| Objective Natural killer/T cell lymphoma(NK/TCL)is a highly aggressive hematogical malignancy and prevalent in Chinese population.Some NK/TCL patients poorly responsed to existing treatment,and had a very short survival time.Therefore,an urgent need for these patients is to establish some novel and effective therapies.Recently,it has been found that blocking the immune checkpoint by PD-1 antibody has significant anti-tumor activity in patients with refractory NK/TCL,suggesting that immunotherapy may be a promising approach for these patients.This study is designed to observe the expression pattern of TIM3,an another immune checkpoint molecule in NK/TCL cell lines,and the biological effects of its ligand on NK/TCL cell lines.Methods Expression of TIM-3 in normal NK cell from healthy donors and NK/TCL cell lines was detected by Western-blot.After treated with rhGAL-9 at various concentrations,growth-inhibitory effects of rhGAL-9 on SNK-1,SNK-6 and SNT-8 cell lines were analyzed with CCK-8 assay.Cell Apoptosis was determined by flow cytometry.Expression levels of apoptosis-related proteins caspase-3 and PARP were detected by Western-blot.Moreover,phosphorylated levels of JNK in MAPK pathway were also detected by Western-blot,after treated with 2.5 ?g/mL of rhGAL-9 for 15,30,and 60 minutes.Results The expression levels of protein TIM-3 in SNK-1,SNK-6 and SNT-8 cells were higher than that of normal NK cell.TIM-3 protein expression was 13±0.04,2.77±0.64 and 3.3±1.04 fold changes in three NK/TCL cell lines respectively as compared with that in normal NK cell.RhGAL-9 inhibited the viability of NK/TCL cells in a concentration dependent manner,the cells viability decreased in SNK-1 and SNK-6 and increased in SNT-8 as(87.58±7.97)%,(89.21±5.702)%,and(105.86±5.02)% as compared with that of controls at a concentration of 1.0 ?g/mL rhGal-9,respectively.At a concentration of 2.5 ?g/mL,rhGal-9 reduced the viability of three NK/TCL cells to(49.80±7.91)%,(74.09±9.04)%,and(36.05±4.64)% respectively.The viability of cells was further reduced to(25.70±5.70)%,(31.33±9.22)%,and(19.24±4.96)% at a concentration of 5 ?g/mL rhGal-9.RhGAL-9 induced the cell apoptosis in SNK-1,SNK-6 and SNT-8 cells,apoptotic cell rates were(4.43±0.70)%,(4.67±0.43)%,and(4.07±1.20)% respectively at controlled culture condition.RhGAL-9 at 1.0 ?g/mLinduced a significant portion of cells apoptosis(of 26.07±1.75%,14.57±1.15%,and 4.03±1.35% respectively).RhGAL-9 at 2.0 ?g/mL further increased the apoptotic cell rates as compared with that of controls(58.57±2.15%,42.43±0.69% and 76.07±6.97% respectively).RhGAL-9(1.0 ?g/mL)increased protein expression levels of cleaved caspase-3 and PARP,and activated JNK pathway in SNK-1,SNK-6 and SNT-8 cells.The expression of cleaved caspase-3 and cleaved-PARP was 2.26±0.33,2.31±0.24,2.85±0.38 and 3.46±0.86,1.20±0.57,2.18±0.77 fold changes as compared with that of controls.When the cells were incubated with 2.5 ?g/mL rhGal-9,the expression levels of cleaved caspase-3 was 2.28±0.20,2.33±0.07,3.82±0.48,and cleaved-PARP was 3.72±0.69,1.43±0.67,3.45±1.07 fold changes.The phosphorylation levels of JNK in SNK-1,SNK-6 and SNT-8 were significantly increased to 2.0±0.2,1.99±0.38 and 2.54±0.23 fold as compared with that of controls when cells incubated with 2.5 ?g/mL rhGal-9 for 15 minutes.Conclusion TIM-3 protein is over-expressed in NK/TCL cells.The ligand galectin-9 induces significant cell apoptosis,and the activation of JNK kinase pathways is one of the potential underlying mechanisms that contributed to the cell apoptosis. |
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