Abstract: ObjectiveTo investigate the effects and its possible mechanisms of tiopronin (TIP) on interleukin-2 (IL-2) immunotherapy of human leukemia KG-1 cells transplanted in nude mice. MethodsKG-1 cells (1×107/ml) in logarithmic growth phase were injected subcutaneously into the groin of the left hind leg of the 45 5-week-old nude mice. When the subcutaneous tumor diameter was about 8 mm, nude mice were randomly divided into three groups (n=15): Control group (intraperitoneal injection of phosphate buffer), IL-2 group (hypodermic injection of IL-2), IL-2+TIP group (hypodermic injection of IL-2 and intraperitoneal injection of TIP). The therapeutic effect of TIP combined with IL-2 on human leukemia KG-1 cells transplanted in nude mice was observed. The number of nature killer (NK) cells in peripheral blood of nude mice was detected by flow cytometry. Nitrate reductase assay was used to detect reactive nitric metabolite (RNM) levels in peripheral blood of nude mice. Enzyme linked immunosorbent assay (ELISA) was used to detect the levels of tumor necrosis factor-β (TNF-β) and interferon-γ (IFN-γ) in peripheral blood of nude mice. Terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL) assay was used to analyze apoptosis. ResultsBoth IL2 and IL2+TIP could inhibit the growth of transplanted tumor. Compared with IL-2 group ［(54.32±4.32)%］, the tumor inhibition rate of IL-2+TIP group was (90.15±3.75)%, and its inhibition of tumor growth was more obvious (t=11.893, P＜0.001). The tumor weights of Control group, IL-2 group and IL-2+TIP group were (0.95±0.05)g, (0.58±0.03)g and (0.27±0.07)g, and there was statistically significant difference among the three groups (F=52.716, P<0.001). Compared with IL-2 group, the tumor weight of IL-2+TIP group was significantly reduced (P=0.008). The number of NK cells in IL-2+TIP group was (0.658±0.157)/L, which was significantly higher than (0.452±0.124)/L of IL-2 group (P=0.021). The concentration of RNM in IL-2+TIP group was (42.92±4.68)μmol/ml, which was significantly lower than (163.38±5.49)μmol/ml in IL-2 group (P=0.007).The concentrations of TNF-β and IFNγ in IL-2+TIP group were (247.68±8.24)pg/ml and (185.61±7.58)pg/ml, which were significantly higher than (97.48±7.28)pg/ml (P=0.021) and (70.62±8.47)pg/ml (P=0.015) in IL-2 group. The apoptotic rate of tumor cells in IL-2+TIP group was (47.38±4.25)%, which was significantly higher than (21.41±2.79)% in IL-2 group (P＜0.001). ConclusionTIP can increase the sensitivity of leukemia cells to IL-2 immunotherapy by removing RNM, promoting NK cells activity and increasing NK cells-induced tumor cell apoptosis.

Key words: Tiopronin, Leukemia, Interleukin-2, KG-1 cell, Immune sensitization