Huo Tan

Mutations increased overexpression of Notch

BackgroundThe Notch signaling pathway is crucial in T-cell development, Notch 1 mutations are frequently present in T-cell acute lymphoblastic leukemia (T-ALL). To investigate the feature of Notch 1 mutation and its corresponding expression level in Chinese patients with T-ALL, detection of mutation and the expression level of Notch 1 gene was preformed using RT-PCR, sequencing and real-time PCR respectively.ResultsTwo Notch 1 point mutations (V1578E and L1593P) located on HD-N domain were identified in three cases out of 13 T-ALL patients. The mutation on 4733 position (V1578E) found in two cases was a novel mutation. The overexpression of Notch 1 was detected in all samples with T-ALL, moreover, significantly higher expression of Notch 1 was detected in the T-ALL with Notch 1 mutation group compared with T-ALL with WT Notch 1 group (p = 0.0192).ConclusionsHigher expression of Notch 1 was associated with Notch 1 mutation, more novel mutation of this gene might be identified in different populations and its contribution to the molecular pathogenesis of T-ALL is needed further research.

Mutations increased overexpression of Notch 1 in T-cell acute lymphoblastic leukemia

BackgroundThe Notch signaling pathway is crucial in T-cell development, Notch 1 mutations are frequently present in T-cell acute lymphoblastic leukemia (T-ALL). To investigate the feature of Notch 1 mutation and its corresponding expression level in Chinese patients with T-ALL, detection of mutation and the expression level of Notch 1 gene was preformed using RT-PCR, sequencing and real-time PCR respectively.ResultsTwo Notch 1 point mutations (V1578E and L1593P) located on HD-N domain were identified in three cases out of 13 T-ALL patients. The mutation on 4733 position (V1578E) found in two cases was a novel mutation. The overexpression of Notch 1 was detected in all samples with T-ALL, moreover, significantly higher expression of Notch 1 was detected in the T-ALL with Notch 1 mutation group compared with T-ALL with WT Notch 1 group (p = 0.0192).ConclusionsHigher expression of Notch 1 was associated with Notch 1 mutation, more novel mutation of this gene might be identified in different populations and its contribution to the molecular pathogenesis of T-ALL is needed further research.

Targeted delivery and controlled release of Paclitaxel for the treatment of lung cancer using single-walled carbon nanotubes

A new type of drug delivery system (DDS) based on single-walled carbon nanotubes (SWNTs) for controlled-release of the anti-cancer drug Paclitaxel (PTX) was constructed in this study. Chitosan (CHI) was non-covalently attached to the SWNTs to improve biocompatibility. Biocompatible hyaluronan was also combined to the outer CHI layer to realise the specific targeting property. The results showed that the release of PTX was pH-triggered and was better at lower pH (pH5.5). The modified SWNTs showed a significant improvement in intracellular reactive oxygen species (ROS), which may have enhanced mitogen-activated protein kinase activation and further promoted cell apoptosis. The results of western blotting indicated that the apoptosis-related proteins were abundantly expressed in A549 cells. Lactate dehydrogenase (LDH) release assay and cell viability assay demonstrated that PTX-loaded SWNTs could destroy cell membrane integrity, thus inducing lower cell viability of the A549 cells. Thus, this targeting DDS could effectively inhibit cell proliferation and kill A549 cells, is a promising system for cancer therapy.

Notch1 signaling is involved in regulating Foxp3 expression in T-ALL

BackgroundT-cell acute lymphoblastic leukemia (T-ALL) is a highly aggressive hematologic malignancy. Immune tolerance induced by CD4+CD25+ regulatory T cells (Tregs) with high expression of Foxp3 is an important hypothesis for poor therapy response. Notch1 signaling is thought to be involved in the pathogenesis of this disease. Crosstalk between Notch and Foxp3+Tregs induced immune tolerance is unknown in T-ALL. We studied Foxp3 and Notch1 expression in vivo and in vitro, and analyzed the biological characteristics of T-ALL cell line systematically after Notch inhibition and explored the crosstalk between Notch signaling and Foxp3 expression.MethodsIn vivo, we established T-ALL murine model by Jurkat cells transplantation to severe combined immunodeficiency (SCID) mice. Notch1 and Foxp3 expression was detected. In vitro, we used γ-secretase inhibitor N-S-phenyl-glycine-t-butyl ester (DAPT) to block Notch1 signaling in Jurkat cells. Notch1, Hes-1 and Foxp3 genes and protein expression were detected by PCR and western blotting, respectively. The proliferation pattern, cell cycle and viability of Jurkat cells after DAPT treatment were studied. Protein expression of Notch1 target genes including NF-κB, p-ERK1/2 and STAT1 were determined.ResultsWe show that engraftment of Jurkat cells in SCID mice occurred in 8 of 10 samples (80%), producing disseminated human neoplastic lymphocytes in PB, bone marrow or infiltrated organs. Notch1 and Foxp3 expression were higher in T-ALL mice than normal mice. In vitro, Jurkat cells expressed Notch1 and more Foxp3 than normal peripheral blood mononuclear cells (PBMCs) in both mRNA and protein levels. Blocking Notch1 signal by DAPT inhibited the proliferation of Jurkat cells and induced G0/G1 phase cell cycle arrest and apoptosis. Foxp3 as well as p-ERK1/2, STAT1 and NF-κB expression was down regulated after DAPT treatment.ConclusionsThese findings indicate that regulation of Foxp3 expression does involve Notch signaling, and they may cooperatively regulate T cell proliferation in T-ALL.

Correlation between B7-H3 expression and matrix metalloproteinases 2 expression in pancreatic cancer.

BackgroundB7-H3 and matrix metalloproteinases 2 (MMP-2) are reported highly expressed in malignant tumor, we investigate the relationship between B7-H3 expression and MMP-2 on malignant behavior and prognosis predictable value in pancreatic cancer.MethodsWe tested the expressions of B7-H3 and MMP-2 protein in 45 pancreatic surgical resected cancer samples; meanwhile, the clinicopathological data of enrolled patients were obtained for correlation analysis to obtain their relationship with pancreatic cancer progress.ResultsThe expression of B7-H3 was up-regulated with infiltrating depth, lymph node metastasis and TNM stage (P < 0.01). Positive expression rate of MMP-2 in pancreatic cancer tissues was 44.35%, whereas negative in normal pancreatic tissues. Multivariate analysis of Logistic regression showed B7-H3 and MMP-2 expressions were hazardous makers correlated with infiltrating depth (P < 0.05).ConclusionOur study showed combined detections of B7-H3 and MMP2 protein expression could identify patients at high risk in disease recurrence and prognosis more efficiently.

Amplified HMGA2 promotes cell growth by regulating Akt pathway in AML

AbstractPurpose The aim of this study was to investigate how the amplification of HMGA2 contributes to acute myeloid leukemia (AML) cell proliferation.MethodsThe amplification and expression of HMGA2 were examined by FISH, qRT-PCR and Western blot in AML cases. The effect of HMGA2 knockdown on cell proliferation was analyzed with XTT, colony-forming assays and BrdUrd incorporation assays. The effects of HMGA2 knockdown on cell cycle were studied by flow cytometry analysis. The progression of AML cells in vivo was examined by the xenografted tumor model. The interaction between Akt pathway and HMGA2 was examined by Western blot.ResultsHMGA2 is amplified in AML, and the levels of HMGA2 messenger RNA (mRNA) and protein expressed in AML cells were significantly higher than those in normal cells, which may be related to NR and prognosis of AML patients. Reduction in HMGA2 expression in AML cells inhibited cell proliferation through a decrease in the protein expression of pAkt and pmTOR, compared with control cells.ConclusionsHMGA2 is predominantly amplified and expressed in AML cells, and that aberrant expression of HMGA2 induces AML cell proliferation through the PI3K/Akt/mTOR signaling pathway. Inhibition of HMGA2 expression represents an attractive target for AML therapy.

Hypoxia-induced secretion of IL-10 from adipose-derived mesenchymal stem cell promotes growth and cancer stem cell properties of Burkitt lymphoma.

In this study, we explored how the altered paracrine of adipose mesenchymal stem cells (ADSCs) contributed to the growth and cancer stem cell (CSC) properties of the Burkitt lymphoma cells. Condition mediums from normoxia or hypoxia cultured ADSC (CM-ADSC-N or CM-ADSC-H) were collected, and their effects on growth, colony formation, and apoptosis of Burkitt’s lymphoma cells were investigated. Differentially expressed cytokines and inflammatory factors were compared between CM-ADSC-N and CM-ADSC-H. The involvement of differentially expressed IL-10 in growth and CSC properties of Burkitt lymphoma was investigated using both in vitro and in vivo models. Findings of this study showed that hypoxia increased IL-10 secretion from ADSCs, through which the growth and CSC properties of BL2 cells were enhanced. Intratumoral injection of CM-ADSC-H or IL-10 enhanced in vivo Burkitt lymphoma growth in nude mice model at least partly via the JAK2/STAT3 signaling pathway.

Abnormal expression of A20 and its regulated genes in peripheral blood from patients with lymphomas

BackgroundCell-mediated immunity is often suppressed in patients with hematological malignancies. Recently, we found that low T cell receptor (TCR)-CD3 signaling was related to abnormal expression of the negative regulator of nuclear factor kappa B (NF-κB) A20 in acute myeloid leukemia. To investigate the characteristics of T cell immunodeficiency in lymphomas, we analyzed the expression features of A20 and its upstream regulating factor mucosa-associated lymphoid tissue lymphoma translocation gene 1 (MALT1) and genes downstream of NF-κB in patients with different lymphoma subtypes, including T cell non-Hodgkin lymphoma (T-NHL), B cell non-Hodgkin lymphoma (B-NHL) and NK/T cell lymphoma (NK/T-CL).MethodsReal-time PCR was used to determine the expression level of the MALT1, MALT-V1 (variant 1), A20 and NF-κB genes in peripheral blood mononuclear cells (PBMCs) from 24 cases with T-NHL, 19 cases with B-NHL and 16 cases with NK/T-CL, and 31 healthy individuals (HI) served as control.ResultsSignificantly lower A20 and NF-κB expression was found in patients with all three lymphoma subtypes compared with the healthy controls. Moreover, the MALT1 expression level was downregulated in all three lymphoma subtypes. A significant positive correlation between the expression level of MALT1 and A20, MALT1-V1 and A20, MALT1-V1 and NF-κB, and A20 and NF-κB was found.ConclusionsAn abnormal MALT1-A20-NF-κB expression pattern was found in patients with lymphoma, which may result a lack of A20 and dysfunctional MALT1 and may be related to lower T cell activation, which is a common feature in Chinese patients with lymphoma. This finding may at least partially explain the molecular mechanism of T cell immunodeficiency in lymphomas.

Gene therapy for acute myeloid leukemia using sindbis vectors expressing a fusogenic membrane glycoprotein

AML has a dismal prognosis. It was previously shown that the expression of gene coding for the hyperfusogenic gibbon ape leukemia virus envelope glycoprotein (GALV.fus) can efficiently kill leukemic cells. However, target killing effect of GALV.fus on leukemia cells may be limited. Viral vectors, such as retroviruses and adenoviruses, have been developed to deliver heterologous genes into tumors in vivo, but these vectors have some limitations for gene therapy of leukemia. Another virus that has drawn interest as a gene transfer vector is the Sindbis virus. Sindbis virus efficiently infects human tumor cells through the high-affinity 67 kDa Laminin receptor. We found that Laminin-R was obviously expressed in HL-60 and primary human AML cells，but weakly expressed in K562 cells and blood samples of normal human. So we reasoned that Sindbis-virus-based vectors might be ideal for target gene transfer of GALV.fus to acute myeloid leukemic cell. It was shown that Sindbis virus efficiently transduced human acute myeloid leukemic cells with high expression of Laminin-R and exhibited potent cytopathic potential. What is more, we found that CFU-GMs were significantly reduced after Sindbis virus carrying GALV.fus transduced human primary AML cells. Sindbis virus carrying GALV.fus was active against human AML xenografts in vivo. Taken together, we concluded that Sindbis virus carrying GALV.fus may be an useful strategy for gene therapy of acute myeloid leukemia.

Notch pathway plays a novel and critical role in regulating responses of T and antigen-presenting cells in aGVHD

Graft-versus-host disease (GVHD) induced by host antigen-presenting cells (APCs) and donor-derived T cells remains the major limitation of allogeneic bone marrow transplantation (allo-BMT). Notch signaling pathway is a highly conserved cell-cell communication that is important in T cell development. Recently, Notch signaling pathway is reported to be involved in regulating GVHD. To investigate the role of Notch inhibition in modulating GVHD, we established MHC-mismatched murine allo-BMT model. We found that inhibition of Notch signaling pathway by γ-secretase inhibitor in vivo could reduce aGVHD, which was shown by the onset time of aGVHD, body weight, clinical aGVHD scores, pathology aGVHD scores, and survival. Inhibition of Notch signaling pathway by DAPT ex vivo only reduced pathology aGVHD scores in the liver and intestine and had no impact on the onset time and clinical aGVHD scores. We investigated the possible mechanism by analyzing the phenotype of host APCs and donor-derived T cells. Notch signaling pathway had a broad effect on both host APCs and donor-derived T cells. The expressions of CD11c, CD40, and CD86 as the markers of activated dendritic cells (DCs) were decreased. The proliferative response of CD8+ T cell decreased, while CD4+ Notch-deprived T cells had preserved expansion with increased expressions of CD25 and Foxp3 as markers of regulatory T cells (Tregs). In conclusion, Notch inhibition may minimize aGVHD by decreasing proliferation and activation of DCs and CD8+ T cells while preserving Tregs expansion.