| The myeloid leukemias are a heterogeneous group of diseases characterized by infiltration of the blood, bone marrow, and other tissues by neoplastic cells of the hematopoietic system. Based on their untreated course, the myeloid leukemias have traditionally been designated acute or chronic. Acute myelocytic leukemia (AML) is a clonal expansion of any one of several nonlymphoid hematopoietic progenitors that retain the capacity of self-renewal, but are severely limited in their ability to differentiate into functional mature cells. These various progenitors include cells of granulocytic, monocyte/macrophage, erythroid, and megakaryocytic lineage. The disordered growth in the myeloid stem cell compartment leads to the patient’s death from bone marrow failure, unless a successful therapeutic strategy is employed. The incidence of AML is～3.7per100,000people per year, and increases with age; it is1.9/100000in individuals<65years and18.6/100000in those>65. The age-adjusted incidence is higher in men than in women (4.6/100000versus3.0/100000). A significant increase in AML incidence has occurred over the past10years. Once the diagnosis of AML is suspected, a rapid evaluation and initiation of appropriate therapy should follow. In addition to clarifying the subtype of leukemia, initial studies should evaluate the overall functional integrity of the major organ systems, including the cardiovascular, pulmonary, hepatic, and renal systems. Factors that have prognostic significance, either for achieving complete remission (CR) or for predicting the duration of CR, should also be assessed before initiating treatment. The length of CR, and the curability of AML.Although considerable progress has been made over the past3decades,, such as new cytotoxic agentsã€differentiation therapyã€singnal transduction inhibitors〠immunotherapyã€anti-angiogenesis and stem cell transplantation (HSCT),20%-40%AML patients still do not achieve CR with standard therapy and continue to have a poor prognosis, furthermore50%～70%patients achieved CR are likely to relapse after CR. Once patients have relapsed, with current therapies, the chance of long-term disease free survival is less than10%. The cause of leukemia refractoriness or relapse is often not clear but likely relates to multiple factors including resistance mechanisms to chemotherapy drugs and minimal resitdual disease(MRD). The treatment of patients with refractory or relapsed AML remains a major challenge for the leukemia community and it is not possible to identify a single regimen or approach as the standard of care in relapsed and refractory AML. New and promising approaches are being explored, however.Epidermal growth factor receptor pathway substrate number8(Eps8), a substrate for the tyrosine kinase activity of the epidermal growth factor receptor (EGFR), was first discovered by Fazioli in NIH3T3murine fibroblasts in1993. It was tyrosine-phosphorylated by EGFR, and also by several other receptor tyrosine kinases (RTKs). The human eps8gene locus was mapped to chromosome12q23-q24. There are two eps8isoforms (p97eps8and p68eps8) and other three eps8family members (Eps8-related genes:Eps8L1-3). Eps8was expressed in all epithelial and fibroblastic lines and in some hematopoietic cells. Scita et al studied, in serum-starved fibroblasts, Eps8displays a punctuate, cytoplasmic perinuclear distribution with some staining at sites of cortical actin accumulation of plasma membrane.Eps8is a structural organization typical of a signaling molecule, contains (from N to C terminus) a putative N-terminal phosphotyrosine binding protein (PTB) domain, a SH3domain and a C-terminal "effector region"(Fiure.l). The PTB domain is a protein-protein interaction module, which binds to a variety of peptides both in a phosphotyrosine-dependent and-independent fashion. SH3binds to proline-X-X-asparate-tyrosine(PXXDY) motifs both specifically and selectively. The C-terminal effector region of Eps8directly binds to F-actin, by activating the GTPase, Rac, and directs Eps8to localization within the cell where actin polymerization occurs, such as membrane ruffles.Eps8participanted in several signaling pathways. The complexs of Eps8-E3b1-Sosl, Eps8-Abil-p85-Sos-l and IRSp53-Eps8are endowed with Rac-specific activity in vitro and are required for Rac activation leading to actin cytoskeletal remodeling. According with the gene structure of Eps8, it participates in EGFR signaling, trafficking and mitogenic signaling. Disanza, et al. demonstrate that Eps8controls actin-based motility by capping the barbed ends of actin filaments. It is also proved that Eps8might participate in non-canonical Wnt signaling to control the movements of cells during vertebrate development and motility of neuronal progenitor cells expressing ErbB4. Overexpression of Eps8facilitates increased mitogenesis and transforming ability in response to EGF, indicating its participating in RTK-activated signaling pathways(including Ras/MAPK signaling pathway and PI3K signaling pathway) leading to tumorigenesis and tumor proliferation.Recently, many studies observed that Eps8is overexpressed in several human cancer samples and in human cancer cell lines, and it contributed to the tumorigenicity, tumor proliferation and tumor metastasis of them:(a) Eps8was overexpressed in human cancers, including cervical carcinomas, pancreatic cancers, papillary thyroid carcinomas, breast cancers, colon cancers and head and neck squamous cell carcinomas (HNSCC), and oral squamous cell carcinoma (OSCC);(b) Eps8contributed to thetumorigenicity and aggressiveness of colon cancers, cervical cancers, HNSCC, pancreatic cancer and OSCC, to the motility and mitogenesis of colon cancer cells;(c) Eps8could be a predictor factor of the survival of cervical cancer patients, breast patients and thyroid cancerpatients, identified Eps8as a novel putative oncogene. So Eps8is probably to be important in the pathogenesis of human cancers, may have potential to be a therapeutic target for inhibition of the cancer progression.Eps8has essential role in many human solid cancers and it contributed to their tumorigenicity, tumor proliferation and tumor metastasis, but there is no study about the interaction between the eps8and AML. In the present study, we detect four parts to investigate the relationship between Eps8and AML:1. The expression of Eps8in AML bone marrow cells and AML cell lines;2. The PCR array was used to detect the expression of Eps8-related genes in AML cell line KG1a;3. Effects of PI3K/Akt inhibitor perifosine for the expression of Eps8-related genes in AML cell line KG1a;4. Effects of traditional chemotherapy medicine daunorubicin for the expression of Eps8-related genes in AML cell line KG1a.The aim of the study was to explore the function of Eps8in AML, and to provide theoretical evidence for therapeutic target of Eps8in AML.The present study includes four parts:Part1The expression of Eps8in AML bone marrow and AML cell linesObjective:The aim of the part is to explore the possible expression of Eps8and in AML patients and cell lines.Methods:Twenty-one patients with do novo AML and10healthy volunteers (as the control) were enrolled in this study. All patients and volunteers were informed about the study and signed a form consenting to the procedure. The institutional pathologist examined the bone marrow samples simultaneously by review of the Wright-Giemsa-stained slide, enzyme histochemistry, and standard immunophenotype classified into the different types. About3-5mL bone marrow aspirate was collected into a tube containing EDTA by iliac crest puncture of each anesthetized patient before treatment and healthy volunteer. The expression of Eps8was detected by quantitative reverse transcription polymerase chain reaction (qRT-PCR) in AML patients and by western blot in AML cell lines.Results:1.1Expression of Eps8in AML patientsqRT-PCR analysis showed that the expression of Eps8was detected in all the AML patients and healthy volunteers, but Eps8was expressed significantly higher in AML patients(t=3.055, P=0.006). Furthermore, we have explored the correlation between Eps8and the patients’clinicopathological characteristics. The result showed that there was no significant association between the expression level of Eps8and sex, age, the WBC count, platelet count, hemoglobin, presence of splenomegaly and hepatomegaly (P>0.05), but we found that the patients with AML achieved CR or not after one course of chemotherapy significantly correlated with the expression of the Eps8(P=0.021). All the patients with AML were subgrouped as either Eps8high expression group (compared with control group, Eps8fold change≥5) or Eps8low expression group (compared with control group, Eps8fold change<5). The result showed that the CR rate in Eps8high expression group was significantly lower than low expression group (P=0.024). This indicated that Eps8may have broader implication in clinical prognostic value.1.2Expression of Eps8in AML cell linesWestern blot analysis showed that the expression of Eps8was detected in all the6AML cell lines, the breast cancer cell line MCF-7was used as the internal control. The result showed that Eps8was highly expressed in KG1a AML cell line.Conclusion:The expression of Eps8was significantly increased in AML patients compared with the healthy volunteers (t=3.055, P=0.006), and correlated with the AML patients achieved CR or not after one course of chemotherapy (P=0.021). The CR rate in Eps8high expression group was significantly lower than low expression group (P=0.024). Eps8was obviously high expressed in KG1a cell line, indicating Eps8may play an important role in the pathogenesis of AML and KG1a maybe a good study model for the investigation of the function of Eps8in the AML. Part2The PCR array was used to detect the expression of Eps8-related genes in AML cell line KGlaObjective:The aim of the part is to explore the expression of Eps8-relsted genes in AML cell line KG1a.Methods:We measured84representative Eps8-related genes using PCR array.Results:2.1The high and low expression genes in KGla cells detected by PCR assayThe result showed that there are64genes (including Eps8) high expressed in KG1a cells and12genes low expressed.2.2The releted signaling pathway in the KGla cellsFurthormore, we found that almost all the high expressed genes were intracellular signaling molecules, participating in the PI3K/Akt signaling pathway, Erkl/Erk2MAPK signaling pathway, IκB Kinase/NFκB cascade, JAK/STAT cascade, JNK cascade, small GTPase mediated signal transduction, and some of them can affect cell survival and growth, through the cell apoptosis, cell cycle, cell differentiation, cell growth, cell motility and cell proliferation.Conclusion:Genes (such as AKT1, BCL2, CASP3, PIK3CA, TP53) which participant in the PI3K/Akt signaling pathway and small GTPase mediated signal transduction high expressed in KG1a cells.Part3Effects of AKT/PI3K inhibitor perifosine for proliferation, the expression of Eps8, cell apoptosis and cell cycle in AML cell line KGlaObjective:The aim of the part is to explore the effects of AKT/PI3K inhibitor perifosine for proliferation, the expression of Eps8, cell apoptosis and cell cycle in AML cell line KG1a.Methods:The growth of KG1a cells was determined using Trypan Blue assay, then the IC50of the perifosine for KG1a cells was calculated by the SPSS. The effect of perifosine for the clone formation of KG1a cells after treated with different concentration perifosine was detected by methylcellulose colony-forming assay. The effect of perifosien for the distribution of cell cycle and the apoptosis rate were measured with flow cytometry. The effect of perifosine for the expression of Eps8and the cell apoptosis and cell cycle regulators (bcl-2, caspase-3, p21, cyclin E) were detected by western blot. The expression of CD34and CD38in the surface of KG1a cells were measured with flow cytometry.Results:3.1Effects of perifosine on the survival rate in KGla cellsThe results of the Trypan Blue assay showed the difference of the survival rate of KG1a cells between different concentration perifosine groups was significant (F=741.027, P=0.000), the difference of the survival rate of KGla cells between different treatment times of perifosine groups(24h,48h and72h) was significant (F=175.399,P=0.000), the interaction between the perifosine treatment concentration and time for the survival rate of KG1a cells was existed(F=12.742, P=0.000). The difference of the survival rates of KGla cells on24h,48h and72h after treated with different concentration perifosine was significant (F=129.905, P=0.000; F=226.088, P=0.000; F=1033.382, P=0.000), the survival rates were decreased significantly comparing with the control group when the concentration of perifosine was increased (P<0.05). Along with the1.25μmol/L perifosien group(F=4.143, P=0.074), the perifosien treatment groups for the survival rate of KGla cells were significantly different between the different treatment times(24h,48h and72h)(F=46.282, P=0.000; F=122.721,P=0.000; F=11.352, P=0.000; F=81.475,P=0.000; F=240.642, P=0.000). The half inhibition rates (IC50) of perifosine on the KGla cells were13.28±3.63μmol/L,4.25±1.35μmol/L and3.65±0.85μmol/L for24h,48h and72h, separately.3.2Effects of perifosine on the clone formation of KGla cellsThe results showed the difference of the clone formation rate of KG1a cells between different concentration perifosine groups was significant (F=82.180, P=0.000), the difference of the clone formation rate of KG1a cells between different treatment times of perifosine groups (24h,48h and72h) was significant (F=55.779,P=0.000), the interaction between the perifosine treatment concentration and time for the clone formation rate of KGla cells was existed (F=8.008, P=0.002). The difference of the clone formation rates of KGla cells on day14after treated with different concentration perifosine for24h and72h was significant (F=19.960, P=0.000; F=70.748, P=0.000), the clone formation rates were decreased significantly comparing with the control group when the concentration of perifosine was increased (P<0.05).. the perifosien treatment groups for the clone formation rate of KG1a cells on day14were significantly different between the different treatment times (F=8.595, P=0.043; F=78.191, P=0.000; F=63.146,.P=0.000).3.3Effects of perifosine on the expression of Eps8in the KGla cellsThe western blot assay showed that the expression of Eps8was decreased allowing with the concentration of perifosien and the treated time increasing.3.4Effects of perifosine on the apoptosis of KGla cells3.4.1Morphological analysis of the effects of perifosien by Wright-Giemsa stainKGla cells with characteristics of apoptosis was confirmed by Wright-Giemsa stain, and the results showed the percentage of apoptosis were increased after treatment with10μmol/L perifosine for72h.3.4.2Perifosien induced apoptosis in KGla cells The results showed the difference of the total apoptosis rate, early apoptosis rate and late apoptosis rate of KG1a cells between different concentration perifosine groups was significant (F=548.501,P=0.000; F=103.168,P=0.000; F=23.608, P=0.000); the difference of the total apoptosis rate and early apoptosis rate of KG1a cells between different treatment times of perifosine groups (24h,48h and72h) was significant (F=5.198, P=0.037; F=6.713, P=0.020), but the diference of the late apoptosis rate was not (F=1.670, P=0.215); the interaction between the perifosine treatment concentration and time for the total apoptosis rate, early apoptosis rate and late apoptosis rate of KGla cells was existed(F=45.352, P=0.000; F=29.159, P=0.000; F=4.594,P=0.017).The difference of the total apoptosis rates of KGla cells after treated with different concentration perifosine for24h and72h were significant (F=334.868, P=0.000; F=233.698, P=0.000), the total apoptosis rates were increased significantly comparing with the control group when the concentration of perifosine was increased (P<0.05). Only the10μmolL and40μmol/L perifosien treatment groups for the total apoptosis rate of KGla cells were significantly different between the different treatment times (F=25.122, P=0.007; F=103.933, P=0.001).The difference of the early apoptosis rates of KGla cells after treated with different concentration perifosine for24h and72h were significant (F=67.738, P=0.000; F=56.817, P=0.000), the early apoptosis rates of KGla after treated with2.5μmol/L,10μmol/L and40μmol/L perifosine were (4.18±0.44)%ã€(6.09±0.35)%,(24.60±4.11)%, respectively, but only the40μmol/L perifosine group was increased significantly comparing with the control group (P<0.05); when the treatment time was72h,10μmol/L and40μmol/L perifosine group was increased significantly comparing with the control group (F<0.05). The difference of the late apoptosis rates of KGla cells after treated with different concentration perifosine for24h and72h were significant (F=7.942, P=0.000; F=19.213, P=0.000), the late apoptosis rates were increased significantly comparing with the control group when the concentration of perifosine was increased (P<0.05). All the2.5μmol/L,10μmol/L and40μmol/L perifosine treatment groups for the early apoptosis rate of KG1a cells were significantly different between the different treatment times (F=10.236, P=0.033: F=67.800, P=0.001; F=23.270,P=0.008), but only the40μmol/L perifosien treatment groups for the late apoptosis rate of KGla cells were significantly different between the different treatment times (F=10.164, P=0.033).3.4.3Effects of perifosine on the expression of caspase-3and bcl-2in KG1a cellsTo study the underlying apoptotic mechanisms, expression of apoptosis regulators, caspase-3and bcl-2, were assessed after treatment with1.25μmol/L,2.5μmol/L,5μmol/L,10μmol/L,20μmol/L and40μmol/L perifosine for72h by western blot. The caspase-3and bcl-2was detected in all the groups treated with or without perifosine. According with the concentration of perifosine increasing, the expression of caspase-3was increased, and the expression of bcl-2was decreased. The result indicated that perifosine can induce the apoptosis of KG1a cells by activating caspase-3and decreasing bcl-2.3.5Effects of perifosine on the cell cycle distribution of KGla cells3.5.1Effects of perifosine on the cell cycle distribution of KGla cellsThe difference of the percentage of Go/G1, S and G2/M phase cells in KG1a cells after treating wirh2.5μmol/L,10μmol/L and40μmol/L perifosine for72h were significant (F=741.027, P=0.000; F=380.969,P=0.000; F=42.050, P=0.000). The percentage of Go/G1and G2/M phase cells were increased first and then decreased and the percentage of S phase cells was decreased first and then increased comparing with the control group when the concentration of perifosine was increased (P<0.05). These results indicated that perifosine could affect the cell cycle distribution and arrest KG1a cells at S phase.3.5.2Effects of perifosine on the expression of p21and cyclinE in KGla cellsTo study the underlying mechanisms of the cell cycle redistribution, expression of cell cycle regulators, p21and cyclinE, were assessed after treatment with2.5μmol/L,10μmol/L and40μmol/L perifosine for72h by weastern blot. P21and cyclinE were detected in all the groups treated with or without perifosine for72h. According with the concentration of perifosine increasing, the expression of p21was decreased and cyclin E was increased. The result indicated that perifosine can induce the cell cycle redistribution in KG1a cells by decreasing p21and increasing cyclin E.3.6The expression of the CD34and CD38in KGla cellsThe expression of CD34and CD38in the surface of KGla cells were measured with flow cytometry, and the result showed that the percentage of CD34+CD38-in KG1a cells was (98.40±1.52)%.Conclusion:Perifosine can significantly inhibite the proliferation, the clone formation of KGla cells and the expression of Eps8in KGla cells on a dose-and time-depended matter; Perifosine can induce the apoptosis of KGla cells by activating caspase-3and decreasing bcl-2, and induce the cell cycle redistribution of KGla cells by decreasing p21and increasing cyclin E; the percentage of CD34+CD38-in KG1a cells was(98.40±1.52)%.Part4Effects of traditional chemotherapy medicine daunorubicin (DNR) for proliferation, the expression of Eps8, cell apoptosis and cell cycle in AML cell line KGla Objective:The aim of the part is to explore the effects of traditional chemotherapy medicine daunorubicin(DNR) for proliferation, the expression of Eps8, cell apoptosis and cell cycle in AML cell line KG1a.Methods:The growth of KG1a cells was determined using Trypan Blue assay, then the IC50of the perifosine for KG1a cells was calculated by the SPSS. The effect of DNR for the clone formation of KGla cells after treated with different concentration DNR was detected by methylcellulose colony-forming assay. The effect of DNR for the apoptosis rate and the distribution of cell cycle were measured with flow cytometry. The effect of DNR for the expression of Eps8and the cell apoptosis and cell cycle regulators (bcl-2, caspase-3, p21, cyclin E) were detected by western blot.Results:4.1Effects of DNR on the survival rates in KGla cellsThe results showed the difference of the survival rate of KGla cells between different concentration DNR groups was significant (F=517.643, P=0.000), the difference of the survival rate of KGla cells between different treatment times of DNR groups (24h,48h and72h) was significant (F=68.968, P=0.000), the interaction between the DNR treatment concentration and time for the survival rate of KG1a cells was existed (F=25.673, P=0.000). The difference of the survival rates of KG1a cells on24h,48h and72h after treated with different concentration DNR was significant (F=88.555, P=0.000; F=145.190, P=0.000; F=1052.825,P=0.000), the survival rates were decreased significantly comparing with the control group when the concentration of perifosine was increased (P<0.05). Along with the0.1μmol/L DNR group (F=3.171, P=0.115), the DNR treatment groups for the survival rate of KGla cells were significantly different between the different treatment times (24h,48h and72h)(F=14.020,P=0.005; F=19.8345,PP=0.002; F=96.066, P=0.000; F=295.952, P=0.000; F=79.127,P=0.000). The IC50of DNR on the KGla cells were (0.53±0.10)μmol/L,(0.26±0.07)μmol/L and (0.19±0.04)umol/L for24h,48h and72h, separately.4.2Effects of DNR on the clone formation of KG1a cellsThe results showed when treated with different concentration of DNR for24h, the clone formation of KGla cells was founded only in the0.05μmol/L, O.lμmol/L and0.2μmol/L NDR treatment groups, not in0.4μmol/L,0.8μmol/L and1.6μmol/L DNR goups. The difference of the clone formation rate of KGla cells on day24between0.05μmol/L,0.1μmol/L and0.2μmol/L DNR groups was significant (F=1437.188, P=0.000), the difference of the clone formation rate of KGla cells between different treatment times of DNR groups (24h,48h and72h) was significant (F=24.175, P=0.000), the interaction between the DNR treatment concentration and time for the clone formation rate of KG1a cells was existed(F=5.189, P=0.002). The difference of the clone formation rates of KG1a cells on day14after treated with different concentration DNR for24h,48h and72h was significant (F=168.525, P=0.000; F=1239.212,P=0.000; F=2698.777, P=0.000), the clone formation rates were decreased significantly comparing with the control group when the concentration of DNR was increased (P<0.05). Along with0.05μmol/L DNR group (F=1.998,P=0.216),0.1μmol/L and0.2μmol/L DNR treatment groups for the clone formation rate of KGla cells on day14were significantly different between the different treatment times (F=58.899,P=0.003; F=16.840,P=0.003).4.3Effects of DNR on the expression of Eps8in the KGla cellsThe western blot assay showed that the expression of Eps8was decreased accompanied with the concentration of DNR and the treated time increasing.4.4Effects of DNR on the apoptosis of KGla cells4.4.1Morphological analysis of the effects of DNR by Wright-Giemsa stainKG1a cells with characteristics of apoptosis were confirmed by Wright-Giemsa stain, and the results showed the percentage of apoptosis were increased after treatment with10μmol/L DNR for72h.4.4.2DNR induced apoptosis in KGla cellsThe results showed the difference of the total apoptosis rate, early apoptosis rate and late apoptosis rate of KG1a cells between different concentration DNR groups was significant (F=1002.926, P=0.000; F=520.100,P=0.000; F=611.588, P=0.000), the difference of the total apoptosis rate, early apoptosis rate and late apoptosis rate of KGla cells between different treatment times of DNR groups (24h and72h) was significant (F=3903.148, P=0.000; F=1164.274, P=0.020; F=1259.918, P=0.000), the interaction between the DNR treatment concentration and time for the total apoptosis rate, early apoptosis rate and late apoptosis rate of KG1a cells was existed(F=627.257, P=0.000; F=324.245,P=0.000; F=541.471,P=0.000).The difference of the total apoptosis rates of KGla cells after treatment with different concentration DNR for24h and72h were significant (F=34.403, P=0.000; F=1280.583,P=0.000), the total apoptosis rates were increased significantly comparing with the control group when the concentration of DNR was increased (P<0.05).0.2μmol/L,0.8μmol/L and1.6μmol/L DNR treatment groups for the total apoptosis rate of KGla cells were significantly different between the different treatment times (F=1638.280,P=0.000; F=1825.615,P=0.000; F=1002.926,P=0.000).The difference of the early apoptosis rates of KGla cells after treated with different concentration DNR for24h and72h were significant (F=12.439, P=0.000; F=918.409, P=0.000), the early apoptosis rates were increased significantly comparing with the control group when the concentration of perifosine was increased (P<0.05); The difference of the late apoptosis rates of KGla cells after treated with0.2μmol/L,0.8μmol/L and1.6μmol/L DNR for24h and72h were significant (F=6.612, P=0.015; F=800.619, P=0.000), the late apoptosis rates of KGla with DNR treatment for24h were (10.60±1.25)%,(11.66±0.83)%,(12.48±0.53)%, respectively, but only the0.8μmol/L and1.6μmol/L DNR group were increased significantly comparing with the control group (P<0.05); when the treatment time was72h, all the three DNR groups were increased significantly comparing with the control group (P<0.05). All the0.2μmol/L,0.8μmol/L and1.6μmol/L DNR treatment groups for the early apoptosis rate of KG1a cells were significantly different between the different treatment times (F=25.773, P=0.007; F=451.418, P=0.000; F=2789.011, P=0.000), and also for the late apoptosis rate of KG1a cells were significantly different between the different treatment times (F=25.878, P=0.007; F=3018.753, P=0.000; F=149.171, P=0.000).4.4.3Effects of DNR on the expression of caspase-3and bcl-2in KGla cellsTo study the underlying apoptotic mechanisms, expression of apoptosis regulators, caspase-3and bcl-2, were assessed after treatment with0.05μmol/l,0.1μmol/L,0.2μmol/L,0.4μmol/L,0.8μmol/L and1.6μmol/L DNR for72h. Caspase-3and bcl-2was detected in all the groups treated with or without perifosine for72h. According with the concentration of DNR increasing, the expression of caspase-3was increased, and the expression of bcl-2was decreased. The result indicated that DNR can induce the apoptosis of KGla cells by increasing caspase-3and decreasing bcl-2.4.5Effects of DNR on the cell cycle distribution of KGla cells4.5.1Effects of DNR on the cell cycle distribution of KGla cellsThe results showed the difference of the percentage of Go/G1, S and G2/M phase cells in KGla cells between different concentration DNR groups was significant (F=83.353, P=0.000; F=163.987, P=0.000; F=392.806, P=0.000), the difference of the percentage of G0/G1, S and G2/M phase cells in KGla cells between different treatment times of DNR groups (24h and72h) was significant (F=315.691, P=0.000; F=35.385, P=0.020; F=536.652, P=0.000), the interaction between the DNR treatment concentration and time for the percentage of Go/G1, S and G2/M phase cells in KG1a cells was existed (F=57.981,P=0.000; F=163.987, P=0.000; F=86.004, P=0.000).The difference of the percentage of Go/G1phase cells in KG1a after treatment with different concentration DNR for24h and72h were significant (F=6.081, P=0.018; F=84.500, P=0.000), the percentage of Go/G1phase cells were decreased with DNR treatment for24h, and significantly decreased with DNR treatment for72h, comparing with the control group when the concentration of DNR was increased (P<0.05).0.2μmol/L,0.8μmol/L and1.6μmol/L DNR treatment groups for the percentage of Go/G1phase cells were significantly different between the different treatment times (F=175.145, P=0.000; F=72.017, P=0.001; F=869.490, P=0.000). DNR induced the percentage of Go/G1phase cells decreased.The difference of the percentage of S phase cells in KG1a after treatment with different concentration DNR for24h and72h were significant (F=43.251, P=0.000; F=126.041,.P=0.000), the percentage of S phase cells were increased first and then decreased significantly comparing with the control group when the concentration of DNR was increased (P<0.05), except the0.2μmol/L DNR goup for24h.0.2μmol/L,0.8μmol/L and1.6μmol/L DNR treatment groups for the percentage of S phase cells were significantly different between the different treatment times (F=13.336, P=0.022; F=100.644, P=0.001; F=256.210, P=0.000). DNR induced the percentage of S phase cells increased finally.The difference of the percentage of G2/M phase cells in KGla after treatment with different concentration DNR for24h and72h were significant (F137.7651, P=0.000; F=270.731, P=0.000), the percentage of G2/M phase cells were decreased first and then increased significantly comparing with the control group when the concentration of DNR was increased (P<0.05), except the1.6μmol/L DNR goup for 24h.0.2μmol/L,0.8μmol/L and1.6μmol/L DNR treatment groups for the percentage of G2/M phase cells were significantly different between the different treatment times (F=280.308, P=0.000; F=238.568, P=0.000; F=59.312, P=0.002). DNR induced the percentage of G2/M phase cells increased.These results indicated that DNR could affect the cell cycle distribution and arrest KG1a cells at S phase.4.5.2Effects of DNR on the expression of p21and cyclin E in KGla cellsTo study the underlying the cell cycle redistribution mechanisms, the expression of cell cycle regulators, p21and cyclin E, were assessed after treatment with0.05μmol/L,0.1μmol/L,0.2μmol/L,0.4μmol/L,0.8μmol/L,1.6μmol/L DNR for72h. P21and cyclin E were detected in all the groups treated with or without DNR treatment for72h. According with the concentration of DNR increasing, the expression of both p21was decreased and cyclin E was increased. The result indicated that DNR can induce the cell cycle redistribution of KG1a cells by decreasing p21and increasing cyclin E.Conclusion:DNR can significantly inhibite the proliferation, the clone formation of KG1a cells and in the expression of Eps8in KGla cells on a dose-and time-depended matter; DNR can induce the apoptosis of KGla cells by increasing caspase-3and decreasing bcl-2, and induce the cell cycle redistribution of KG1a cells by decreasing p21and increasing cyclin E.Conclusions:1.The expression of Eps8was significantly increased in AML patients compared with the healthy volunteers(t=3.055, P=0.006), and correlated with the AML patients achieved CR or not after one course of chemotherapy (P=0.021). The CR rate of high expression group was significantly lower than low expression group (P=0.024). Eps8was obviously high expressed in KG la cell line, indicating Eps8may play an important role in the pathogenesis of AML and KG1a maybe s good study model.2. Genes (such as AKT1, BCL2, CASP3, PIK3CA, TP53) which participant in the PI3K/Akt signaling pathway and small GTPase mediated signal transduction highly expressed in KG1a cells.3. Perifosine can significantly inhibite the proliferation, the clone formation and the expression of Eps8in KGla cells in a dose-and time-depended matter. Perifosine can induc... |
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