[Expression of Netrin-1 in Patients with Acute Lymphoblastic Leukemia and Its Clinical Significance].

OBJECTIVE: To investigate the correlation of the Netrin-1 expression level with the clinical characteristics in children with acute lymphoblastic leukemia (ALL) and to explore its possible regulatory mechanism. METHODS: ELISA was used to detect the expression level of Netrin-1 in peripheral blood serum from 48 child ALL patients (newly diagnosed, recurrent), and its relevance with clinical indicators was statistically analyzed. The blood serum samples from 27 children with non malignant hematological diseases were choosen as controls. Leukemia cell lines of Jurkat,Molt-4,SUP-B15 and Raji were cultivated in vitro, after treated with different concentrations of recombinant human Netrin-1 protein， the invasive ability of the cells was detected by Transwell method； the effect of Netrin-1 to the proli feration of cells was detected by CCK-8 method； The expression and phosphorylation level of key molecules, such as FAK,Erk1/2,PI3K and Akt signaling pathway were detected by Western blot. RESULTS: The expression of Netrin-1 in child patients was significantly higher than that of the control group (P<0.05). With the increasing of Netrin-1 level, the level of Plt (r=0.483, P<0.05) increased, while the level of WBC (r=-0.290, P<0.05) decreased, and there were no significant correlation with age, Hb level and the proportion of immature cells in bone marrow. When the concentration of Netrin-1 was 25-50 ng/ml, the level of Netrin-1 positively correlated with WBC (r=0.886, P<0.05) ; the level of Netrin-1 significantly decreased when the patient's WBC was ＞50×109/L and Plt ＞20×109/L(P=0.042，P=0.001); The expression level of Netrin-1 was significantly different in the risk group(P=0.017), and level of Netrin-1 in high-risk group was significantly higher than that in low risk group and middle risk group, but there was no significant difference of Netrin-1 expression in sex, hepatosplenomegaly, MRD, recurrence and chromosome abnormality. Netrin-1 could promote the invasiveness of the four kinds of cells (P<0.05). With the increase of Netrin-1 concentration, the number of cells increased at first and then decreased, and the number of cells in the invading chamber was the highest when the concentration of Netrin-1 was 100 ng/ml; the survival rate of the four kinds of cells significantly increased when the concentration of Netrin-1 was 25 ng/ml(P<0.05), and SUP-B15 cells showed the highest cell survival rate at a concentration of 100 ng/ml; The survival rate of the four kinds of cells showed a tendency : survival of cells increased at low concentration of Netrin-1 and survival of cells decreased at high concentration of Netrin-1. The results of Western blot showed that Netrin-1 activated the phosphorylation level of key molecules such as FAK,Erk1/2,PI3K,Akt signaling pathway (P<0.05). CONCLUSION: There is abnormal expression of Netrin-1 in serum of children with ALL. Netrin-1 may affect the occurrence and development of ALL by increasing the proliferation and invasiveness of leukemia cells, and may become a risk factor of ALL or a potential target in biotherapy.

[E-cadherin Expression in Children with Acute Leukemia and Its Clinical Significance].

OBJECTIVE: To investigate the correlation of E-cadherin expression level with the clinical characterastics in children with acute leukemia (AL), and to explore the possible regulatory mechanism. METHODS: Real-time quantitative RT-PCR was applied to detect the expression level of E-cadherin in bone marrow samples from 135 child patients diagnosed as AL, and its relevance with clinical indicators was statistically analyzed. The expression levels of E-cadherin, ß-catenin, and Akt/p-Akt were detected by using Western blot. The bone marrow samples from 22 children with non-malignant hematological diseases were used as controls. RESULTS: The expression level of E-cadherin significantly decreased in newly diagnosed patients with all 3 types of AL as compared with bone marrow samples from control group (P<0.01). In B-ALL group, compared with standard risk group, E-cadherin expression level significantly decreased in intermediate risk group (P<0.05). Moreover,the expression level of E-cadherin mRNA was also reduced in splenomegaly group (P<0.01). However, the correlation of E-cadherin level with clinical characteristics was not found in T-ALL and AML (P>0.05). The expression level of E-cadherin in the patients from Common-B-ALL group was higher than B-ALL patients with other immunophenotypes (P<0.01), while no significant difference was found among patients grouped by FAB classification. By the correlation analysis of measured data, lower E-cadherin expression level was found to be related with high WBC count and serum lactic dehydrogenase level (LDH) (r=-0.419, r=-0.269), but with low blood platelet count in B-ALL (r=0.335). In T-ALL, expression of E-cadherin was found to be negatively correlated with LDH and percentage of immature cells in the bone marrow (r=-0.567, r=-0.557). In addition, the lower expression of E-cadherin was also found to be related with WBC count and percentage of immature cells in the bone marrow in newly diagnosed AML patients (r=-0.368, r=-0.391). Compared with control group, the expression of E-cadherin was down-regulated significantly (P<0.01), while ß-catenin, Akt significantly was up-regulated in 3 types of AL patients (P<0.01). The expression of p-Akt and p-Akt/Akt was up-regulated significantly in T-ALL (P<0.01). CONCLUSION: Lower expression of E-cadherin is related factor of unfavourable prognosis in children with acute leukemia. The expression deficiency or down-regulation of E-cadherin may activate Wnt/ß-catenin and PI3K/ Akt signaling pathways to promote the genesis and progress of haematological malignancies, thus resulting in a series of malignant biological behaviors in cells. E-cadherin may be a new prognostic indicator for pediatric acute leukemia, thus to guide individualized hemotherapy.

[Effect of 4' -Hydroxywogonin on Proliferation and Apoptosis of Human Acute Lymphoblastic Leukemia Cells].

OBJECTIVE: To investigate the effect of 4' -hydroxywogonin on proliferation and apoptosis of human acute lymphoblastic leukemia SUP-B15 and Jurkat cells, and to analyze its possible mechanism. METHODS: SUP-B15 and Jurkat cells were cultivated in vitro and treated with different concentrations of 4' -hydroxywogonin, the inhibitory effect of 4' -hydroxywogonin on the proliferation of SUP-B15 and Jurkat cells was detected by CCK-8 method; the cell apoptosis was examined by the flow cytometry with Annexin V-APC/7-AAD donble staining; the expression of C-MYC, BCL-2 and cleaved caspase 3 in SUP-B15 and Jurkat cells were measured with Western blot. RESULTS: 4' -hydroxywogonin inhibited the proliferation of SUP-B15 and Jurkat cells in a dose-dependent manner (r=0.78, r=0.89), with IC50 value of (6.32± 0.53) µg/ml in SUP-B15 cells and (12.04± 0.42) µg/ml in Jurkat cells at 24 h. The early apoptotic rate of cell was also enhanced with the increase of 4' -hydroxywogonin concentrations. The results of Western blot showed that 4' -hydroxywogonin could down-regulate the expression of proliferation-related molecule C-MYC(P<0.01) and apoptosis-related molecule BCL-2(P<0.01), the expression of apoptosis-related molecule cleaved caspase 3 was up-regulate(P<0.01). CONCLUSION: 4' -hydroxywogonin shows the effects of anti-tumor by inducing cell apoptosis and inhibiting cell proliferation, its molecular mechanism maybe relate with down-regulation of C-MYC and BCL-2 expression and up-regulation of the cleaved caspase 3 expression.