Inhibition of RPMI-8226 myeloma cell xenografted tumor angiogenesis by down-regulation of Notch1

Objective To investigate the effects of Notch1 siRNA on VEGF and angiogenesis of myeloma cell line RPMI-8226 in vitro and in vivo. Methods In vitro, Notch siRNA was transfected into RPMI-8226 cells, and then cell supernatant VEGF secretion was detected using ELISA method. Expression levels of Notch1 and VEGF proteins were assayed by Western blotting. RPMI-8226 cells were subcutaneously transplanted in NOD/SCID mice, and then the tumor mice were divided into three groups randomly: NS group (Notch1 siRNA-transfected group), CS group (Control siRNA-transfected group) and UN group (Untransfected group), and the changes of tumor volume were observed. Immunohistochemical staining was used to detect the changes in expression levels of Notch1, VEGF and CD34. Results Notch1 and VEGF proteins expressions of RPMI-8226 cells were significantly decreased by Notch1 siRNA. At 48 h and 72 h, VEGF secretion level in NS group was significantly different with CS group [(120±25)ng/L∶(175±15)ng/L, t=3.27, P<0.05; (145±24)ng/L∶(295±17)ng/L, t=8.83, P<0.01]. At 13 d, 17 d and 21 d, tumor volume in NS group was significantly reduced, that was significantly different with CS group [(1 548±218)mm3∶(1 820±64)mm3,t=2.68, P<0.05; (1 200±75)mm3∶(2 180±84)mm3,t=19.46, P<0.01; (1 150±88)mm3∶(2 250±145)mm3,t=14.50, P<0.01]. The expression levels of Notch1 and VEGF protein were decreased by Notch1 siRNA. The expression levels of Notchl and VEGF in NS group were different with CS group [(16.33±2.52)%∶(75.33±2.52)%, t=28.71, P<0.01; (5.00±1.00)%∶29.67±2.08 %, t=18.50, P<0.01]. Notch1 siRNA reduced the number of transplanted tumor neovascularization in NS group. Microvascular density in NS group was significantly less than that in CS group [(14.67±2.52)∶(30.00±5.00), t=4.74, P<0.01]. Conclusion In vitro, Notch siRNA reduces human myeloma cell RPMI-8226 cell supernatant VEGF secretion. In vivo, Notch siRNA can reduce tumor volume and the number of new blood vessels in transplanted-multiple myeloma mice. Thus, Notch1 is an effective molecular target for anti-angioge-nesis in myeloma. Key words: Multiple myeloma; Vascular endothelial growth factors; RNA, small interfering; Receptor, Notch1