Study On The Effects And Mechanisms Of Dasatinib Induced Differentiation In Acute Mveloid Leukemia Cells

Objective:Acute myeloid leukaemia (AML) is the generic term for a group of myeloid leukaemias that are characterized by clonal expansion of immature myeloid progenitor, therefore, differentiation therapy is a effective treatment strategy for AML in clinic. All-trans retinoic acid (ATRA), the biologically active form of vitamin A, bypasses this arrest in differentiation, which is central to the extremely successful clinical application of ATRA for the differentiation-based treatment of acute promyelocytic leukemia (APL). However the therapeutic efficacy of ATRA is still burdened by problems such as toxicity and resistance. So the development of new compounds that are more powerful and easily tolerated than the individual compounds is critical. Recent years, tyrosine kinase inhibitors attract widespread attention, some of which have been reported to induce differentiation of AML cells. Dasatinib is a Food and Drug Administration (FDA)-approved multitargeted kinase inhibitor of BCR/ABL and Src kinases. Studies on the effects and mechanisms of dasatinib induced differentiation in AML cells were rarely reported.Here we reported a novel effect of dasatinib on inducing the differentiation of AML cells by acting through a MEK/ERK-dependent activation of signal transducer and activator of transcription1(STAT1).Moreover, we observed the occurrence of autophagy in AML cells, studies have shown that autophagy can affect the process of cell differentiation, however, the relationship between autophagy and differentiation in AML cells has not been reported. Therefore, we want to explore the molecular mechanisms of dasatinib induced differentiation in AML cells by the experimental method.Methods:Human acute myeloid leukemia cells HL60and NB4were treated with dasatinib. Cell growth curve was performed by trypan blue staining and cells counting. Cell surface marker CD11b, cell apoptosis profile and cell cycle profile were detected by flow cytometry. Morphologic changes in the cells were assessed by Wright-Giemsa staining. Cell lysates were subjected to western-blot analysis for the detection of protein level (PARP、Caspase3、Cleaved-Caspase3、c-My、p21/p27、PU.1、 C/EBPβ). The transcriptional level of PU.1, C/EBPa and C/EBPβ was measured through Real-time PCR. NBT-reducing activity as a potent differentiation marker of leukemia cells was used and the blue cells were counted by light microscopy for at least200cells. Xenografted athymic mice model was used to assessment the efficacy of dasatinib in vivo.Human acute myeloid leukemia cells HL60and NB4were used to examine the molecular mechanisms of dasatinib induced differentiation in AML cells. Cell surface marker CD11b and cell cycle profile were detected by flow cytometry. NBT-reducing activity as a potent differentiation marker of leukemia cells was used and the blue cells were counted by light microscopy for at least200cells. Morphologic changes in the cells were assessed by Wright-Giemsa staining. Cell lysates were subjected to western-blot analysis for the detection of protein level (PU.1、p-STAT1(S727)、p38、 p-p38、 JNK、p-JNK、 RARα、p-STAT1(Y701)、 STAT1、 MEK、 p-MEK、 ERK、 p-ERK、 AKT、p-AKT、 LC3). The transcriptional level of CXCL-10, RIG-G and IRF-1was measured through Real-time PCR. Immunofluorescence was carried out to detect the colocalisation of STAT1in cells. Luciferase reporter gene system was applied to detect RARa transcriptional activity in cells. MDC staining was used to detect intracellular distribution of autophagic vacuoles.Results:1) Trypan blue exclusion staining showed that dasatinib can significantly inhibit proliferation of HL60cells, which was a concentration-dependent and time-dependent process. Cell apoptosis profile was detected by flow cytometry, the results showed that the high concentration of Dasatinib can induce apoptosis of HL60cells (30μM) and NB4(20μM). Dasatinib could also induce G1/G0phase arrest in HL60and NB4cells. By the using of xenografted athymic mice model, efficacy of dasatinib in vivo was proved obvious.2) The number of HL60and NB4cells expressing CD11b, a marker of myeloid differentiation, increased significantly in a concentration-dependent and time-dependent manner. Of note, dasatinib-enhanced expression of CD11b was already detectable in the early time, albeit slightly, with appropriately50%of CD11b positive cells. Morphologic changes assessed by Wright-Giemsa staining revealed that the dasatinib-treated HL60and NB4cells acquired a myeloid morphology. The results of NBT reduction experiment confirmed the differentiation inducing ability of dasatinib in HL60cells. The results of real-time PCR and western blot further confirmed that dasatinib could induce myeloid differentiation in AML cells.3) Western blot showed that RARa protein levels decreased significantly over time process in AML cells with the treatment of dasatinib. By using the luciferase reporter gene system to detect the activity of RARa, data showed that dasatinib didn’t activate the transcription of RARa. RARa-shRNA was transfected into HL60cells, resulting in dramatic reduction in RARa at protein level.Cells were treated with dasatinib for72h, the CDl lb marker was detected by flow cytometry, data showed that the expression of CD11b had no changed. These results suggested that RARa may not play a major role in dasatinib induced differentiation in AML cells.4) The endogenous expression of STAT1was knocked down by shRNA. Notably, the silence of STAT1expression significantly inhibited dasatinib-induced cell differentiation. In detail, the population of NBT positive cells after dasatinib treatment decreased appropriately from80%to50%by the deletion of STAT1. In line with the fact, dasatinib-induced CD11b expression also decreased from54%to39%after STAT1knockdown. The important role of STAT1in dasatinib’s differentiating effect was also corroborated by the changes of morphology features. Compared with HL60-Vector cells, HL60-shSTAT1cells exhibited less myeloid morphology, with a decreased cytoplasm to nuclear ratio. Results of western blot showed that the phosphorylation of MEK was augmented within1h and increased to quite a high level at72h of dasatinib treatment. In consistence with p-MEK, the enhanced phosphorylation of ERK was also detectable as early as1h and then maintained in quite a high level at least for72h. HL60and NB4cells were treated with dasatinib in the presence of MEK inhibitor PD98059and then analyzed for CDllb expression. PD98059significantly inhibited dasatinib-induced CD11b expression in both cells. In detail, the population of CD11b-positive cells decreased from50%to16%in HL60cells and from57%to12%in NB4cells in the presence of PD98059. The activation of MEK and ERK was successfully inhibited by PD98059, both the phosphorylation of STAT1(Y701and S727) were also strikingly inhibited by PD98059both in HL60and NB4cells. Taken together, dasatinib-induced activation of STAT1is dependent on MAPK activation, thus leading to differentiation of leukemic cells.5) HL60and NB4cells were treated with dasatinib for0,6,12,24and48hours, then western blot were used to detect the change of autophagy marker protein LC3, results showed that the LC3Ⅱ protein expression gradually increased over time, optical density analysis showed that LC3Ⅱ/Ⅰ ratio increased as well. Cells were stained by MDC after treating with dasatinib for6,12and24h, some cells appeared fluorescent punctate distribution of autophagic vacuoles in6h, and this phenomenology was more evident after24hours. Dasatinib induced differentiation of AML cells is suppressed by autophagy inhibitor3MA, Wortmannin and LY294002. In contrast, rapamycin promoted dasatinib induced differentiation of AML cells. Lower concentrations of dasatinib significantly promoted ATRA induced differentiation of AML cells. MDC staining of autophagic vacuoles formation in HL60and NB4cells speculated that autophagy may be involved in dasatinib-collaborative ATRA induced differentiation of AML cells. These results showed that dasatinib could induce autophagy in AML cells, and the autophagy is conducive to the dasatinib induced differentiation of AML cells.Conclusion:The paper discussed the effects and mechanisms of dasatinib induced differentiation in AML Cells. Data presented here demonstrated that dasatinib could induce myeloid differentiate in AML cells, meanwhile the MEK/ERK-dependent activation of STAT1and the upregulation of autophagy might be conducive to the differentiation process. We provided the new views about the molecular mechanisms of dasatinib induced differentiation in AML cells, which might help us developing new treatments for human myeloid leukemia.