Abstract
Objective: To evaluate the effects of endothelial cell-targeted soluble Notch ligand hD1R protein on the proliferation of acute myeloid leukemia (AML) cells. Methods: The expression levels of Notch1, Notch2, Notch3, Notch4, Hes1 in bone marrow CD34(+) cells from 24 cases of untreated AML (AML group) and 9 healthy controls (control group) were determined by real time quantitative polymerase chain reaction (PCR) . Recombinant hD1R protein was first induced and purified. Bone marrow CD34(+) cells were co-cultured on human umbilical vein endothelial cells (HUVEC) supplemented with a cocktail containing 5 types of human cytokines (5GF) and soluble hD1R. The cultured cells were tested under different culture conditions including PBS group (PBS replaces HUVEC) , hD1R group, 5GF group, GSI group (hD1R plus GSI) . Proliferation and apoptosis of cultured cells were also analyzed. Real time quantitative PCR was used to test the expression levels of Hes1 and Bcl-2 in cultured cells. Results: The expression levels of Notch1 and Hes1 in primary AML patients were significantly lower, and Notch4 expression was higher compared to the control group (P<0.05) . Cell counting showed a remarkable decrease of AML cells number in the culture with hD1R compared with that in the PBS group[ (0.74±0.13) ×10(5) vs (2.16±0.21) ×10(5), P<0.01]. FACS analysis showed that the percentage of AML cells was (18.48±2.51) % in apoptosis, which was higher than that of control cells (3.19±0.58) % after co-culture with hD1R. AML cells in the hD1R group underwent significantly increased apoptosis compared with those in the PBS one (P<0.05) . Moreover, apoptosis of AML cells in the GSI group was lower than that in the hD1R one (P<0.05) . Apoptosis in the PBS group also decreased compared with that in the 5GF one (P<0.05) . Finally, hD1R up-regulated Hes1 expression and inhibited Bcl-2 one in the AML cells. Conclusion: hD1R effectively activated Notch signaling and down-regulated Bcl-2 mRNA in AML cells, which lead to cell apoptosis.