Effects of XPO1 Inhibitor KPT-8602 on Proliferation and Apoptosis in U937 cells / 中山大学学报(医学科学版)

@#【Objective】To explore the effects and the possible mechanism of KPT- 8602，a novel selective inhibitor of nuclear export protein （XPO1），on proliferation，cell cycle and apoptosis in human histiocytic lymphoma cell line U937 cells.【Methods】U937 cells were treated with different concentrations of KPT- 8602. Cell viability was assessed by CCK-8 assay. The cell cycle distribution and the apoptosis rate were analyzed by flow cytometry. The proteins expression of XPO1，p-AKT，AKT，Cleaved Caspase-3，p21 were determined by Western blot. Fluorescence microscope was used in observing the intracellular location of XPO1. 【Results】 KPT- 8602 inhibited the growth of U937 cells in a dose- dependent（P<0.001）and time- dependent manner（P<0.001），but normal PBMC were unaffected. 48 h after treatment with KPT-8602，a higher proportion of cells in G1 phase was observed（P<0.001）and the apoptosis rate increased（P=0.016）with drug concentration in U937 cells. XPO1 protein expression of U937 cells was significantly higher than normal PBMC（P=0.003）. 48 h after treatment with KPT- 8602，the protein expression of XPO1 decreased（P=0.011），p-AKT decreased（P=0.011），and Cleaved Caspase- 3 increased（P=0.009）. In addition，the protein expression of p21，the cargo protein of XPO1，increased in both the nuclei and the cytoplasm（P<0.05）after treatment with KPT- 8602. XPO1 decreased in both the nuclei and the cytoplasm under the fluorescence microscope after treatment with KPT- 8602.【Conclusion】KPT- 8602 can inhibit the proliferation of U937 cells，block the cell cycle at G1 phase，and induce cell apoptosis，which may partially be attributed to the down-regulation of XPO1 and inhibition of PI3K/AKT signaling.

Expression and Clinical Significance of mPGES-1 and NF-κB p65 in Diffuse Large B Cell Lymphoma / 中山大学学报(医学科学版)

[Objective]Examine the expression of microsomal prostaglandin E synthase-1(mPGES-1)and nuclear transcription factor kappa B p65(NF-κB p65)in diffuse large B cell lymphoma(DLBCL),assessing their correlation with clinical variables,prognosis and potential clinical valve.[Methods]The immunohistochemistry was uesd to investigate the expression of mPGES-1 and NF-κB p65 in 83 DLBCL patiens'tissues.The relationship between these two proteins and the clinical variables and prognosis of these patients was evaluated.[Results]The high expression of mPGES-1 and NF-κB p65 were observed in 65.1%(54/83)and 73.5%(61/83)cases of DLBCL,respectively.The expression level of NF-κB p65 was positively correlated with mPGES-1 expression(P<0.05).The expression of these two proteins was found to be significantly associated with B cell lymphoma/leukemia-2 protein(BCL-2),higher expression of Ki67,higher lactate dehydrogenase (LDH),more extranodal lesions,advanced Ann Arbor stage and higher international prognostic index(IPI)score(P<0.05). In addition,NF-κB p65 was related with multiple myeloma oncogene 1(MUM1),pathological type(P<0.05). Both mPGES-1 and NF-κB p65 overexpression was correlated with worse overall survival(OS)while NF-κB p65 was an in-dependent prognostic factor for OS of DLBCL(P<0.05).[Conclusions]mPGES-1 and NF-κB p65 were highly expressed in DLBCL and closely linked with each other. The overexpression of mPGES-1 and NF-κB p65 was correlated with tumor progression and unfavorable prognosis in patients with DLBCL.

Effects of shRNA Targeting mPGES-1 on Tumorigenicity of K562 Cells in Nude Mice In Vivo / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To investigate the effects of shRNA targeting mPGES-1 on tumorigenicity of human acute leukemia K562 cells in nude mice in vivo and its mechanisms.</p><p><b>METHODS</b>For experiment 3 groups including KD group(expression of mPGES-1 in K562 cells was down-regulated by shRNA), CON (cells without any treatment) and NC group (cells treated with nonspecific-sequence shRNA) were set-up. Western blot was used to test the expression of β-catenin and cyclinD1 in cells. Then the cells of 3 groups were implanted into BALB/c nude mice subcutaneously to establish murine xenograft model. The growth state of the mice and the size of the xenograft tumor were recorded. HE staining was used to observe the morphology of xenograft tumor. Expressions of β-catenin and cyclinD1 in xenograft tumor were detected by immunohistochemical staining.</p><p><b>RESULTS</b>In vitro the expression of β-catenin and cyclinD1 in KD group were lower than the CON group and NC group (P<0.05). In vivo the tumor volume and weight of KD group were significant smaller than the other two groups (P<0.01). HE staining showed that tissues in the KD group were relatively looser in arrangement with smaller cell nucleus and less cytoplasm. The expression of β-catenin and cyclinD1 in the KD group were remarkable weak as compared with that in CON group and NC group (P<0.05).</p><p><b>CONCLUSION</b>Down-regulating the expression of mPGES-1 by shRNA may significantly inhibit the tumorigenicity of K562 cells in nude mice in vivo and its mechanism may be related with the inhibition of expression of β-catenin and cyclinD1.</p>

Effect of mPGES-1 inhibitor MK886 on cell cycle of leukemia HL-60 cells / 中国实验血液学杂志

To investigate the effect of a microsomal prostaglandin E synthase-1 (mPGES-1) inhibitor MK886 on cell cycle of the human acute myeloid leukemia HL-60 cells. HL-60 cells were treated with different concentration of MK886 (10, 25, 50 µmol/L) for 24 h. Flow cytometry, Western blot and ELISA were used to measure cell cycle, cyclin D1, mPGES-1, PGE(2), Akt, P-Akt and C-MYC. The results indicated that after treated with MK886, the percentage of HL-60 cells decreased in G(0)/G(1) phase and increased in S phase, and expressions of mPGES-1, cyclin D1, P-Akt and C-MYC and synthesis of PGE(2) decreased significantly. It is concluded that MK886 can arrest HL-60 cells in G(0)/G(1) phase, the mechanism of which is possibly associated to inhibition of mPGES-1 expression, reduction of PGE(2) synthesis, suppression of Akt phosphorylation and C-MYC expression, down-regulation of cyclin D1 expression.

Effect of human bone marrow mesenchymal stem cell transplantation on hematopoietic recovery of irradiated mice / 中国实验血液学杂志

This study was aimed to investigate the effect of human bone marrow mesenchymal stem cells (hBMMSC) on the hematopoietic recovery of sublethally irradiated mice. Female BALb/c mice irradiated with (60)Co γ-ray at a single dose of 6 Gy received graded doses of hBMMSC (1×10(5), 1×10(6) and 5×10(6)) by intravenous infusion. The counts of leukocytes, platelets, erythrocytes and hemoglobin level in peripheral blood, the amount of bone marrow hematopoietic progenitors, and the serum levels of human TPO, SCF and G-CSF as well were evaluated at different time points after transplantation. The results showed that hBMMSC infusion had little protective effect on the survival of irradiated mice. Compared with the control mice, the peripheral blood cell counts of hBMMSC-treated mice were not obviously elevated during 3 weeks after infusion, however, blood cell counts were significantly greater at 4 weeks after cell treatment (P < 0.05). The amount of colony-forming unit of mononuclear cells and granulocyte/monocytes in bone marrow of mice that received middle and high doses of hBMMSC were dramatically greater than that in control mice (P < 0.05). Two days after hBMMSC administration, human G-CSF and SCF could be detected in the sera from hBMMSC-treated mice, and the G-CSF concentration of mice that received high-dose hBMMSC was significantly higher than that in other groups (P < 0.01). Nevertheless, human TPO was undetectable in the sera of all mice tested and serum human G-CSF and SCF could not be detected on days 9 and 16 in all groups. It is concluded that hBMMSC may promote the hematopoietic recovery of irradiated mice, probably by transient secretion of hematopoiesis-associated factors by the implanted cells.

Protective effects of human bone marrow mesenchymal stem cells on hematopoietic organs of irradiated mice / 中国实验血液学杂志

The objective of this study was to explore the protective effects of human bone marrow mesenchymal stem cells (MSC) on hematopoietic organs of irradiated mice. Human bone marrow MSC were isolated, ex vivo expanded, and identified by cell biological tests. Female BALB/c mice were irradiated with (60)Co γ-ray at a single dose of 6 Gy, and received different doses of human MSC and MSC lysates or saline via tail veins. The survival of mice was record daily, and the femurs and spleens were harvested on day 9 and 16 for pathologic examination. The histological changes were observed and the cellularity was scored. The results showed that the estimated survival time of MSC- and MSC lysate-treated mice was comparable to that of controls. The hematopoiesis in the bone marrow of mice that received high-dose (5×10(6)) of MSC or MSC lysates was partially restored on day 9 and the capacity of hemopoietic tissue and cellularity scorings were significantly elevated as compared with that of controls (P < 0.05). Proliferative nudes were also obviously observed in the spleens of mice that received high-dose of MSC or MSC lysates on d 9 after irradiation. The histological structures of the spleen and bone marrow of the mice that received high-doses (5×10(6)) of MSC or MSC lysates were restored to normal, the cell proliferation displayed extraordinarily active. Further, the cellularity scores of the bone marrow were not significantly different between the high-dose MSC and MSC lysate-treated mice. It is concluded that the bone marrow MSC can promote the hematopoietic recovery of the irradiated mice, which probably is associated with the bioactive materials inherently existed in bone marrow cells.

Effect of mPGES-1 inhibitor MK886 on apoptosis and drug resistance of HL-60/A cells / 中国实验血液学杂志

This study was aimed to investigate the effect of MK886, a mPGES-1 inhibitor, on apoptosis and drug resistance of leukemia HL-60/A cell line. Expression of mPGES-1 was assayed by QT-PCR and Western blot. The effect of MK886 on HL-60/A cell proliferation was assayed by CCK-8 method, and flow cytometry was used to detect cell apoptosis. The expression of Akt and P-Akt was detected by Western blot. PGE2 was measured by ELISA. Effect of MK886 (10 µmol/L) on the chemotherapeutic sensitivity of HL-60/A cells and expression of mdr-1 mRNA and P170 protein were investigated too. The results indicated the expression of mPGES-1 was higher in HL-60/A cells. MK886 inhibited HL-60/A cell proliferation and induced apoptosis in a time- and concentration-dependent manner. Expression of mPGES-1 and P-Akt and synthesis of PGE2 decreased significantly. MK886 reduced expression of mdr-1 and P170 protein and enhanced the sensitivity of HL-60/A cells to chemotherapeutic drugs. It is concluded that MK886 can inhibit HL-60/A cell proliferation, induce apoptosis and enhance sensitivity to chemotherapeutic drugs, the mechanism of which possibly associates to down-regulation of mPGES-1/PGE2 synthesis, reduction P-Akt expression and decreasing mdr-1 and P170 protein expression.

Cyclin D1, hTERT expression and telomerase activity in HL-60 and HL-60A cell lines and their significance / 中国实验血液学杂志

To observe the expression of cyclin D1, hTERT, and telomerase activity in MNC, HL-60, HL-60A and to explore their effects on leukemogenesis and drug-resistance, normal human peripheral blood mononuclear cells, HL-60 cells sensitive to adriamycin and HL-60A cells resistant to adriamycin were investigated. The cell cycle was analyzed by flow cytometry, and the apoptosis was analyzed by Annexin V-FITC(+) PI staining. Expressions of cyclin D1 and hTERT were determined by real-time PCR and Western blot. Telomerase activity was detected by TRAP-ELISA. The results indicated that the percentage of MNC, HL-60 and HL-60A in S phase was (10.21 + 2.11)%, (44.93 + 3.00)%, and (51.38 + 1.10)% respectively; the percentage of apoptosis cells was (16.14 + 2.13)%, (7.53 + 0.92)%, (4.15 + 0.96)% respectively; the expression of mRNA and protein for cyclin D1 and hTERT increased; the telomerase activities of HL-60 and HL-60A were higher (p = 0.000), whereas the difference between HL-60 and HL-60A was no statistically significant (p = 0.232); positive correlation between cyclin D1, hTERT and telomerase activity had been found (p < 0.01). It is concluded that the cells of S phase increased while the apoptotic cells decreased in HL-60 and HL-60A, especially in HL-60A, which may be due to the up-regulation of cyclin D1, hTERT and telomerase activity.

Effect of valproic acid on apoptosis of leukemia HL-60 cells and expression of h-tert gene / 中国实验血液学杂志

This study was aimed to clarify whether valproic acid (VPA) induces apoptosis of leukemia HL-60 cell line and its possible mechanism. The effect of different concentrations and treatment time of VPA on HL-60 cell proliferation was assayed by cytotoxicity test (CCK-8 method) and fluorescence microscopy, and flow cytometry was used to detect cell apoptosis. The expressions of telomerase subunit h-tert mRNA and apoptosis-related protein as well as caspase-3 activity were detected by real time-quantitative PCR, Western blot and ELISA respectively. The results indicated that VPA inhibited proliferation of HL-60 cells and induced cell apoptosis in a dose dependent manner (r = -0.87). The expressions of anti-apoptotic protein BCL-2 and h-tert mRNA were significantly decreased while the pro-apoptotic protein BAX and caspase-3 activity increased after treatment with VPA. The apoptosis rate of HL-60 cell was negatively correlated with expression of h-tert mRNA. It is concluded that VPA can inhibit leukemia HL-60 cell proliferation and induce apoptosis. The VPA displays anti-leukemia activity possibly through reducing h-tert mRNA and BCL-2 protein expression, increasing BAX expression and activity of caspase-3.

Effect of valproic acid on the expression of P27(Kip1) and P170 and drug resistance of HL-60/HT cells / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the effect of valproic acid on the expression of P27(Kip1) and P170 and drug resistance of leukemia HL60/HT cell line and explore its possible mechanisms.</p><p><b>METHODS</b>HL-60/HT cells were derived from HL-60 cells induced by harringtonine (HT) in gradient concentrations. The inhibitory effect of valproic acid on the proliferation of HL-60 and HL-60/HT cells was evaluated by MTT assay, and the P27(Kip1) expression, P170 expression and cell cycle of the cells were analyzed with flow cytometry.</p><p><b>RESULTS</b>The multidrug-resistant HL-60/HT was acquired, which showed a stable drug-resistant index with increased IC(50) of HT, VCR, DNR and Ara-c by 9.30, 5.20, 4.91 and 3.65 folds, respectively, as compared with those of HL60 cells. The expression of P27(Kip1) in HL-60/HT cells was significantly lower but P170 expression significantly higher than that of HL-60 cells and normal mononuclear cells (P<0.05). The expressions of P27(Kip1) and P170 showed no significant difference between normal mononuclear cells and HL-60 cells. The growth inhibition rate of VPA combined with Ara-C was significantly higher than that of valproic acid or Ara-C alone in HL-60/HT cells and HL-60 cells (q=1.37 and 1.51, respectively). HL-60/HT and HL-60 cells cultured in the presence of VPA resulted in a significant increase in the expression of P27(Kip1) and the G(1)-phase cells (P<0.05), but the expression of P170 underwent no significant changes (P>0.05).</p><p><b>CONCLUSION</b>HL-60/HT cells have lower P27(Kip1) expression compared with HL-60 cells. Valproic acid can inhibit the growth of HL-60/HT cells and enhance their Ara-C sensitivity possibly by increasing P27(Kip1) expression and causing cell cycle arrest in G(1) phase.</p>