OBJECTIVETo investigate the effect of SH2-containing inositol phosphatase-1 (SHIP-1) on the proliferation, invasion and migration of human leukemia cells as well as phosphatidylinositol-3 kinase (PI3K) / protein kinase B (AKT) signaling pathway.

METHODSThe overexpression vector pCDNA3.1-SHIP1 was transfected into THP-1 cells by Lipofectamine 2000. The experiment was divided into 3 groups: control group (untreated cells) and empty vector group (transfected with empty vector pCDNA3.1-NC) and overexpression group (transfected with overexpression vector pCDNA3.1-SHIP1). The cell proliferation was tected by CCK-8 assay, Transwell assay was used to evaluate the cell invasion and migration capabilities. The expressions of SHIP-1, AKT, phosphorylated AKT (pAKT), matrix metalloproteinase-9 (MMP-9) protein were analyzed by Western blot.

RESULTSThe expression of SHIP-1 in overexpression group was significantly higher than that in the control group(P<0.05). Compared with the control group, the absorbance of the cells in the empty vector group was not statistically different (P>0.05), and the absorbance in overexpression group decreased significantly(P<0.05). The cell numbers of invasion and migration were not significantly different between empty and control groups(P>0.05), but those in overexpression group were significantly lower than those in the control group(P<0.05). Compared with the control group, the expression of AKT, pAKT and MMP-9 in the empty vector group was not statistically different (P>0.05); the AKT protein in overexpression group was not significantly different (P>0.05), but the pAKT and MMP-9 significantly decreased(P<0.05).

CONCLUSIONSHIP-1 plays a role in inhibiting the proliferation, invasion and migration of leukemia cells, the mechanism probably relates with supressing the expression of MMP-9 by regulating PI3K/AKT signaling pathway.

Cell Line, Tumor ; Cell Movement ; Cell Proliferation ; Humans ; Neoplasm Invasiveness ; Phosphatidylinositol 3-Kinases ; Phosphatidylinositol-3,4,5-Trisphosphate 5-Phosphatases ; Proto-Oncogene Proteins c-akt ; Signal Transduction

Brain ; pathology ; Genome-Wide Association Study ; Humans ; Parkinson Disease ; genetics ; Phosphatidylinositol-3,4,5-Trisphosphate 5-Phosphatases ; genetics ; Proto-Oncogene Proteins c-crk ; genetics

The SH2 domain containing inositol 5'-phosphatase (SHIP) was initially described as a 145 kD protein phosphorylated on tyrosines upon growth factor and cytokine stimulation. SHIP is predominately expressed in hematopoietic cells, and is a crucial negative regulator in the development of hematopoietic cells. To evaluate the role of the SHIP gene in human leukemogenesis, expression and mutation of SHIP gene in bone marrow and/or peripheral blood from 32 patients with acute myeloid leukemia (AML), 9 patients with acute lymphoblastic leukemia (ALL), as well as human hematopoietic cell lines were analyzed by reverse transcription-polymerase chain reaction (RT-PCR), single strand conformational polymorphism (SSCP) and sequencing. The RT-PCR showed that all samples expressed SHIP gene. Mutations of SHIP gene were detected in 7 out of 32 AML patients (22%) and one out of 9 ALL patients (12%). Interestingly, two missense mutations that had been observed in one AML patient at diagnosis disappeared after complete remission (CR). In addition, Akt phosphorylation was prolonged and increased following IL-3 stimulation in this patient sample. In conclusion, data of this study demonstrate the mutation of the SHIP gene in acute leukemia for the first time and suggest a possible role of the mutation of this gene in the development of acute leukemia. SHIP serves as a tumor suppressor by negatively regulating the PI3K/Akt signaling pathway in hematopoietic cells.
Cell Line ; Humans ; Leukemia, Myeloid, Acute ; genetics ; Mutation ; PTEN Phosphohydrolase ; Phosphatidylinositol-3,4,5-Trisphosphate 5-Phosphatases ; Phosphoric Monoester Hydrolases ; genetics ; physiology ; Phosphorylation ; Polymorphism, Genetic ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; genetics ; Protein-Serine-Threonine Kinases ; metabolism ; Proto-Oncogene Proteins ; metabolism ; Proto-Oncogene Proteins c-akt ; Tumor Suppressor Proteins ; physiology

OBJECTIVE: To investigate the anti-inflammatory effect and mechanisms of interleukin-35 (IL-35) in inflammatory bowel disease. METHODS: BALB/c mice were divided into three groups with 10 mice in each group:control group, model group (oral administration of 4% glucan sodium sulfate for 7 d) and IL-35-treated group (oral administration of 4% glucan sodium sulfate for 7 d, intraperitoneal injection of 2 μg IL-35 at d2-5). Disease activity index (DAI) was scored every day. After 7 d, the mice were sacrificed, and the serum and intestinal tissue samples were collected. The gross morphology of the colon was observed; HE staining was used to observe the pathological changes of colon tissue; flow cytometry was employed to detect the change of macrophage polarization ratio in colon tissue; the mRNA expression levels of cytokines IL-6, TNF-α, IFN-γ, IL-10 and SHIP1 in colon tissue were determined by real-time quantitative RT-PCR; the expression and distribution of SHIP1 in colon tissue was measured by immunohistochemistry; Western blotting was adopted to detect the expression level of SHIP1 protein in colonic intestinal tissues of each group. RESULTS: The DAI scores of the mice in the model group were higher than those in the control group, while the DAI scores in the IL-35-treated group were lower than those in the model group (all <0.01). Compared with the control group, the colon length was significantly shortened in the model group (<0.05), while the colon length of the IL-35-treated group had an increasing trend compared with the model group, but the difference was not statistically significant (>0.05). Compared with the model group, microscopic inflammatory infiltration score was decreased and microscopic crypt destruction and score was significantly lower in IL-35-treated group (all <0.05). The relative expression of proinflammatory cytokines IL-6, TNF-α and IFN-γ in the colon tissue of IL-35-treated group was decreased compared with the model group, while the relative expression of IL-10 mRNA was higher than that of the model group (all <0.05). Compared with the control group, the proportion of M1 macrophages in the model group increased (<0.05), while the proportion of M1 macrophages in the IL-35-treated group was lower than that in the model group (<0.05). The relative expression of SHIP1 mRNA and protein in the colon tissue of IL-35-treated group was higher than that in the model group (all <0.05). CONCLUSIONS IL-35 can inhibit the polarization of M1 macrophages and regulate inflammatory cytokines to promote anti-inflammatory effect on mice with colitis.
Animals ; Anti-Inflammatory Agents ; pharmacology ; Colitis ; drug therapy ; physiopathology ; Colon ; drug effects ; Cytokines ; genetics ; Disease Models, Animal ; Gene Expression Regulation ; drug effects ; Glucans ; pharmacology ; Interleukin-6 ; genetics ; Interleukins ; pharmacology ; Macrophages ; drug effects ; Mice ; Mice, Inbred BALB C ; Phosphatidylinositol-3,4,5-Trisphosphate 5-Phosphatases ; genetics