Study On The Role Of FOXO1 In The Apoptosis Of Non-Hodgkin's Lymphoma Cells

ObjectsForkhead box O 1 is a kind of transcription factor that exists extensively,acts diversely and is related to differentiation,apoptosis and DNA repair.Recent studies have indicated that abnormal phosphorylation of FOXO1 is closely related to the tumorigenesis,development and prognosis in multiple tumor types,which notifies that FOXO1 is a tumor suppressor.So it has become a hotspot to reactivate FOXO1 factor to promote the apoptosis of tumor cells,which is greatly potential and significant.PI3K/Akt pathway plays a key role in the regulation of activation of FOXO1.Inhibition of PI3K/Akt pathway causes dephosphorylation and activation of FOXO1 which inhibits proliferation and promotes apoptosis.Wortmannin is a potent and selective inhibitor of PI3K/Akt within a range of certain concentration. Wortmannin could induce the inhabitation of proliferation and apoptosis in a certain tumor cells and promote the apoptosis induced by the chemotherapy.Etoposide is commonly used in the chemotherapy of Non-Hodgkin lymphoma and in experimental studies.Recent studies show that etoposide may directly interfere with the signal transmission.In vitro studies confirmed that the inhibition of the phosphorylation of FOXO1 in prostate cancer,breast cancer,colorectal cancer and other tumor cells inhibited the proliferation of tumor cells and promoted the apoptosis of tumor cells. However,the role of FOXO1 in Non-Hodgkin lymphoma is not clear.The experiment intents to investigate the relation between the phosphorylation of FOXO1 and the apoptosis of lymphoma cells induced by wortmannin and to clarify its specific mechanism.Methods1.To detect the cell growth inhibition rate(CGIR) of Namalwa cells and Jurkat cells treated by wortmannin and wortmannin plus etoposide by XTT assay.2.To detect the apoptosis rates(AR) of Namalwa cells and Jurkat cells treated by wortmannin,etoposide and wortmannin plus etoposide by FCM method.3.After the protein of Namalwa cells and Jurkat cells had been extracted, Western blot was performed to detect the change of p-Akt,p-FOXO1, FOXO1,Bim in the cells treated by wortmannin,etoposide and wortmannin plus etoposide.Results1.The growth inhibition rate of lymphoma cells:Wortmannin could inhibit the proliferation of Jurkat cells and Namalwa cells and showed dose and time dependence.The inhibition rate of Jurkat cells treated by 0.1,0.25,0.5,1μmol/L wortmannin for 24 hours was(9.49±0.72)%,(13.38±1.60)%,(16.52±1.79)%,(21.05±1.96)%. The difference of the inhibition rate between the control group and four experimental groups was significant(p＜0.01).And the difference of the inhibition rate among 0.1,0.25,0.5,1μmol/L group was significant(p＜0.05).There was a difference between 24 hours group and 48 hours group(p= 0.015,0.004,0.002,0.003).Administration of etoposide combined with wortmannin improved cytotoxity of etoposide to lymphoma cells.The inhibition rate was only(34.13±3.27)%and (21.93±2.50)%when Jurkat cells and Namalwa cells were treated with 5μmol/L etoposide alone for 24 hours.The inhibition rate increased to(54.19±4.16)%and (42.63±3.47)%when cells were pretreated with 0.5μmol/L wortmannin before exposed to 5μmol/L etoposide.The difference between both methods were significant at different concentration of etoposide(P_jurkat=0.003,p_namalwa=0.001).2.The apoptosis of lymphoma cells:Apoptosis of Jurkat cells and Namalwa cells induced by wortmannin was confirmed using Annexin V and PI staining to detect externalization of PS on the cell membrane.The apoptosis of cells induced by wortmannin was in a time-dependent manner.The apoptotic rate of Jurkat cells treated by wortmannin for 24 hours and 48 hours was(13.28±1.53)%and(17.17±1.58)%,and the apoptotic rate of control groups were(3.15±0.60)%and(5.48±0.82)%.The apoptotic rate of Namalwa cells for 24 hours and 48 hours was(9.84±1.30)%and(14.65±1.43)%,and the control groups were(2.81±0.49)%and(4.95±0.82)%.In two cells the difference between wortmannin groups and control groups were significant(p＜0.01),and there have difference between 24 hours and 48 hours(p_jurkat=0.038,P_namalwa=0.013).Moreover,the difference of the apoptotic rate between wortmannin and etoposide alone and wortmannin plus etoposide were significant(p＜0.01).These results notify that wortmannin improved the apoptosis induced by etoposide in lymphoma cells.3.The protein expression of the p-Akt,p-FOXO1,FOXO1,Bim in lymphoma cells treated with wortmannin and etoposide.The p-Akt and p-FOXO1 were down- regulated and the level of FOXO1 and Bim protein were up-regulated treated with wortmannin in Jurkat cells.And the change was time dependent.The p-FOXO1 was up-regulated by etoposide with the down-regulation of FOXO1.When Jurkat cell was pretreated by wortmannin before exposed to etoposide,the p-FOXO1 significantly decreased with the increase of FOXO1 and Bim protein.Conclusions1.The level of phosphorylation of FOXO1 is negatively correlated with apoptosis in Non-Hodgkin lymphoma cells2.The deactivation of PI3K/Akt pathway could decrease the level of phosphorylation of FOXO1 and increase the expression of Bim and promote apoptosis.3.The phosphorylation of FOXO1 could be promoted by etoposide in lymphoma cells.Wortmannin inhibit phosphorylation of FOXO1 and promote the apoptosis induced by etoposide.