Effect Of FOXM1 Inhibition By Thiostrepton On Proliferation, Apoptosis And Chemosensitivity Of Jurkat Cells

Objective:To investigate the effect of FOXM1 inhibition by thiostrepton (TST) on proliferation, apoptosis and chemosensitivity of human T-cell acute lymphoblastic leukemia (T-ALL) Jurkat cell line.Methods:1. Jurkat cells were randomly divided into experimental group (cells with various concentrations of TST treatment) and control group (cells without TST treatment). Cells were treated with TST at various concentrations for 24 h, then CCK-8 method was used to detect the inhibition rate, and IC50 was obtained.2. Jurkat cells were treated with various concentrations of TST (0,1.0,2.0 and 3.0 μmol/L) for 24 h, the mRNA and protein expression levels of FOXM1 were quantitatively measured by Real-Time PCR and Western blot, the cell cycle and apoptosis were detected using flow cytometry.3. To investigate whether Jurkat cells treated with TST were more sensitive to doxorubicin (Dox), cells were incubated with various concentrations of Dox with or without TST (1.0 μmol/L) for 24 h, then cell proliferation was measured by performing CCK- 8 assays, and IC50 of Dox was obtained.4. Jurkat cells were treated with the concentration of either TST (1.0 umol/L) or Dox (0.2μmol/L), or treated with the two drugs in combination for 24 h, the cell apoptosis were detected using flow cytometry, the Survivin and Caspase-3 protein expression were analyzed by Western blot.5. Jurkat cells were cultured with TST at various concentrations (0,1.0,2.0 and 3.0 μmol/L) for 24 h, then the cells were incubated with Dox (0.2 μmol/L) for 1 h. The cell- associated mean fluorescence intensity (MFI) of intracellular Dox was determined using flow cytometry, the mRNA and protein expression levels of GSTπ were quantitatively measured by Real-Time PCR and Western blot.Results:1. Jurkat cells were treated with various concentrations of TST (1-4 μmol/L) for 24 h, compared with the control group, TST inhibited cell growth in a dose- dependent manner, and the differences were statistically significant (P <0.05). The IC50 value for TST in the Jurkat cells at 24 h was 2.03±0.12μmol/L.2. Jurkat cells were cultured with TST at various concentrations (0,1.0,2.0 and 3.0 μmol/L) for 24 h, the results revealed that TST inhibited the the mRNA and protein expression of FOXM1 in a dose-dependent manner, and induced a gradual dose- dependent G2/M arrest and apoptosis in the Jurkat cells. The differences were all statistically significant (P<0.05).3. Jurkat cells were incubated with various concentrations of Dox with or without TST (1.0 μmol/L) for 24 h, the results showed that TST enhanced the antiproliferative effects of Dox and reduced the IC50 value of Dox from 0.295±0.026 μmol/L to 0.198±0.021μmol/L in the Jurkat cells (P<0.05).4. Jurkat cells were treated with the concentration of either TST (1.0 μmol/L) or Dox (0.2 μmol/L), or treated with the two drugs in combination for 24 h, the results demonstrated that treatment with a combination of TST and Dox significantly increased cell apoptosis, decreased the protein expression of Survivin and increased the protein expression of Caspase- 3 compared with treatment with either drug alone (P<0.05).5. Jurkat cells were treated with various concentrations of thiostrepton (0,1.0,2.0 or 3.0 μmol/L) for 24 h, the results indicated that TST increased the Dox-associated MFI and downregulated the mRNA and protein expression levels of GSTπ in a dose-dependent manner. The differences were all statistically significant (P<0.05).Conclusions:1. Inhibition of FOXM1 by TST inhibited proliferation and induced apoptosis in the Jurkat cells.2. Inhibition of FOXM1 by TST enhanced the chemosensitivity of the Jurkat cells to Dox, which might involve the following molecular mechanisms:the inhibition of FOXM1 by TST enhanced Dox-induced apoptosis, possibly through a synergistic effect of TST and Dox on decreasing the protein expression of Survivin and increasing the protein expression of Caspase- 3, and increased the accumuation of intracellular Dox, possibly through downregulating the mRNA and protein expression of GSTπ.3. FOXM1 gene may be a potential therapy target for human leukemia.