Euphorbia ebracteolata Hayata (EEH) inhibited SIV ectopic hyperplasia induced by melanoma cells in transgenic Tg (flk1:GFP) zebrafish embryos. Human melanoma cells were microinjected into zebrafish embryos at 48 hpf, and the resulting xenografts were imaged with a fluorescence microscope after 24 and 48 h (hpi), respectively. (a, d) SIV ectopic vessels in green fluorescence (red arrows) at 24 hpi zebrafish embryo (control). (b, e) SIV ectopic vessels (red arrows) at 48 hpi in zebrafish embryo (control). (c, f) SIV ectopic vessels after treatment with 50 µg/mL EEH for 48 (h). (g) The length of ectopic blood vessels in A2058 or HUVEC xenografts at 24 and 48 hpi and the effects after treatment with Euphorbia ebracteolata Hayata (EEH) (p<0.05). (h) The number of ectopic blood vessels in A2058 or HUVEC xenografts at 24 hpi and 48 hpi and the effects after treatment with EEH (p<0.05). Scale bar = 200 µm.

Euphorbiaceae compound B (ECB) inhibited SIV ectopic hyperplasia induced by melanoma cells in transgenic Tg (flk1:GFP) zebrafish embryos. Human melanoma cells were microinjected into zebrafish embryos at 48 hpf, and the resulting xenografts were imaged with a fluorescence microscope after 24 and 48 h (hpi), respectively. (a, e) SIV ectopic vessels in green fluorescence (red arrows) at 24 hpi zebrafish embryo (control). (b, f) SIV ectopic vessels (red arrows) at 48 hpi zebrafish embryo (control). (c, g) SIV ectopic vessels after treatment with 20 µg/mL ECB for 48 h. (d, h) SIV ectopic vessels after treatment with 1 µM PTK for 48 h. (i) The length of ectopic blood vessels in A2058 or HUVEC xenografts at 24 and 48 hpi and the effects after treatment with ECB and PTK (p<0.05). (j) The number of ectopic blood vessels in A2058 or HUVEC xenografts at 24 hpi and 48 hpi and the effects after treatment with ECB or PTK (p<0.05). Scale bar = 200 µm.

Effect of ECB on SIV and ISV angiogenesis in zebrafish embryos. Zebrafish embryos were imaged after treatment with ECB or PTK from 4 hpf to 72 hpf. (a, b) Control group. (b, e) ECB group. (c, f) PTK group. Red arrows point to SIV vessels above the yolk region (a–c). Yellow arrows show ISVs in the tail region (d–f). (g) The area covered by SIV vessels in zebrafish embryos (p<0.001). (h) The perimeter (length) of SIV vessels in zebrafish embryos (p<0.001). Scale bar = 200 µm.

Effect of Euphorbiaceae compound B (ECB) on metastasis of melanoma cells in zebrafish xenografts. Zebrafish embryos were injected with A2058 cells at 48 hpf and observed at 6 hpi, 24 hpi, and 48 hpi, respectively. (a) Not injected. (b–d) A2058 cells were injected into zebrafish embryos at 48 hpf and observed at 6 hpi, 24 hpi, and 48 hpi, respectively. (e) A2058 cells were injected into zebrafish embryos and treated with 20 µg/mL ECB for 24 h and observed at 48 hpi. (f) A2058 cells were injected into zebrafish embryos and treated with 1 µM PLX4720 (PLX) for 24 h and observed at 48 hpi. (g) The area of CM-DiI labeling (melanoma cell) in the zebrafish embryos. Scale bar = 200 µm. indicates a significant difference (p<0.001).

Effect of Euphorbiaceae compound B (ECB) on zebrafish vegfa, vegfr2, and vegfr3 mRNA expression in xenografts using real-time PCR: (a) vegfa mRNA expression, (b) vegfr2 mRNA expression, and (c) vegfr3 mRNA expression. ∗∗ and ∗∗∗ indicate a significant difference (p<0.01; p<0.001).

Euphorbiaceae compound B (ECB) affected the expression of zebrafish casp3a and p53mRNA in xenografts using real-time PCR. A2058 cells were labeled with CM-DiI, injected into zebrafish embryos at 48 hpf, and treated with 20 µg/mL ECB and 1 µM PLX or the medium only control at 72 hpf (24 hpi), respectively. Casp3a (a) and p53 (b) mRNA expressions were quantified. p<0.05, p<0.01, and p<0.001.