Transfection of tumor metastasis suppressor gene nm23-H1 up-regulates the activity of glycogen synthase kinase -3β in lung cancer cell line L9981

Abstract

Objective To investigate the influence of tumor metastasis suppressor gene nm23-H1 on the activity of glycogen synthase kinase -3β (GSK-3β) in lung cancer cell line L9981. Methods The levels of GSK-3β expression in cytoplasm and nucleus were determined with anti-GSK-3β antibody in lung cancer cell line control group (cell line with nm23-H1 gene deletion), nm23-H1 transfection group (cell line with nm23-H1 transfected) and blank transfection group (cell line with vector transfected) by Western blotting. The activity of GSK-3β among those three groups was detected by immunoprecipitation and analyzed by a radioactive isotope scintillation counter before and after treatment with 20 mmol/L LiCl. Results (1) The expression indensity of GSK-3β in cytoplasm and nucleus was (6 639±503) and (4 481±446) A in nm23-H1 transfection group, (645±352) and (726±68) A in blank transfection group, and (763±267) and (683±49) A in control group, respectively. There was very significance in GSK-3β expressive indensity of both cytoplasm and nucleus among nm23-H1 transfection group, blank transfection group and control group (P=0.025). There was significant difference between nm23-H1 transfection group and blank transfection group or control group (P=0.017), but no significant difference was observed between blank transfection group and control group (P=0.094). (2) The GSK-3β activity in cytoplasm and nucleus was (29 371±2 492) and (9 647±859) counts per minute in nm23-H1 transfection group, (11 241±1 495) and (1 492±176) counts per minute in blank transfection group, and (14 271±1 137) and (1 853±113) counts per minute in control group, respectively. There was very significant difference in GSK-3β activity of both cytoplasm and nucleus among nm23-H1 transfection group, blank transfection group and control group (P=0.032). The GSK-3β activity in nm23-H1 transfection group was significantly higher than that in blank transfection group and control group (P=0.027), but no significant difference was observed between the blank transfection group and control group (P=0.131). (3) After treatment with 20 mmol/L LiCl, the expression indensity of GSK-3β in cytoplasm and nucleus was (5 037±427) and (823±350) A in nm23-H1 transfection group, (2 174±126) and (248±12) A in blank transfection group, and (2 273±128) and (253±14) A in control group. No significant difference in GSK-3β expression indensity existed before and after treatment with LiCl in nm23-H1 transfection group (P=0.168). However, the GSK-3β expression indensity in cytoplasm and nucleus before treatment was remarkably higher than that after treatment in both blank transfection group and control group (P=0.012, 0.015). (4) After treatment with 20 mmol/L LiCl, the GSK-3β activity in cytoplasm and nucleus was (12 837±1 042) and (748±215) counts per minute in nm23-H1 transfection group, (4 739±401) and (947±126) counts per minute in control group, and (4 638±401) and (1 162±127) counts per minute in blank transfection group, respectively. The GSK-3β activity in cytoplasm and nucleus after treatment with LiCl was remarkably lower than that before treatment in nm23-H1 transfection group, blank transfection group and control group (P=0.006, 0.029, 0.010). Conclusion Transfection of nm23-H1 gene can significantly up-regulate the expression level and activity of GSK-3β in lung cancer cell line. The inhibitory effects of nm23-H1 gene on the signal transduction of Wnt pathway might be carried out through up regulating GSK-3β expression and activity in lung cancer cell line. Key words: Lung cancer cell lines; nm23-H1 gene; Wnt signal pathway; Glycogen synthase kinase -3β