harvested and western blotting was performed to detect pTyr¹¹⁷⁵-VEGFR-2 pTyr¹²¹³-VEGFR-1 and total VEGFR-2, VEGFR-1 expressions. B. HUVECs were pretreated for 24 h with various concentrations (25, 50 and 100 nM) of EriB before exposure to VEGF (10 ng/mL) for 120 min. Then whole cell extracts were extracted for western blotting analysis to detect modulation of VEGFR-2-mediated signaling pathway. C. The histograms showed quantified results of protein levels, which were adjusted with corresponding GAPDH protein level. Each value was expressed as fold of control mean + S.D. (n=3). * p < 0.05, ** p < 0.01 compared with control, ^# p < 0.05 compared with VEGF group (one-way ANOVA).

Figure 6. EriB-mediated anti-angiogenesis effect in Matrigel plug assay

A. C57 mice were injected with Matrigel in the presence or absence of EriB (10 and 20 μM) for 7 days and sacrificed to obtain the Matrigel plugs. The appearance of Matrigel plugs from each group was presented. B. Hemoglobin content of Matrigel plugs from each group was measured using Drabkin's reagent kit. Each value was presented as means + SEM (n=3). * p < 0.05, ** p < 0.01 compared with control (one-way ANOVA). C. Quantification of the number of blood vessels in frozen sections of the Matrigel plugs were presented as means + SD (n=2). ** p < 0.01, *** p < 0.001 compared with control (one-way ANOVA). D. Representative photos of blood vessels stained with H&E (magnification, ×100), scale bar: 100 μm.

Figure 7. EriB inhibited tumor angiogenesis and suppressed tumor growth in mouse 4T1 breast tumor model

Female BALB/c mice were injected subcutaneously with 4T1 cells for 6 days before EriB treatment A. The growth of 4T1 xenograft tumors in mice after control (vehicle), EriB (5 mg/kg) treatments. The final tumor weight B. and body weights C. were measured. Each value was presented as means + SEM (n=13). * p < 0.05, *** p < 0.001 compared with control (Student's t-test). Representative tumor image D. and tumor size E. was shown. F. 4T1 tumor tissues were stained with anti-Ki67 (brown staining), anti-VEGFR-2 (brown staining) anti-VEGF (brown staining), and anti-CD 31(blood vessels) antibodies (magnification, ×100, ×400), scale bar: 100 μm, 25 μm. G. Results are shown as the relative percentages of the positive cells in the total number of cells per section. Each value was presented as means + SD (n=4). ** p < 0.01, *** p < 0.001, compared with control (Student's t-test).