Bufalin suppresses tumour microenvironment-mediated angiogenesis by inhibiting the STAT3 signalling pathway

Abstract

Background: Antiangiogenic therapy has increasingly become an important strategy for the treatment of colorectal cancer. Recent studies have shown that the tumour microenvironment (TME) promotes tumour angiogenesis. Bufalin is an active antitumour compound whose efficacy has been indicated by previous studies. However, there are very few studies on the antiangiogenic effects of bufalin.

Methods: Herein, human umbilical vein endothelial cell (HUVEC) tube formation, migration and adhesion tests were used to assess angiogenesis in vitro. Western blotting and quantitative PCR were used to detect relevant protein levels and mRNA expression levels. A subcutaneous xenograft tumour model and a hepatic metastasis model were established in mice to investigate the influence of bufalin on angiogenesis mediated by the TME in vivo.

Results: We found that angiogenesis mediated by cells in the TME was significantly inhibited in the presence of bufalin. The results demonstrated that the proangiogenic genes in HUVECs, such as VEGF, PDGFA, E-selectin and P-selectin, were downregulated by bufalin and that this downregulation was mediated by inhibition of the STAT3 pathway. Overexpression of STAT3 reversed the inhibitory effects of bufalin on angiogenesis. Furthermore, there was little reduction in angiogenesis when bufalin directly acted on the cells in the tumour microenvironment.

Conclusion: Our findings demonstrate that bufalin suppresses tumour microenvironment-mediated angiogenesis by inhibiting the STAT3 signalling pathway in vascular endothelial cells, revealing that bufalin may be used as a new antiangiogenic adjuvant therapy medicine to treat colorectal cancer.

Keywords: Angiogenesis; Bufalin; Colorectal cancer; STAT3; Tumour microenvironment.

Fig. 1

Bufalin suppresses angiogenesis induced by cells in the tumour microenvironment cells. a Molecular structure of BU. b Cell viability of HUVECs after treated with BU for 24 h. c Cell proliferation of HUVECs after treated with different TME-CMs for 24 h. The effect of bufalin (BU) on the tube formation (d), migration (e) and adhesion (f) of HUVECs in response to different TME-CMs. *P < 0.05, **P < 0.01, ***P < 0.001. Data are shown as mean s.e.m. BU, bufalin

Fig. 2

Bufalin suppresses TME-mediated angiogenesis by inhibiting angiogenic factors. Relative PDGFA, VEGF, E-selectin and P-selectin mRNA expression in HUVECs after treatment with CT26-CM (a), CAF-CM (b) or TMA-CM (c) with or without bufalin (BU). d–f WB showing the protein expression of VEGF, p-STAT3 and STAT3 in HUVECs treated with different TME-CMs in the presence or absence of BU, with total β-actin as a control. g–i The concentration of VEGFA in HUVECs supernatant after treatment with different TME-CMs in the presence or absence of BU. j Immunofluorescence analysis showing p-STAT3⁺ HUVECs after treatment with different TME-CMs in the presence or absence of BU. *P < 0.05, **P < 0.01, ***P < 0.001. Data are shown as mean s.e.m. BU, bufalin

Fig. 3

Bufalin suppresses TME-mediated angiogenesis by the STAT3 signalling pathway. Tube formation (a), migration (b) and adhesion (c) of HUVECs after treatment with TME-CMs and BU with or without the STAT3-OE plasmid. *P < 0.05, **P < 0.01, ***P < 0.001. Data are shown as mean s.e.m. BU, bufalin

Fig. 4

Bufalin suppresses TME-mediated angiogenesis by directly affecting vascular endothelial cells. Tube formation (a), migration (b), and adhesion (c) of HUVECs treated with TME-CMs and TME + BU-CMs for 24 h. d–f WB showing the protein expression of p-STAT3 and STAT3 in HUVECs treated with different TME-CMs and TME + BU-CMs, membranes were stripped and re-probed with total β-actin as a control. N.S. indicates no significant difference between the two groups, P > 0.05. Data are shown as mean s.e.m. BU, bufalin

Fig. 5

Bufalin inhibits CRC cell growth via an antiangiogenic mechanism in vivo. a Scheme and schedule of imaging and treatments. b Tumour volumes from day 0 to day 28. c Tumour growth was visualized by an in vivo imaging system from day 7 to day 28. d Tumour weights and images. e IHC analysis of CD31 and Ki67 in tumours. f VEGF expression level in serum. g Immunofluorescence analysis of CD31 and p-STAT3 in tumours. **P < 0.01, ***P < 0.001. Data are shown as mean s.e.m. IHC, immunohistochemistry

Fig. 6

Bufalin inhibits CRC cell metastasis via an antiangiogenic mechanism in vivo. a Scheme and schedule of imaging and treatments. b Tumour metastasis was visualized by an in vivo imaging system from day 7 to day 21. c Representative images of liver and H&E-stained liver tissue. d Tumour number and area in liver. e IHC analysis of CD31 in spleen and liver tissue. f VEGF expression level in serum. g Immunofluorescence analysis of CD31 and p-STAT3 in liver. **P < 0.01, ***P < 0.001. Data are shown as mean s.e.m. IHC, immunohistochemistry

Fig. 7

The mechanism of BU inhibiting tumor microenvironment-mediated angiogenesis