Study On The Function Of FoxM1 Gene In Human Burkitt Lymphoma Cell Line Raji

Objective:Forkhead box protein M1 (FoxM1) belongs to the forkhead superfamily of transcription factors. It regulates a wide spectrum of biological processes including the proliferation and apoptosis in cells. FoxM1 has been demonstrated to be over-expressed in many human solid malignancies. FoxM1 has captured great attention in blood malignancies recently. Our study aims at exploring the expression level of FoxM1 in Burkitt lymphoma cell line Raji to investigate its role in the tumorigenesis of Burkitt lymphoma.Methods:The mRNA levels of FoxM1 were quantified using real-time quantitative reverse transcription-polymerase chain reaction (qPCR) in Raji cell and the B cells of 10 healthy donors (normal control, NC), extracted proteins and performed Western blot to analyze the expression of FoxM1 proteins. FoxM1 inhibitor thiostrepton (TST), a natural product with antibiotic properties isolated from Streptomyces azureus, was added after the Raji cells were seeded in 96-well plates containing complete medium. After incubated for 24 and 36 h,20 μ1 of CCK-8 solution was added to each well and the absorbance was measured using microplate reader. The half maximal inhibitory concentration of TST in Raji cell in 24 and 36 h was measured. Cells were treated with TST at IC50 concentration for 24 h and for detection of apoptosis, cells were harvested and percentage apoptosis was measured by flow cytometry after staining with fluoresce in-conjugated annexin V and propidium iodide (PI). At the meantime, the mRNA and protein expression were detected by qPCR, Western blot to validate the down-regulation of FoxM1 expression after the treatment of TST.Results:The mRNA and protein level of FoxM1 is obviously higher in Raji cell (p<0.05 and p<0.001, respectively) compared with healthy donors. Over-expression of FoxMl in Raji cell was down-regulated by thiostrepton in a dose- and time-dependent manner. While the IC50 concentration in 24 h was 5.39 μmol/L. TST can significantly induce the apoptosis in Raji cells (p<0.05) compared with NC. At the meantime the mRNA and protein level was down-regulated (p<0.001 and p<0.05, respectively) compared to NC.Conclusion:The expression of FoxM1 is high in Raji cell. The inhibition of FoxM1 by TST can induce Raji cell apoptosis through down-regulating the expression of FoxM1. The result indicates FoxM1 acted an important role in the proliferation of Raji cells and might serve as a therapy target of Burkitt lymphoma.Objective:To investigate whether the utilization of thiostrepton (TST) could sensitize Raji cells to doxorubicin by inhibiting FoxMl expression.Methods:CCK8 and flow cytometry analysis were used to test the impact of TST and doxorubicin on Raji cell proliferation and apoptosis. To explore the concentration of each drug and combine the two drugs in their lower concentration to test their synergistic effect on Raji cells.Results:Our results showed the impact of TST and doxorubicin on Raji cells was in a dose- and time-dependent manner. The 24 h IC50 of TST in Raji cells was 5.39 μmol/L, while a concentration of 3 μmol/L was considered to be non-cytotoxic dose. The mean apoptosis rate of Raji cells was 10.82%. While the apoptosis rate of Raji cells treated with 0.08 μmol/L and 0.04 μmol/L doxorubicin was 23.52% and 9.32% respectively, suggesting a concentration of 0.03 μmol/L to be non-cytotoxic dose. After the combination of 3 μmol/L TST and 0.03 μmol/L doxorubicin, the 24 h IC50 of doxorubicin dropped from 0.08 μmol/L to 0.04 μmol/L. The mean apoptosis rate significantly increased to 21.41% compared with treatment with either drug alone (p<0.05).Conclusion:TST and doxorubicin had both exerted an impact on surpressing the proliferation and inducing the apoptosis on Raji cells in a dose-and time-dependent way. The combination of the two drugs in a low concentration had a synergistic effect, suggesting TST could increase the chemosensitivity of Raji cells to doxorubicin.