The Necroptosis Induced By Chidamide In T-ALL Cells Via Inhibiting The Expression Of C-FLIP_L And Its Molecular Mechanism

Objective:T cell acute lymphoblastic leukemia(T-ALL)is a hematopoietic clonal malignancy caused by the malignant transformation of T lymphocyte driven by gene mutation.T-ALL comprises 15%in pediatric acute lymphoblastic leukemia(ALL),and 25%in adult ALL.In the past decades,a tendency towards increasing incidence of T-ALL was observed.The prognosis of T-ALL is poor and early relapse is common.Complete remission(CR)is hardly achieved by chemotherapy alone in relapsed disease.T-ALL is considered as moderate to high risk ALL and often treated by aggressive chemotherapy.It's crucial to find a new treatment approach.In the present study,we aimed at looking for specific and effective therapeutic target for T-ALL and eventually cure this form of leukemia by targeted therapy.Methods:1.Bone marrow mononuclear cells(BMMC)are collected from bone marrow samples of T-ALL patients,including at initial presentation(n=46),during first CR(n=23)and at relapse(n=6).The expression level of mRNA encoding L-cellular Fas-associated death domain-like interleukin-1?converting enzyme inhibitory protein(c-FLIP)was assessed by realt time PCR.Immunophenotype assay was conducted using flow cytometry with monoclonal antibody.Real-time PCR was used to detect fusion genes.Bone marrow cytogenetic test was conducted both directly and after 24-hour incubation.The relation between the expression level of c-FLIP_L and clinical manifestation and response to induction chemotherapy was analyzed.2.Histone deacetylase expression level in BMMCs of T-ALL patients as well as Jurkat and HUT-78 cell lines were assessed by western blot.Changes in the expression level of HDAC before and after chidamide treatment were also assessed by western blot.3.Necroptosis and apoptosis after chidamide treatment were assessed by flow cytometry.Changes in expression level of c-FLIP_L mRNA were assessed by real-time PCR.Changes in expression level of c-FLIP_L protein before and after treatment were assessed by western blot.Expression level of early apoptotic protein,key proteins of necroptosis were assessed by western blot.And changes in mitochondrial membrane were observed.4.The effect of chidamide on NF-?B signaling pathway activity and expression of key molecules when inducing necroptosis were assessed by western blot.The regulating effect of chidamide on downstream genes of NF-?B pathway including cyclinD1,TNF?,lL-2,IL-8 were assessed by real-time PCR.Thus,the molecular mechanism of necroptosis induced by chidamide in T-ALL cell lines could be clarified.Results:1.The expression level of c-FLIP_L mRNA is significantly higher in patients at initial presentation and relapse,compared to those at complete remission and healthy control.The expression level of c-FLIP_L mRNA is associated with patient risk stratification,white blood cell count at initial presentation,serum level of lactate dehydrogenase(LDH),serum level of hydroxybutyrate dehydrogenase(HBDH),CD45,HLA-DR,SIL-TAL1 fusion gene and complex karyotype,and is not associated with age,sex,plasma fibrinogen level,and the chromosomal aberration 6q-.Patients who did not achieve CR during first chemotherapy had a higher c-FLIP_L mRNA level than those who did(p<0.05).2.The expression level of histone deacetylase is higher in bone marrow mononuclear cells of T-ALL patients,Jurkat and HUT-78 cell lines.After treatment with chidamide,the expression level of histone deacetylase was significantly decreased in both cell lines.3.Chidamide induced necroptosis and apoptosis in Jurkat and HUT-78 cell lines.After apoptosis inhibitor was applied,chidamide mainly exert its effect of inducing cell death by inducing necroptosis.Chidamide inhibits the transduction and translation to c-FLIP_L gene.When apoptosis is inhibited,chidamide upregulates the expression level of receptor-interacting protein 3(RIP3)and the phosphorylation level of mixed lineage kinase domain-like(MLKL).Furthermore,decreased mitochondrial membrane potential was observed in necroptosis induced by chidamide in Jurkat and HUT-78cell lines.4.After treatment with chidamide,the phosphorylation level of I-?B and p65 protein were both significantly decreased.Chidamide down regulated the expression level of cyclin D1,and upregulate the cell inflammatory factors including TNF?,lL-2 and IL-8.Conclusion:1.c-FLIP_L mRNA expression level is abnormally high in T-ALL patients both at initial presentation and at relapse.The expression level of c-FLIP_L is associated with risk stratification,white blood cell count,serum LDH level,serum HBDH level,CD45,SIL-TAL1 fusion gene,complex karyotype and disease outcome.c-FLIP_L could be used as a prognostic marker in T-ALL.2.Chidamide suppresses histone deacetylation in Jurkat and HUT-78 cell lines.3.Chidamide induces necroptosis in Jurkat and HUT-78 cell lines by down regulating the transcription and translation of c-FLIP_L gene.4.Chidamide induces necroptosis in Jurkat and HUT-78cell lines via the classical NF-?B signaling pathway.