Key Laboratory of Developmental Diseases in Childhood

Chongqing, China

Key Laboratory of Developmental Diseases in Childhood

Chongqing, China

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Wu Y.,Key Laboratory of Developmental Diseases in Childhood | Wu Y.,Key Laboratory of Pediatrics in Chongqing | Wu Y.,Cooperation Technology | Xu Y.,Key Laboratory of Developmental Diseases in Childhood | And 14 more authors.
Brazilian Journal of Medical and Biological Research | Year: 2012

The objective of this study was to evaluate the effects of tetramethylpyrazine (TMP) in combination with arsenic trioxide (As2O3) on the proliferation and differentiation of HL-60 cells. The HL-60 cells were treated with 300 μg/mL TMP, 0.5 μM As2O3, and 300 μg/mL TMP combined with 0.5 μM As2O3, respectively. The proliferative inhibition rates were determined with MTT. Differentiation was detected by the nitroblue tetrazolium (NBT) reduction test, Wright's staining and the distribution of CD11b and CD14. Flow cytometry was used to analyze cell cycle distribution. RT-PCR and Western blot assays were employed to detect the expressions of c-myc, p27, CDK2, and cyclin E1. Combination treatment had synergistic effects on the proliferative inhibition rates. The rates were increased gradually after the combination treatment, much higher than those treated with the corresponding concentration of As2O3 alone. The cells exhibited characteristics of mature granulocytes and a higher NBT-reducing ability, being a 2.6-fold increase in the rate of NBT-positive ratio of HL-60 cells within the As2O3 treatment versus almost a 13-fold increase in the TMP and As2O3 group. Cells treated with both TMP and As2O3 expressed far more CD11b antigens, almost 2-fold compared with the control group. Small doses of TMP potentiate As2O3 -induced differentiation of HL-60 cells, possibly by regulating the expression and activity of G0/G1 phase-arresting molecules. Combination treatment of TMP with As2O3 has significant synergistic effects on the proliferative inhibition of HL-60 cells.


Yang G.-C.,Key Laboratory of Developmental Diseases in Childhood | Xu Y.-H.,Chongqing Medical University | Chen H.-X.,Cooperation Technology | Wang X.-J.,Key Laboratory of Developmental Diseases in Childhood
Stem Cells International | Year: 2015

The disruption of normal hematopoiesis has been observed in leukemia, but the mechanism is unclear. Osteoblasts originate from bone mesenchymal stem cells (BMSCs) and can maintain normal hematopoiesis. To investigate how leukemic cells inhibit the osteogenic differentiation of BMSCs and the role of Notch signaling in this process, we cocultured BMSCs with acute lymphoblastic leukemia (ALL) cells in osteogenic induction medium. The expression levels of Notch1, Hes1, and the osteogenic markers Runx2, Osteopontin (OPN), and Osteocalcin (OCN) were assessed by real-time RT-PCR and western blotting on day 3. Alkaline phosphatase (ALP) activity was analyzed using an ALP kit, and mineralization deposits were detected by Alizarin red S staining on day 14. And then we treated BMSCs with Jagged1 and anti-Jagged1 neutralizing Ab. The expression of Notch1, Hes1, and the abovementioned osteogenic differentiation markers was measured. Inhibition of the expression of Runx2, OPN, and OCN and reduction of ALP activity and mineralization deposits were observed in BMSCs cocultured with ALL cells, while Notch signal inhibiting rescued these effects. All these results indicated that ALL cells could inhibit the osteogenic differentiation of BMSCs by activating Notch signaling, resulting in a decreased number of osteoblastic cells, which may impair normal hematopoiesis. © 2015 Gui-Cun Yang et al.


Wang X.-J.,Key Laboratory of Developmental Diseases in Childhood | Wang X.-J.,Key Laboratory of Pediatrics in Chongqing | Wang X.-J.,Cooperation Technology | Xu Y.-H.,Key Laboratory of Developmental Diseases in Childhood | And 11 more authors.
Oncology Reports | Year: 2015

Tetramethylpyrazine (TMP) has been proven to be an anticancer agent in many studies. However, its effectiveness in acute lymphoblastic leukemia (ALL) and its molecular mechanisms are still unclear. The present study aimed to evaluate the effect of TMP against Jurkat and SUP-B15 ALL cell lines and to investigate the possible detailed mechanism of action of TMP. A Cell Counting Kit-8 (CCK-8) assay was employed to examine the proliferation of Jurkat and SUP-B15 cells. Flow cytometric analysis was conducted to detect the cell cycle distribution and apoptotic rate. The expression of total glycogen synthase kinase-3β (GSK-3β), cox-2, survivin, bcl-2 and p27 RNA and protein levels was detected by quantitative real-time PCR and western blot assay, respectively. Additionally, western blot analysis was used to determine the whole-cell and nuclear protein levels of GSK-3β downstream transcription factors, NF-κB (p65) and c-myc. TMP inhibited the proliferation of Jurkat and SUP-B15 cells in a dose- and time-dependent manner, with IC50 values of 120 and 200 μg/ml, respectively at 48 h. TMP induced the apoptosis of Jurkat and SUP-B15 cells and synergistically blocked cell cycle progression at the G0/G1 phase. Cells treated with TMP exhibited significantly attenuated GSK-3β, NF-κB (p65) and c-myc expression, followed by downregulation of bcl-2, cox-2 and survivin and an upregulation of p27. The results showed that TMP induced apoptosis and caused cell cycle arrest in Jurkat and SUP-B15 cells through the downregulation of GSK-3β, which may have further prevented the induced translocation of NF-κB and c-myc from the cytoplasm to the nucleus.


Su Y.,Chongqing Medical University | Xu H.,Chongqing Medical University | Xu Y.,Chongqing Medical University | Yu J.,Chongqing Medical University | And 2 more authors.
Hematology | Year: 2012

Background: Azacytidine (Aza) was the first demethylation agent identified that may inhibit DNA methyltransferases and reverse DNA hypermethylation, restoring the expression of silenced tumor suppressor genes in patients with myelodysplastic syndromes (MDS). It is unclear whether azacytidine can alter the proliferative and apoptotic changes in myeloid leukemia cells, and methylation changes induced by this drug have remained poorly characterized in therapy-related models. Methods: The proliferation rate of azacytidine on HL60 cells was determined by the MTT protocol. Methylation-specific PCR (MSP) and RT-PCR were used respectively to detect gene methylation status changes and expression levels of p16, Death associated protein kinase (DAPK) and O6-methylguanine-DNA methyltransferase (MGMT) before and after treatment with azacytidine. Results: Azacytidine inhibited HL60 cell proliferation and showed a time- and dose-dependent effect. MSP showed hypermethylated p16, DAPK, and MGMT genes before azacytidine treatment. Complete demethylation was seen in p16 and DAPK genes and partial demethylation in the MGMT gene after coculture with azacytidine. The expression level of p16, DAPK and MGMT genes in HL60 cells was upregulated after treatment with azacytidine. Conclusions: The CpG islands of p16, DAPK and MGMT genes are hypermethylated in HL60 cells. Azacytidine inhibits proliferation of leukemic cells by hypomethylation of p16, DAPK and MGMT genes. © W. S. Maney & Son Ltd 2012.


PubMed | Key Laboratory of Developmental Diseases in Childhood
Type: Journal Article | Journal: Oncology reports | Year: 2015

Tetramethylpyrazine (TMP) has been proven to be an anticancer agent in many studies. However, its effectiveness in acute lymphoblastic leukemia (ALL) and its molecular mechanisms are still unclear. The present study aimed to evaluate the effect of TMP against Jurkat and SUP-B15 ALL cell lines and to investigate the possible detailed mechanism of action of TMP. A Cell Counting Kit-8 (CCK-8) assay was employed to examine the proliferation of Jurkat and SUP-B15 cells. Flow cytometric analysis was conducted to detect the cell cycle distribution and apoptotic rate. The expression of total glycogen synthase kinase-3 (GSK-3), cox-2, survivin, bcl-2 and p27 RNA and protein levels was detected by quantitative real-time PCR and western blot assay, respectively. Additionally, western blot analysis was used to determine the whole-cell and nuclear protein levels of GSK-3 downstream transcription factors, NF-B (p65) and c-myc. TMP inhibited the proliferation of Jurkat and SUP-B15 cells in a dose- and time-dependent manner, with IC values of 120 and 200 g/ml, respectively at 48 h. TMP induced the apoptosis of Jurkat and SUP-B15 cells and synergistically blocked cell cycle progression at the G0/G1 phase. Cells treated with TMP exhibited significantly attenuated GSK-3, NF-B (p65) and c-myc expression, followed by downregulation of bcl-2, cox-2 and survivin and an upregulation of p27. The results showed that TMP induced apoptosis and caused cell cycle arrest in Jurkat and SUP-B15 cells through the downregulation of GSK-3, which may have further prevented the induced translocation of NF-B and c-myc from the cytoplasm to the nucleus.

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