The Effects Of 4-azido-2’-deoxy-2’-fluoroacytidine On Proliferation And Invasion Of Acute T Lymphoblastic Leukemia And Its Mechanism

Background and objective:Acute T lymphocytic leukemia（T-ALL）is a kind of malignant clonal disease originating from T lymphocyte progenitor cells.It is a hematological malignant tumor with strong invasiveness and heterogeneity.T-ALL accounts for about 15%of children’s ALL and 25%of adult ALL,which is a highly malignant type of ALL.Although the therapeutic effect of T-ALL has been greatly improved due to the improvement of treatment scheme,the prognosis is still poor.Therefore,there is an urgent need to find efficient and safe therapeutic drugs.4’-azido-2’-deoxy-2’-fluoroacytidine（FNC）is a new cytosine nucleoside analogue with independent intellectual property rights.The previous research results of our group found that FNC can effectively inhibit the proliferation of Burkitt’s lymphoma,mantle cell lymphoma and non-small cell lung cancer,but whether FNC can inhibit acute T lymphocytic leukemia is still unknown.In this study,two kinds of human acute T lymphoblastic leukemia cells（Jurkat and MOLT-4）were selected to study the effect and mechanism of FNC on T-ALL,so as to provide experimental basis for the development of FNC into anti-tumor drugs.Methods:（1）CCK8 method was used to detect the effect of FNC on the proliferation of Jurkat and MOLT-4 cells in acute T lymphoblastic leukemia.（2）The apoptosis rate and cell cycle distribution of Jurkat and MOLT-4 cells treated with FNC were detected by flow cytometry.（3）Acridine orange/ethyl bromide（AO/EB）double fluorescence staining was used to detect the apoptosis of Jurkat and Molt-4 cells after FNC treatment.（4）Transwell assay was used to detect the effect of FNC on the migration and invasion of Jurkat and MOLT-4 cells.（5）The effect of FNC on the expression of PTEN,Bax,Bcl-2 and Caspase-3m RNA in Jurkat and MOLT-4 cells was detected by real-time fluorescence quantitative PCR（RT-q PCR）.（6）Western Blot assay was used to detect the effects of FNC on the expression of PI3K/Akt and JAK/STAT3 signaling pathway related proteins in Jurkat and MOLT-4 cells.（7）Western Blot detects the effect of FNC on the expression of PI3K/AKT pathway related proteins in Jurkat cells after the application of PI3K inhibitor（LY294002）.（8）Western Blot detects the effect of FNC on the expression of JAK/STAT3pathway related proteins in Jurkat cells after the application of JAK1 inhibitor（Ruxolitinib）.（9）Western Blot was used to detect the effect of FNC on the expression of two pathway-related proteins in Jurkat cells treated with PI3K/Akt and JAK/STAT3pathway agonists（IGF-1）.Results:（1）FNC could significantly inhibit the proliferation of Jurkat and MOLT-4cells in a time and dose dependent manner（P<0.01）.（2）After the treatment of FNC,the morphological changes of apoptosis were observed in Jurkat and MOLT-4 cells,and the apoptosis rate was significantly increased（P<0.05）.（3）FNC could block Jurkat and MOLT-4 cells cycle at G₀/G₁ phase（P<0.05）.（4）FNC could significantly inhibit the migration and invasion of Jurkat and MOLT-4 cells,and the cell migration and invasion of each experimental group decreased in a dose-dependent manner（P<0.05）.（5）After FNC treatment,the expression of PTEN,Bax and Caspase-3 genes increased and the expression of Bcl-2 gene decreased in Jurkat and MOLT-4 cells in a dose-dependent manner（P<0.01）.（6）After FNC treatment,the expression of p-PI3K,p-AKT1,p-STAT3,p-JAK1,Bcl-2,BCAT1,Vimentin,MMP2 and MMP9 in Jurkat and MOLT-4 cells decreased,while the expression of Bax and Caspase-3 increased（P<0.05）.（7）The application of PI3K inhibitor LY294002 enhanced the inhibitory effect of FNC on PI3K/AKT signaling pathway in Jurkat and MOLT-4 cells（P<0.01）.（8）The application of JAK1 inhibitor Ruxolitinib enhanced the inhibitory effect of FNC on JAK/STAT3 signaling pathway in Jurkat and MOLT-4cells（P<0.05）.（9）The application of PI3K/AKT and JAK/STAT3 pathway agonist IGF-1attenuated the inhibitory effect of FNC on PI3K/AKT and JAK/STAT3 signaling pathways（P<0.05）.Conclusions:（1）FNC can significantly inhibit the proliferation and invasion of T-ALL in vitro.（2）FNC can inhibit the proliferation and invasion of T-ALL by regulating PI3K/AKT and JAK/STAT3 signal pathways.