Histone deacetylase inhibitors suppress JNK activity-mediated proliferation of glioma cells

Abstract

Objective To investigate the mechanism of histone deacetylase inhibitors in proliferation of glioma cells. Methods (1) Glioma cell line U251 was cultured in vitro and treated with Trichostatin A (TSA) at 0.1, 0.2, 0.5, 1.0 or 2.0 μmol/L for 48 h to determine the IC50 for TSA, and then, cells were treated with TSA at the IC50 concentration (0.5 μmol/L) for 8, 16, 24, 48 h; 1 μmol/L M344, 0.5 μmol/L LBH589, 6 mmol/L NaBu and 6 mmol/L VPA were used to treat the cells for 48 h and same volume of solvent was given to cells for 48 h as control group; MTT assay was performed to determine cell viability. (2) Western blotting was performed to test the Jun N-terminal kinase (JNK) expression and phosphorylated JNK (p-JNK) level in the U251 cells after being treated with 0.5 μmol/L TSA for 8, 16 and 24 h. (3) Western blotting and MTT assay were employed to detect the c-Jun expression and phosphorylated c-Jun (p-c-Jun) level, and cell viability in the U251 cells of control group, 10 μmol/L SP600125 treatment group (JNK inhibitor) and 1 μmol/L CEP11004 treatment group (MLK3, a direct upstream kinase of JNK). (4) Western blotting and MTT assay were employed to detect the c-Jun and Flag expressions, and cell viability in the U251 cells of pcDNA3.1 transfected group, pcDNA3.1 + TSA transfected group, pMKK7-JNK1 transfected group and pMKK7-JNK1 + TSA transfected group. Results (1) The viability of cells of 0.1, 0.2, 0.5, 1.0 or 2.0 μmol/L TSA treated group was significantly lower than that in the control group, and the higher the TSA concentration, the lower the viability of cells; the viability of cells of 0.5 μmol/L TSA treated for 16, 24, 36 and 48 h groups was significantly lower than that in the control group, and the longer the TSA treatment, the lower the viability of cells. (2) The viability of 1 μmol/L M344, 0.5 μmol/L LBH589, 6 mmol/L NaBu and 6 mmol/L VPA treatment groups was significantly lower than that in the control group (P<0.05). (3) As compared with those in the control group, the p-JNK expression was significantly decreased in the cells after 0.5 μmol/L TSA treatment for 16 and 24 h, and the p-c-Jun protein expression and the cell viability were significantly decreased in the cells of 10 μmol/L SP600125 and 1 μmol/L CEP11004 treatment groups (P<0.05). (4) As compared with that in the pcDNA3.1 transfected group, cell viability in the U251 cells of pcDNA3.1+TSA transfected group, pMKK7-JNK1 transfected group and pMKK7-JNK1+TSA transfected group was significantly increased(P<0.05). Conclusion Histone deacetylase inhibitors reduce the proliferation of glioma cell U251 through inhibiting JNK activity. Key words: Glioma; Trichostatin A; JNK; Proliferation