| Objective:Acute T lymphocytic leukemia(T-ALL)is a malignant tumor with abnormal proliferation of T lymphocytes in the bone marrow.Although the response rate of chemotherapy reached about 80%,50% of patients still have recurrence,the treatment of acute T lymphocytic leukemia is still a major problem.Finding high-efficiency and specific therapeutic drugs has also become a hotspot of T-ALL research in recent years.Abnormal activation of PI3K/AKT/mTOR signaling pathway can cause abnormal differentiation of T cells and formation of leukemia stem cells,which induced the occurrence of acute T lymphocytic leukemia.GDC-0349 is an ATP competitive inhibitor of the mTOR signaling pathway,which blocks mTORC1 and mTORC2 activation and induces apoptosis.The role of GDC-0349 in acute T lymphocytic leukemia has not been reported so far.Therefore,the purpose of this study was to determine the effect of GDC-0349 on proliferation and apoptosis of acute T lymphocytic leukemia cell line CCRF-CEM cells and its mechanism of action.Methods:1.Acute T-lymphocytic leukemia cell line CCRF-CEM cell culture;2.CCK8 method was used to detect the proliferative activity of CCRF-CEM cells which cultured by 0.1% DMSO and 10,25,50 and 100 ?M concentrations of GDC-0349 for 12,24,48 and 72 h respectively;3.Apoptosis of CCRF-CEM cells which cultured with 0.1% DMSO,10,50 and 100 ?M of GDC-0349 respectively were detected with Annexin V-FITC/PI double staining by flow cytometry.4.The expression of apoptosis-related proteins in CCRF-CEM cells cultured with 0.1% DMSO and 10,50 and 100 ?M of GDC-0349 respectively,including Bcl2,Caspase3 and Cleaved Caspase3,were detected by Western blot;5.The expression of AKT/mTOR pathway-related proteins were detected by Western blot.The proteins include AKT,p-AKT,mTOR,p-mTOR and 4EBP1 in CCRF-CEM cells which cultured with 0.1%DMSO and 10,50 and 100 ?M GDC-0349 respectively;6.Statistical methods: All data are expressed as mean ± standard deviation.Statistical analysis was performed by SPSS 22.0 analysis software.The difference between the two samples was analyzed by independent sample t test.One-way Anova method was used to analyze the difference of multiple groups.P <0.05 was considered statistically significant.Results:1.After CCRF-CEM cells cultured with 0.1% DMSO and GDC-0349 at concentrations of 10,25,50 and 100 ?M for 12,24,48 and 72 h,the results showed that GDC-0349 inhibited the proliferation of CCRF-CEM cells time-dose dependently.There are significant differences between the groups(P <0.05);2.After CCRF-CEM cells cultured with 0.1% DMSO and 10,25,50,and 100 ?M concentrations of GDC-0349 for 48 h respectively and adding Annexin V-FITC /PI double staining,the rate of apoptosis was detected by flow cytometry.The results showed that GDC-0349 promoted apoptosis of CCRF-CEM cells in a concentration-dependent manner.The apoptotic rates were(8.74±3.23)%,(13.84±2.48)%,(19.66±1.73)% and(35.98±5.87)% respectively.The apoptotic rate of each group was statistically different from the control group(P <0.05).3.Western blot results showed that GDC-0349(concentration of 10,50 and 100 ?M)promoted apoptosis.With the concentration increasing,withering Caspase3 and anti-apoptotic protein Bcl2 expression were decreased gradually,and Cleaved Caspase3 gradually was increased(P <0.05).4.Western blot results showed after CCRF-CEM cells cultured with GDC-0349(concentration of 10,50 and 100 ?M)for 48 hours,the AKT/mTOR pathway-related proteins,including AKT,mTOR,4EBP1 and phosphorylated proteins p-AKT and p-mTOR,were decreased gradually in a concentration-dependent manner(P < 0.05).Conclusions:1.The mTOR pathway inhibitor GDC-0349 can inhibit the proliferation of CCRF-CEM cells in a time and concentration-dependent manner.2.The mTOR pathway inhibitor GDC-0349 can promote the apoptosis of CCRF-CEM cells in a concentration-dependent manner.3.The mTOR pathway inhibitor GDC-0349 inhibits the proliferation and promotes apoptosis of CCRF-CEM cells by inhibiting phosphorylation of AKT/mTOR pathway-associated proteins. |
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