Effects of Amifostine and Fengling Polysaccharide on Proliferation and Chemoprotection of Human Granulocyte-Macrophage Progenitor Cells.

Objective: This paper is to study the proliferation and chemoprotection effects of amifostine (WR-2721) and fengling polysaccharide (FLPS) on the human granulocyte-macrophage progenitor cells.

Method: (1) Mononuclear cells (MNC) were deparated by Ficoll (1.077g/ml). The MNC of WR-2721 and FLPS treatment were cultured in methylcellulose senisolid medium, the colony forming unit-granulocyte macrophage(CFU-GM) was measured. The effects of WR-2721 and FLPS on CFU-GM were studied. (2) MNC of WR-2721 treatment or containing FLPS were cultured 14h at 37°C with VP16. The cytoprotective activity against VP16 toxic effects of WR-2721 and FLPS on CFU-GM were observed. (3)To study the effects of WR-2721 on proliferation inhibition and apoptosis of HL-60 human leukemia cell line, the cell apoptosis rate of HL-60 was determined by annexinV/PI double staining method, cell proliferation and chemotherapy sensitivity were analyzed with XTT assay, and the changes of cell cycle were observed through flow cytometry.(4) HL-60 cells of containing FLPS were cultured 14h at 37°C with VP16. The cytoprotective activity against VP16 toxic effects of FLPS on HL-60 cells were observed.

Results: (1) The number of CFU-GM was significantly increased in 10 groups by addition of 0.5–25μg/ml FLPS and 12 groups treated with 0.01–5mmol/L WR-2721( 30 min, 37°C), p<0.05. The mean value of CFU-GM in groups of negative control, WR-2721 (1mmol/L), FLPS(5μg/ml) and WR-2721+FLPS were 91.4±50.4,119.8±62.9,143.2±76.4 and 179.2±97.6 per 1×10⁵, respectively, Compared with control, singnificant differences were seen between each group, p<0.05. WR-2721+FLPS group compared with WR-2721 and FLPS group, p<0.01 and p<0.05. (2) The number of CFU-GM was significantly increased in MNC of adding FLPS or WR-2721 treatment. The mean value of CFU-GM in groups of VP-16 control, negative control, WR-2721 + VP16, FLPS + VP-16 and WR-2721 + FLPS + VP16 were 30.9±22.5, 83.2±43.8, 64.6±41.2, 55.3±33.5 and 78.3±48.2 per 1×10⁵, respectively. Compared with VP16 control group, singnificant differences were seen between each group, p < 0.01. (3) After treatment (30min,37°C)with WR-2721, the sensitivity of HL-60 cells to VP-16 was enhanced, and the IC₅₀ descended from 52.5μg/ml to 40.5μg/ml. After 72hours trentment of HL-60 cells with WR-2721, the early apoptotic cells (annexinV-FITC positive/PI negative) were increased from(5.5±1.9)% to (48.5±8.4)%(p<0.001), late apoptotic cells(annexinV-FITC positive/PI positive)were increased from(1.2±0.5)% to (39.0±4.0)% (p<0.001), and HL-60cells were arrested in G₂-M phase. (4) HL60 cells were cultured 14h at 37°C with 20μg/ml VP16 and 50μg/ml FLPS, the cell survival rate was 88.0%±2.3%, negetive control was 90.5%±2.9%, no singnificant difference was seen between two group, n =21, p >0.05.

Conclusion :(1) WR-2721 and FLPS can increase the prolification of CFU-GM. Combination of WR-2721 and FLPS show stronger effect than the WR-2721 or FLPS alone. (2) WR-2721 and FLPS selectiveiy protect human peripheral blood CFU-GM from the cytotoxicity of VP16 without decreasing its cytotoxic effect on HL60 cells. (3) WR-2721 treatment can enhance HL-60 cells chemotherapy sensitivity to VP-16, inhibit proliferation, induce HL-60 cells apoptosis and accumulation of cells in G₂-M phase.