The Differentiation Induced By Rabdosin B,An Ent-Kaurane Diterpene,in Human Promyelocytic Leukemia Cells HL-60

In this paper, the differentiation of HL-60 to mature granulocyte caused by rabdosin B were studied, meanwhile,if it mediated by NADPH oxidase-derived reactive oxygen species was explored.The effects of rabdosin B, a kind of ent-kaurane diterpenoids, on thegrowth, cellular morphology, NBT-reducing ability, cellular uptake, cell ROS and cellsurface markers CD11 b of HL-60 cells were determined by trypan blue exclusionmethod, giemsa stainning and flow cytometry. Further detection displayed that the inducement of differentiation by rabdosin B was inhibited by N-acetyl-L-cysteine(NAC, an antioxidant) andapocynin/diphenyleneiodonium(APO/DPI, the inhibitors of NADPH oxidase). Namely, the increase of ROS was associated with NADPH oxidase, it revealed NADPH oxidase-derived reactive oxygen species mediated HL-60 cells differentiation caused by rabdosin B.The results as follows:1.The results of trypan blue staining method showed that 1.0 ?mol/L, 3.0 ?mol/L and 5.0 ?mol/L rabdosin B had a dose- and time-dependent growth inhibitioneffect on HL-60 cells. 1.0 ?mol/L rabdosin B and 1.2 ?mol/L ATRA treatment group had similar results at 48 h.2. The results of PI staining flow cytometry indicated that G0/G1 phase arrest and size, granularity changes of HL-60 cells caused by rabdosin B in a dose-dependent manner.3.The results of giemsa stainning, NBT-reducing ability, cellular uptake and flowcytometry showed that metamyelocytes and lobate nucleus increased, the ratio ofnuclear/cytoplasm decreased; NBT-reducing ability, cellular uptake and theexpression of CD11 b of HL-60 cells enhanced markedly in a obvious dose-dependentmanner. These indicated that differentiation of human HL-60 cells into granulocytecould be induced by rabdosin B.4. The results of DCFH-DA staining and flow cytometry indicated that the level of ROS increased obviously in cells treated with1.0 ?mol/L and 3.0 ?mol/L rabdosin B for6 h and 9 h;the whole level of ROS in cells treated for 24 h and 48 h was lower than in cellstreated for 6h and 9h,these indicated that rabdosin Bcould result in the increasing of ROS in a short period. After 0.3 mmol/L NAC and 3.0?mol/Lrabdosin B were added and cultured for 6 h, the level of ROS in cells was lowerthan in cells treated with 3.0 ?mol/L rabdosin B alone, the results indicated that NACcould decrease the produce of ROS in cells caused by rabdosin B.5.The level of ROS increased obviously in cells treated with 3.0 ?mol/L rabdosin B, NACcould decrease the produce of ROS in cells caused by rabdosin B;The activity of NADPH oxidase increased obviously in cells treated with 3.0 ?mol/L rabdosin B, DPI and APO could inhibit the increasecaused by rabdosin B, it revealed NADPH oxidase-derived reactive oxygen species mediated HL-60 cells differentiation caused by rabdosin B.6. Further detection displayed that the inducement of differentiation by rabdosin B was inhibited by NAC?APO?DPI. The results of giemsa stainning, NBT-reducing ability, cellular uptake and flowcytometry showed, NAC?APO?DPIinhibite the increase of the number of kidney-shaped nuclei and segmented nuclei, NBT-reducing ability, cellular uptake and the expression of CD11 b in HL-60 cells.Namely, the increase of ROS was associated with NADPH oxidase, it revealed NADPH oxidase-derived reactive oxygen species mediated HL-60 cells differentiation caused by rabdosin B.