Purpose To observe the effects of midkine ( MK) on human breast cancer cell line MDA-MB-231 angiogenesis in vitro, and to explore its mechanism. Method shRNA interference was performed to silence the expression of MK in MDA-MB-231 cells, and Western blot was used to identify the expression of MK and EPCR. After MK and EPCR knockdown, or treated with anti protease-activated receptor 1 (PAR1) antibody, the culture medium of MDA-MB-231 cells were collected and the conditioned medium were pre-pared. Human umbilical vein endothelial cells ( HUVECs) were cultured with conditioned medium, and the endothelial cells prolifera-tion was detected by CCK-8 assay, cell migration was detected by transwell method, vasculogenic activity was assessed by Matrigel-based tube formation assay. Results After knockdown of MK, the protein level of EPCR was decreased in MDA-MB-231 cells. Com-pared with control, knockdown of MK and EPCR decreased the proliferation, migration and angiogenesis ability of HUVECs significant-ly (P<0. 05), and the effect of EPCR knockdown group was stronger than MK knockdown group (P<0. 05). After treated with anti-PAR1 antibody, the proliferation, migration and angiogenesis ability of HUVECs were decreased compared with control and EPCR knockdown group (P<0. 05). Conclusion MK promotes human breast cancer cell line MDA-MB-231 angiogenesis through EPCR /PAR1 signaling pathway in vitro.

Objective: To investigate the effect of cinobufacin combined with As₂O₃ on the angiogenesis of subcutaneous transplantation tumor in colorectal carcinoma BALB/C nude mice. Methods: The colorectal carcinoma transplantation tumor model of BALB/C nude mice was established and divided into 4 groups, 5 mice in each group. The growth state of nude mice was observed by injecting the corresponding reagents into the tumor in As₂O₃ group, cinobufacin group, cinobufacin combined As₂O₃ group and the blank control group (replaced by phosphate buffer), respectively. The nude mice were killed two weeks later, and the tumor tissues, liver and kidney tissues and orbital vein blood were taken. The tumor volume inhibition rate and mass inhibition rate of nude mice were calculated. The histomorphology of tumor, liver and kidney and blood routine were detected. The expressions of vascular endothelial growth factor (VEGF), epidermal growth factor receptor (EGFR), fibroblast growth factor-basic (b-FGF) and CD105 in transplanted tumor tissues were detected by using immunohistochemistry method, and the microvascular density (MVD) of transplanted tumor in nude mice was evaluated. Western blot method was used to detect the protein expression levels of VEGF, EGFR and b-FGF. Results: After 2 weeks of administration, the tumor volume and tumor mass in As₂O₃ group, cinobufacin group and cinobufacin combined As₂O₃ group were lower than those in the blank control group. The volume inhibition rate was 16%, 17%, 72%, and the mass inhibition rate was 31%, 33%, 78%, respectively, and the difference was statistically significant (all P < 0.01); there was no statistical difference between As₂O₃ group and cinobufacin group in the tumor volume and tumor mass (P > 0.05), and cinobufacin combined As₂O₃ group had the most obvious therapeutic effects (all P < 0.05). The immunohistochemistry method showed that the expressions of VEGF, EGFR, b-FGF and MVD were decreased in As₂O₃ group, cinobufacin group and cinobufacin combined As₂O₃ group compared with the blank control group, and there were statistical differences of the four groups (all P < 0.05). There was no statistical difference of the marker changes in As₂O₃ group and cinobufacin group (all P > 0.05), and cinobufacin combined As₂O₃ group had the most significant decrease in the marker changes, and there was a significant difference compared with the other groups (all P < 0.05). Western blot showed that compared with the blank control group, the other three groups could downregulate the protein expressions of VEGF, EGFR and b-FGF in the transplanted tumor of nude mice. And the decreasing expression of each protein in cinobufacin combined As₂O₃ group was the most significant. Pathomorphology examination showed that the histomorphology of liver and kidney of the four groups of nude mice was normal. Blood routine examination showed that compared with the blank control group, the white blood cell count of nude mice in other groups was decreased, and the difference was statistically significant (all P < 0.05). The white blood cell count of cinobufacin combined As₂O₃ group was decreased most significantly; there was no statistically significant difference in hemoglobin and platelet count among the four groups (all P > 0.05). Conclusions Cinobufacin and As₂O₃ show synergistic effects in the tumor angiogenesis and inhibition of transplantation tumor growth of colorectal cancer BALB/C nude mice. Moreover, cinobufacin and As₂O₃ have no obvious toxicity to the hepatic, kidney and hematopoietic tissues.