| Background and ObjectivesAcute myeloid leukemia(AML) is a highly heterogeneous hematologic malignancy in cytogenetics and molecular biology, which is characterized by proliferation of an abnormal clone of myeloid hematopoietic stem/progenior cell and blockage of cell differentiation and anti-apoptosis. Reproducibility of genetic abnormality can be detected in approximately 30% of AML patients by modern cytogenetics technology, and the t (8; 21) (q22; q22) translocation is a common chromosome abnormality. The t(8;21)(q22;q22) translocation involves the AML1 gene on chromosome 21 and the ETO gene on chromosome 8 generating the AML1/ETO fusion gene, which is found in 12% to 20% of AML patients. The AML1/ETO fusion gene occurs in about 40% of M2 subtype according to the French-American-British (FAB) classification, and occasionally occurs in other subtypes, such as M1 and M4. The AML-M2b subtype proposed by Chinese scholars in the late 1950s is commonly characterized by highrate of extramedullary infiltration, good treatment response, high rate of complete response and long remissionn duration. In addition, the AML1/ETO fusion gene is detected more than 90% of AML-M2b patients. AML with t (8; 21) (q22; q22) (AML1/ETO) was defined as an independent subtype by the world health organization (WHO) in 2001, and patients with the tipical cytogenetic abnormality can be diagnosed as AML regardless of the percentage of blasts in bone marrow. AML with AML1/ETO-positive is generally considerated as a favorable prognosis according to the risk stratification, which can obtain good curative effect by standard chemotherapy, particularly with high-dose cytosine arabinoside (Ara-C) consolidation chemotherapy. However, the treatment outcomes are not satisfactory for a high relapse rate within one year about 30% or more. As for the assessment of prognosis of AML patients with AML1/ETO-positive, many other elements including additional chromosomal aberrations, individual genes mutations, and clinical course should be considerated.In recent years, with the improvement and development of the classification of acute leukemia MICM, more and more molecular markers associated with the prognosis of AML patients have been found. The discovery of these molecular which could not been detected by markers could not be detected by cytogenetics method indicate that estimation of prognosis in AML patients at a molecular level. The gene for human amyloid precursor protein (APP) is on chromosome 21q21.3-22.05 and encode a type Ⅰ integral membrane glycoprotein. APP is expressed in most human cells, which plays important roles in several physiological processes, such as regulating growth of neuron, synaptogenesis and cell adhesion. Many recent studies have indicated that APP is overexpressed commonly in solid tumors and mainly involved in promoting proliferation of tumor cells, including oral squamous cell carcinoma, pancreatic cancer cells, parathyroid tumor and breast cancer. In addition, overexpression of APP gene indicates poor prognosis in these tumors. However, reports on research of the clinical significance in leukemia have been rarely reported so far. APP is also found significantly higher in AML with abnormal chromosome 21 compared to a control group of AML with normal cytogenetics using oligonucleotide arrays, which is verified by quantitative real-time polymerase chain reaction(qRT-PCR) subsequantly. We previously found that the expression levels of APP gene is significantly higher in bone marrow mononuclear cells(BMMNCs) of AML patients with AML1/ETO fusion gene than other AML subtypes. Therefore, we speculate that APP gene may be closely related to the development and prognosis of AML1/ETO-positive AML. However, the clinical significance and function of APP gene in AML patients with AMLl/ETO-positive remain unclear.In this study, we detected the expression levels of APP gene in BMMNCs from 44 cases of AML1/ETO-positive AML patients, then analyzed the relationship of APP expression levels and cinical efficacy, as well as clinical prognosis to make clear the clinical significance of APP gene in this type of AML. We next examined the roles of APP gene in AML1/ETO-positive leukemia cells via an RNA-interference (RNAi) lentivirus system in human AML-M2b cell line Kasumi-1 carrying AML1/ETO fusion gene, and studied the effects of APP gene silencing on biological behavior of Kasumi-1 cells such as cell proliferation, apoptosis and migration. Besides, we further explored the possible mechanisms of APP gene in AML1/ETO-positive leukemia cells and investigated that whether the pan-histone deaceylase inhibitor panobinostat could inhibite APP expression, which may provide a new strategy for the treatment of AMLl/ETO-positive AML patients.Research methods and contents1. Detection of expression levels of APP gene in BMMNCs from AML1/ETO-positive AML patientsBone marrow samples were collected from 44 cases of initial diagnosed AML patients from June 2007 to December 2013 in our hosipital, and all the 44 patients are confirmed with t (8; 21) (q22; q22) chromosome translocation and AML1/ETO fusion gene positive by chromosome karyotype analysis, fluorescence in situ hybridization (FISH) and PCR detection. BMMNCs from AMLl/ETO-positive AML patients were isolated using lymphocyte separation medium. The APP mRNA levels relative to P-actin in BMMNCs were determined using qRT-PCR method.2. Clinical curative effect and prognosis of APP gene overexpression in AMLl/ETO-positive AML patientsPatients were divided into highly expressed APP and low expressed APP groups according to mRNA median expression level of APP gene. The incidence rate of extramedullary infiltration(EMI) and complete response(CR) rate receiving no more than two courses of chemotherapy between the two groups were analyzed. The follow-up was completed on Feb 18th 2013, with a median follow-up of 21 months(range 2-88 months). Overall survival (OS) rate and recurrence free survival (RFS) rate were analyzed between the two groups. We also evaluated the clinical prognosis of the two groups using Kaplan-Meier survival analysis.3. APP expression in Kasumi-1 cell line was inhibited by lentivirus-mediated shRNAHuman AML-M2b cell line Kasumi-1, carrying t (8; 21) (q22; q22) chromosome translocation and AML1/ETO fusion gene positive, is an ideal cell line for studying the roles of APP gene in AML1/ETO-positive leukemia cells. We explored the function of APP gene via inhibiting APP expression in Kasumi-1 cells using RNAi technology. First of all, the Short hairpin RNAs (shRNAs) designed directly from APP specific and non-specific small interference RNA(siRNA) sequences, and then were packaged into lentivirus particles by Shanghai Genechem. Lentivirus transfection experiment was divided into three groups, including non-treated Kasumi-1 cells(Con), transfected APP non-specific shRNA(Lv-shCon) and transfected APP specific shRNA(Lv-shAPP). After Kasumi-1 cells were transducted with lentivirus particles, green fluorescent protein (GFP) positive cells were separated by flow cytometry and then were incubated expandedly. The suppressive effect of shRNA on endogenous APP gene of Kasumi-1 cells were determined by qRT-PCR and western blotting.4. Effects of APP gene silencing on proliferation, cell-cycle, apoptosis, differentiation and migration of Kasumi-1 cellsWe next studied the effects of stable APP gene silencing on proliferation, cell-cycle, apoptosis, differentiation and migration of Kasumi-1 cells. The proliferation effect of APP gene silencing on Kasumi-1 Cells was determined using Cell Counting Kit-8 (CCK8). The percentage of apoptotic cells was analyzed using the Annexin V-APC/propidium iodide (PI) Apoptosis Detection Kit. The cell-cycle distribution was analyzed by flow cytometer with PI staining. Cell differentiation was analyzed by flow cytometer with CDllb-PE staining. Cell migration assay was performed in transwell chambers with 8μm pore size polycarbonate filter.5. Research on the possible molecular mechanism of APP gene in mediating migration of Kasumi-1 cellsMatrix metalloproteinases(MMPs) are the main enzymes which involve in the degradation of extracellular matrix(ECM). Many studies have shown that MMPs play important roles in migration of tumor cells, and MMP1, MMP2 and MMP9 are closely related to migration of many tumor cells. We first detected the protein levels of MMP1, MMP2 and MMP9 by western blotting. The transcription factors c-Myc and Etsl that regulating MMPs were analyzed. As upstream regulatory genes of c-Myc, the expression levels of p-ERK and ERK were also determinedso as to explore the possible molecular mechanism of APP gene in mediating migration of Kasumi-1 cells.6. Analysis of Panobinostat treatment on APP signalingAfter treatment with Panobinostat at different concentritions for 48 hours, the concentration of PS that induced inhibition in 50% of the treated Kasumi-1 cell population (IC50 value) was determined by CCK8 method. After treatments with PS at different concentrations(5,10,20,40 and 80nM) for 48 hours, the expression levels of APP, p-ERK, c-Myc and MMP2 were analyzed by western blotting, so as to make clear whether Panobinostat could intervene APP-mediated signaling pathways.7. Statistical analysisStatistical analyses were performed with SPSS 16.0 softwore. The data with non-normal distribution were described with the median. Nonparametric test was used for comparison between the groups (the Mann-Whitney U test for two groups). The comparison of rate between groups was performed with chi-square(χ2) test. Kaplan-Meier curve and log-rank test were used to analyze OS and RFS.The data on normal distribution were presented as mean±S.D. from at least three independent experiments. One-way ANOVA was used for comparison among multi-groups if the variance was homogeneous, while comparison between two groups was performed with the student’s test test A P-value of less than 0.05 was considered statistically significant.Results1. Expression levels of APP gene in BMMNCs of AMLl/ETO-positive AML patientsThe median relative expression levels of APP mRNA in BMMNCs from 44 cases of AML1/ETO-positive AML patients was 0.009397(0.0000413-0.08367). All the patients were divided into highly expressed APP group(n=22) and low expressed group(n=22) according to the below and above the median levels of mRNA, and the difference of APP mRNA between the two groups was confirmed significant statistically by Mann-Whitney U test(Z=-5.680, P<0.001). There were 18 cases(81%) achieved CR after no more than two courses of chemotherapy in highly expressed APP group, while 4 cases(19%) were partial remission or non-remission. However, all the 22 cases in low expressed APP group achieved CR after no more than two courses of chemotherapy. The CR rate in highly expressed APP group was significantly less than low expressed APP group by Chi-square analysis(x2=4-400, P=0.036). The incidence of EMI in highly expressed APP group was 9 cases, which was higher than that 3 cases in low expressed APP group(x2=4.125, P=0.042). The median follow-up time was 21 months (2-88 months) before closing date of follow-up. The median OS and RFS were 16.5 and 13 months in highly expressed APP group,24.5 and 22.5 months in low expressed APP group. The rate of OS and RFS in highly expressed APP group were both significantly less than that in low expressed APP group(54.5% vs 90.9%, x2=7.333, P=0.007; 36.4% vs 77.3%, χ2=7.503, P=0.006).2. Lentivirus-mediated shRNA decreased APP expression in Kasumi-1 cellsThe protein levels of APP relative to GAPDH in Kasumi-1 was 1.3423±0.1152, which was dramatically higher than that in K562(0.6254±0.0846,P<0.001) and HL-60 (0.8227±0.0974, P< 0.001). The results demonstrated that APP is overexpressed in AMLl/ETO-positive leukemia cells specifically. After tranfected with lentivirus particles for 72 hours, GFP-positive cells were separated by flow cytometry under aseptic conditions and then were incubated expandedly. The GFP-positive rate of expandedly cultured Kasumi-1 cells was (96.04±0.17)% in Lv-shCon group and (95.82±0.18)% in Lv-shAPP group, which indicated that Kasumi-1 cells were transfected with lentivirus successfully. The suppressive effect of shRNA on APP expression in Kasumi-1 cells were analyzed by qRT-PCR and western blotting. The results of qRT-PCR showed that the relative mRNA levels of APP in Lv-shAPP infected cells was 0.0116±0.0016, which was dramatically reduced when compared with Lv-shCon group(1.0350±0.0557, P<0.001). Compared with con group(set as 1), no significant difference was observed between Con group and Lv-shCon group(95%CI,0.933-1.112). Western blotting analysis showed that relative protein levels of APP was 0.0084±0.0007 in Lv-shAPP group, which was significantly decreased when compared with Con group(1.2465±0.0480, P<0.001) and Lv-shCon group(1.2366±0.0421, P< 0.001). No significant difference was observed between Con group and Lv-shCon group(P>0.05).3. Inhibition expression of APP gene mainly impairs migration ability of Kasumi-1 cellsIn comparision with untreated (Con) and Lv-shCon transfected Kasumi-1 cells, the OD450nm values at different timepoints(24,48,72 and 96 hours) were slightly lower after APP gene was inhibited in Kasumi-1 cells. Therefore, depletion of APP gene inhibited the ability of cell proliferation slightly. However, no statistical significance was found in these distinctions (P>0.05). Flow cytometry analysis of three groups of cell-cycle distribution showed that there was no statistical significance of the proportions of G0/G1, S and G2/M phases between Lv-shAPP group and Con group(36.37±3.73% vs 36.25±9.01%, P=0.985; 28.19±6.08% vs 26.21±5.13, P=0.663; 33.07±2.74% vs 34.25±2.92%, P=0.771). Flow cytometry analysis of cell apoptosis showed that the mean proportion of apoptotic cells in Lv-shAPP group was slightly higher than that in control groups. However, no statistical significance was found in these distinctions (8.40±0.96% vs 6.80±0.46%, P=0.065; 8.40±0.96% vs 7.10±1.06%, P=0.116). The proportion of CD11b-positive Kasumi-1 cells in Lv-shAPP group had no statistical difference when compared to con group (6.63± 0.66% vs 5.69±0.50%, P>0.05) and Lv-shCon group (6.63±0.66% vs 6.29± 0.50%, P>0.05). The effect of APP silencing on migration ability of Kasumi-1 cells was assessed using transwell chamber by counting cells in the lower chamber. It was worth noting that the proportion of cells in lower chamber was much less than that in Con group(7.50±0.83% vs 12.78±2.1%, P=0.004). The results demonstrated that APP silencing in Kasumi-1 cells mainly impairs the ability of cell migration rather than inhibites cell proliferation, induces apoptosis or arrests cell-cycles.4. Molecular mechanism of APP gene in mediating migration of Kasumi-1 cellsWestern blotting analysis showed that the protein levels of MMP2 was significantly reduced after APP gene silencing in Kasumi-1 cells(P<0.001). However, the protein levels of MMP1 and MMP9 changed little in Lv-shAPP group when compared with Con and Lv-shCon groups(P>0.05). We next detected the expression levels of transcription factors c-Myc and Etsl, which are the upstream regulations of MMPs. Compared to Con group, the protein levels of c-Myc in Lv-shAPP group was significantly decreased(0.0896±0.0160 vs 0.7024±0.0179, P<0.001), whereas the protein levels of Etsl in Lv-shAPP group was almost no change (0.0996±0.0097 vs 0.1035±0.0177, P=0.727). In addition, the differences of expression levels of c-Myc and Etsl in Con group and Lv-shCon group had no statistical significance(P>0.05). As upstream regulations of c-Myc, the protein expression levels of p-ERK and ERK were further analyzed. The protein levels of p-ERK in Lv-shAPP group was significantly decreased when compared with Con group(0.01838±0.007 vs 0.1666± 0.01624, P<0.001). The differences of ERK expression levels in the three groups had no statistical significance(P>0.05).5. Panobinostat treatment inhibites APP signalingThe suppressive effect of panobinostat on Kasumi-1 cells was proved to be a concentration- and time-dependent manner. The concentration of panobinostat that induced inhibition in 50% of the treated cell population (IC50 value) was 20 nM. The protein levels of APP, p-ERK, c-Myc and MMP2 in Kasumi-1 cells were all depleted after treatment with panobinostat for 48 hours with increasing concentrations (5,10, 20,40 and 80nM). Taken together, these results indicated that panobinostat could inhibite APP expression and suppress APP signaling effectively. The concentrations of panobinostat that surpressed APP signaling pathway also significantly inhibited proliferation, induced apoptosis and necrosis of Kasumi-1 cells.Conclusions1. AML1/ETO-positive AML patients with overexpression of APP gene have a significant higher incidence of EMI than patients with low expressed APP. In addition, the rate of CR, OS and RFS in patients with highly expressed APP are lower than that in patients with low expressed APP, while OS and RFS are both shorter in highly expressed APP. As a result, overexpression of APP gene indicates a poor prognosis in AMLl/ETO-positive AML patients.2. Lentivirus-mediated shRNA could inhibite expression of APP gene in Kasumi-1 cells stably. APP silencing in Kasumi-1 cells mainly impairs the ability of cell migration rather than inhibites cell proliferation, arrests cell-cycles, induces cell apoptosis and differentiation.3. The protein levels of p-ERK, c-Myc and MMP2 are significantly down-regulated after APP gene silencing, which indicates that ERK pathway is involved in APP-mediated cell migration. Therefore, ERK/c-Myc/MMP2 pathway is involved in the mechanism of APP mediating migration of Kasumi-1 cells.4. The protein levels of APP, p-ERK, c-Myc and MMP2 in Kasumi-1 cells are all depleted after Panobinostat treatment with increasing concentrations. These results indicated that Panobinostat could inhibite APP expression and suppress APP signaling effectively. The concentrations of panobinostat that surpressed APP signaling pathway also significantly inhibited proliferation, induced apoptosis and necrosis of Kasumi-1 cells, which indicates an effective drug against leukemia patients with AMLl/ETO-positive. |
PDF Full Text Request