sincalide and Precursor-Cell-Lymphoblastic-Leukemia-Lymphoma

B-cell acute lymphoblastic leukemia (B-ALL) comprises over 85% of all acute lymphoblastic leukemia (ALL) cases and is the most common childhood malignancy. Although the 5 year overall survival of patients with B-ALL exceeds 90%, patients with relapsed or refractory B-ALL may suffer from poor prognosis and adverse events. The axon guidance factor netrin-1 has been reported to be involved in the tumorigenesis of many types of cancers. However, the impact of netrin-1 on B-ALL remains unknown.. The expression level of netrin-1 in peripheral blood samples of children with B-ALL and children without neoplasia was measured by enzyme-linked immunosorbent assay (ELISA) kits. Then, CCK-8 cell proliferation assays and flow cytometric analysis were performed to detect the viability and apoptosis of B-ALL cells (Reh and Sup B15) treated with exogenous recombinant netrin-1 at concentrations of 0, 25, 50, and 100 ng/ml. Furthermore, co-immunoprecipitation(co-IP) was performed to detect the receptor of netrin-1. UNC5B expression interference was induced in B-ALL cells with recombinant lentivirus, and then CCK-8 assays, flow cytometry assays and western blotting assays were performed to verify that netrin-1 might act on B-ALL cells via the receptor Unc5b. Finally, western blotting and kinase inhibitor treatment were applied to detect the downstream signaling pathway.. Netrin-1 expression was increased in B-ALL, and netrin-1 expression was upregulated in patients with high- and intermediate-risk stratification group of patients. Then, we found that netrin-1 induced an anti-apoptotic effect in B-ALL cells, implying that netrin-1 plays an oncogenic role in B-ALL. co-IP results showed that netrin-1 interacted with the receptor Unc5b in B-ALL cells. Interference with UNC5B was performed in B-ALL cells and abolished the antiapoptotic effects of netrin-1. Further western blotting was applied to detect the phosphorylation levels of key molecules in common signaling transduction pathways in B-ALL cells treated with recombinant netrin-1, and the FAK-MAPK signaling pathway was found to be activated. The anti-apoptotic effect of netrin-1 and FAK-MAPK phosphorylation was abrogated by UNC5B interference. FAK inhibitor treatment and ERK inhibitor treatment were applied and verified that the FAK-MAPK pathway may be downstream of Unc5b.. Taken together, our findings suggested that netrin-1 induced the anti-apoptotic effect of B-ALL cells through activation of the FAK-MAPK signaling pathway by binding to the receptor Unc5b. Video Abstract.

Topics: Child; Humans; MAP Kinase Signaling System; Netrin Receptors; Netrin-1; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Receptors, Cell Surface; Sincalide; Tumor Suppressor Proteins

Microrna-143 (miR-143) has been suggested to be a tumor suppressor, yet its role in hematological tumors has not been determined. Thus, we aimed to explore the expression and function of miR-143 in leukemia cells. miR-143 expression was assessed in bone marrow samples from 63 leukemia patients and 15 healthy controls using q-PCR, and its correlation with DNMT3A expression was determined. In addition, after lentiviral-mediated miR-143 overexpression, K562 cell proliferation was evaluated using CCK-8 analysis; cell cycle progression and apoptosis were determined using flow cytometry. The expression of Bcl-2 and pro-caspase-3 and -9 was assessed by q-PCR and western blot analysis, respectively. Leukemia patients had significantly lower relative miR-143 expression than healthy controls (P=0.004), and the expression levels of miR143 and DNMTA3A were negatively correlated (r=-0.663, P=0.001). Overexpression of miR-143 decreased DNMT3A mRNA and protein expression, and significantly reduced K562 cell proliferation at 72 and 96 h (both P ≤ 0.018). In addition, reduced colony formation and cell cycle progression were observed upon miR-143 overexpression. Flow cytometric analysis revealed that the early apoptosis rate was higher in the miR-143 group than the rate in the NC group. Bcl-2 mRNA expression and pro-caspase-3 and -9 protein expression were reduced in the miR-143-expressing cells. These findings suggest that miR-143 plays an important role in leukemia cell proliferation and apoptosis, possibly through silencing of DNMT3A. Further studies are necessary to determine the prognostic value and therapeutic potential of targeting miR-143.

Topics: Adolescent; Adult; Aged; Apoptosis; Bone Marrow Cells; Caspase 3; Caspase 9; Cell Line, Tumor; Cell Proliferation; Child; DNA (Cytosine-5-)-Methyltransferases; DNA Methyltransferase 3A; Female; Gene Expression Regulation, Neoplastic; Genes, Tumor Suppressor; Humans; Leukemia, Myeloid, Acute; Male; MicroRNAs; Middle Aged; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Proto-Oncogene Proteins c-bcl-2; RNA, Messenger; Sincalide; Young Adult