Umbilical cord blood-derived mesenchymal stem cells ameliorate graft-versus-host disease following allogeneic hematopoietic stem cell transplantation through multiple immunoregulations / 华中科技大学学报（医学）（英德文版）

Although mesenchymal stem cells (MSCs) are increasingly used to treat graft-versus-host disease (GVHD), their immune regulatory mechanism in the process is elusive. The present study aimed to investigate the curative effect of third-party umbilical cord blood-derived human MSCs (UCB-hMSCs) on GVHD patients after allogeneic hematopoietic stem cell transplantation (allo-HSCT) and their immune regulatory mechanism. Twenty-four refractory GVHD patients after allo-HSCT were treated with UCB-hMSCs. Immune cells including T lymphocyte subsets, NK cells, Treg cells and dendritic cells (DCs) and cytokines including interleukin-17 (IL-17) and tumor necrosis factor-alpha (TNF-α) were monitored before and after MSCs transfusion. The results showed that the symptoms of GVHD were alleviated significantly without increased relapse of primary disease and transplant-related complications after MSCs transfusion. The number of CD3(+), CD3(+)CD4(+) and CD3(+)CD8(+) cells decreased significantly, and that of NK cells remained unchanged, whereas the number of CD4(+) and CD8(+) Tregs increased and reached a peak at 4 weeks; the number of mature DCs, and the levels of TNF-α and IL-17 decreased and reached a trough at 2 weeks. It was concluded that MSCs ameliorate GVHD and spare GVL effect via immunoregulations.

Angiogenic factors are associated with development of acute graft-versus-host disease after allogeneic hematopoietic stem cell transplantation / 华中科技大学学报（医学）（英德文版）

Acute graft-versus-host disease (aGVHD) is a serious complication after allogeneic hematopoietic stem cell transplantation (allo-HSCT). However, the mechanisms of aGVHD are not well understood. We aim to investigate the roles of the three angiogenic factors: angiopoietin-1 (Ang-1), Ang-2 and vascular endothelial growth factor (VEGF) in the development of aGVHD. Twenty-one patients who underwent allo-HSCT were included in our study. The dynamic changes of Ang-1, Ang-2 and VEGF were monitored in patients before and after allo-HSCT. In vitro, endothelial cells (ECs) were treated with TNF-β in the presence or absence of Ang-1, and then the Ang-2 level in the cell culture medium and the tubule formation by ECs were evaluated. After allo-HSCT, Ang-1, Ang-2 and VEGF all exhibited significant variation, suggesting these factors might be involved in the endothelial damage in transplantation. Patients with aGVHD had lower Ang-1 level at day 7 but higher Ang-2 level at day 21 than those without aGVHD, implying that Ang-1 may play a protective role in early phase yet Ang-2 is a promotion factor to aGVHD. In vitro, TNF-β promoted the release of Ang-2 by ECs and impaired tubule formation of ECs, which were both weakened by Ang-1, suggesting that Ang-1 may play a protective role in aGVHD by influencing the secretion of Ang-2, consistent with our in vivo tests. It is concluded that monitoring changes of these factors following allo-HSCT might help to identify patients at a high risk for aGVHD.

Mechanism of gambogic acid-induced apoptosis in Raji cells / 中国实验血液学杂志

This study was purposed to explore the apoptotic effect of gambogic acid on Raji cells and the role of death inducer-obliterator 1 (DIO-1) in this process. Annexin V-fluorescein-isothiocyanate/propidium iodide was used to detect apoptosis of Raji cells. Western blot was used to determine the expressions of DIO-1, Bcl-xL, pro-caspase 3 and 2 activated subunits: P17 and P20. The subcellular localization of DIO-1 in untreated and treated Raji cells was checked by immunofluorescence and Hoechst 33258 double staining. The results showed that the Gambogic acid dose-dependently induced the apoptosis of Raji cells, downregulated the expression of Bcl-xL, upregulated the expressions of DIO-1 and pro-caspase 3, induced the cleavage of pro-caspase 3 and DIO nuclear translocation. It is concluded that gambogic acid induces the apoptosis of Raji cells through DIO-1 upregulation, nuclear translocation, Bcl-xL downregulation and caspase 3 activation.

Effect of betulinic acid on inducing apoptosis of human multiple myeloma cell line RPMI-8226 / 中国实验血液学杂志

The aim of this study was to investigate the effect of betulinic acid on inducing apoptosis of human multiple myeloma RPMI-8226 cell line. The inhibitory effect of betulinic acid on proliferation and its inducing apoptosis effect, influence on cell cycle and induced morphological changes of RPMI-8226 were evaluated by MTT, flow cytometry Annexin-V/PI double staining, flow cytometry with PI staining and fluorescence microscopy with Hoechst33258 staining, respectively. The transcription level changes of bcl-xl gene and caspase 3 which are two kinds of apoptosis related protein gene were determined by RT-PCR. The results showed that within a certain range of concentration (0, 5, 10, 15, 20 microg/ml), IC50 of betulinic acid to RPMI-8226 at 24 hours was 10.156+/-0.659 microg/ml, while the IC50 at 48 hours was 5.434+/-0.212 microg/ml, and its inhibiting effect on proliferation of RPMI-8226 showed both time-and dose-dependent manners. Flow cytometry with Annexin-V/PI double staining revealed that apoptotic rate of RPMI-8226 cells increased as betulinic acid concentration increased. Flow cytometry with PI staining showed that the ratio of cells in G0/G1 phase increased, while it in S phase decreased, and ratio of cells at G2/M phase did not present a significant change. Morphological differences were typical and obvious between cells in treated and control groups under fluorescence microscope using Hoechst33258 staining. RT-PCR detection of caspase 3 gene indicated that its transcription level showed an increasing trend as the concentration of betulinic acid increased, while the bcl-xl showed the opposite trend. It is concluded that the betulinic acid can induce apoptosis of RPMI-8226 within a certain range of concentration in a time- and dose-dependent manners. This phenomenon may be related to the transcriptional level increase of caspase 3 gene and decrease of bcl-xl. Betulinic acid also affects G1/S in cell cycle which arrests cells at phase G0/G1.

Effect of betulinic acid on proliferation and apoptosis in Jurkat cells and its mechanism / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To investigate the anticancer effects of betulinic acid (BA) on Jurkat cells in vitro and its molecular mechanism.</p><p><b>METHODS</b>The effects of betulinic acid on the growth of Jurkat cells were studied by 3-(4, 5-dimethyl-2-thiazolyl)-2, 5diphenyl-2H-tetrazolium (MTT) assay. Apoptosis was assessed by Hoechst33258 staining and annexin-V/PI double-labeled cytometry. The effect of betulinic acid on the cell cycle of Jurkat cells was studied by propidium iodide staining. RT-PCR and Western blot were used to analyze the changes of cyclin D3, bcl-xl mRNA and protein levels in Jurkat cells after treatment with betulinic acid.</p><p><b>RESULTS</b>The proliferation of Jurkat cells was decreased in betulinic acid-treated group at a 24 h IC50 value of 70.0 micromol/L. The effect of betulinic acid to induce apoptosis in Jurkat cells was in a time- and dose-dependent manner. Jurkat cells treated with betulinic acid showed an increase of G0/G1 phase and decrease of S phase. The Jurkat cells treated with 0, 20, 60, 100 micromol/L betulinic acid for 24 h, showed an increased G0/G1 phase from 31.0% to 58.8%, whereas decreased S phase from 61.5% to 35.8%, respectively. PBMC was less sensitive to the cytotoxic effect of betulinic acid than Jurkat cells. The expression of cyclin D3, bcl-xl mRNA and protein were decreased sharply in Jurkat cells treated with betulinic acid.</p><p><b>CONCLUSION</b>Betulinic acid can inhibit the proliferation of Jurkat cells by regulating the cell cycle that arrests cells at G0/G1 phase and induces apoptosis in Jurkat cells. The antitumor effects of betulinic acid may be related to down-regulation of the expression of cyclin D3 and bcl-xl.</p>

Effect of deguelin on expression of nup98 in K562 cells / 中国实验血液学杂志

The study was purposed to investigate the effect of deguelin on expression of nucleoporin 98 (nup98) in leukemia K562 cells. MTT assay was used to assess the effects of deguelin on cell proliferation. FCM and RT-PCR were used to analyze the changes of nup98 mRNA and protein in K562 cells after treating with deguelin. The results showed that deguelin inhibited the proliferation of K562 cells in a time- and dose-dependent manner; mean fluorescence intensity of nup98 in blank group (34.22 +/- 1.63) was significantly higher than that in control group (2.83 +/- 0.02, P < 0.01), 10 nmol/L deguelin could significantly inhibit the expression of nup98 protein; 10 nmol/L could not inhibit the expression of nup98 mRNA, 20, 40, 80, 160 nmol/L deguelin could significantly inhibit the expression of nup98 mRNA in a dose-dependant manner. It is concluded that deguelin inhibits the proliferation of K562 cell through inhibiting the expression of nup98, and may be considered as a new target for therapy of acute leukemia.

Effect of deguelin on proliferation and apoptosis of lymphoma Daudi cells and its mechanism / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To investigate the anticancer effects and molecular mechanism of deguelin on human Burkitt's lymphoma Daudi cells in vitro and compare the cytotoxicity of deguelin on Daudi cells and human peripheral blood monocular cells (HPBMC).</p><p><b>METHODS</b>The effects of deguelin on the growth of Daudi cells were studied by 3-(4, 5-dimethyl-2-thiazolyl)-2, 5 diphenyl-2H-tetrazolium (MTT) assay. Apoptosis was assessed through Hoechst 33258 staining and annexin V/PI double-labeled cytometry. The effect of deguelin on the cell cycle of Daudi cells was studied by propidium iodide method. The expressions of cyclin D1 and pRb were checked by Western blot.</p><p><b>RESULTS</b>The proliferation of Daudi cells was decreased in deguelin-treated group with a 24 h IC50 value of 51. 55 nmol/L. Deguelin induced Daudi cells apoptosis in a time- and dose-dependent manner. Daudi cells treated with deguelin showed an increase of G0/G1 phase and decrease of S phase. The Daudi cells treated with 0, 5, 10, 20, 40 nmol/L deguelin for 24 h, showed an increased Go/G, phase from 37.34% to 56.56% , whereas decreased S phase from 37.72% to 21.36%, respectively. PBMC was less sensitive to the cytotoxic effect of deguelin than Daudi cells. The expression of cyclin D1 and pRb protein were decreased in Daudi cells treated with deguelin.</p><p><b>CONCLUSION</b>Deguelin can inhibit the proliferation of Daudi cells by regulating the cell cycle that arrest cells at G0/G1 phase and inducing apoptosis. Moreover, deguelin demonstrats low toxicity in PBMC but selectively induces apoptosis of Daudi cells. The antitumor effects of deguelin may be related to down-regulation of the expression of cyclin D1 and pRb protein.</p>

Deguelin regulates cell cycle and nuclear pore complex protein Nup98 and Nup88 in U937 cells in vitro / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate antitumor activity and molecular mechanism of deguelin to the human U937 leukaemia cells and to explore the mechanisms regulating cell cycle and nucleoporin 98 (Nup98) and nucleoporin 88 (Nup88) in vitro.</p><p><b>METHODS</b>The effects of deguelin on the growth of U937 cells were studied by MTT assay, and the cell cycle of U937 cells by a propidium iodide method. The localization of the nuclear pore complex protein Nup98 and Nup88 was checked by immunofluorescence and immunoelectron microscopy. The expressions of Nup98 and Nup88 in U937 cells were checked by flow cytometry (FCM) and Western blot respectively.</p><p><b>RESULTS</b>The proliferation of U937 cells was significantly inhibited in a time-dose dependent manner in deguelin-treated group with a 24 h IC50 value of 21.61 nmol/L and 36 h IC50 value of 17.07 nmol/L. U937 cells treated with deguelin showed reduction in the percentages of cells in G0/G1, whereas accumulation of cells in S and G2/M phase. The ratio of G1/G0 phase cells were 73.01%, 71.15%, 68.42%, 52.45%, 43.99% and 22.82%, and that of S phase cells were 17.18%, 16.30%, 18.09%, 27.56%, 31.21% and 46.85%, and that of G2/M phase cells were 9.75%, 12.31%, 13.09%, 18.99%, 24.83% and 27.79% at deguelin concentrations of 0, 5, 10, 20, 40, 80 nmol/L respectively. Nup88 and Nup98 were found on both the nuclear and cytoplasmic side of the U937 cells. The expression of Nup98 was up-regulated and Nup88 down-regulated in deguelin treated U937 cells.</p><p><b>CONCLUSION</b>Deguelin is able to inhibit the proliferation of U937 cells by regulating the cell cycle. The antitumor activity of deguelin was related to up-regulating the expression of Nup98 and down-regulating Nup88 protein.</p>

Effect of betulinic acid on proliferation,apoptosis,and cell cycle of human lymphoma cell line Raji / 中草药

Objective To investigate the anticancer effects and molecular mechanism of betulinic acid (BA)on Raji cells in vitro.Methods The effects of BA on the growth of Raji cells were studied by MTT assay.Apoptosis was assessed by Annexin-V/PI double-labeled cytometry.The influence on cell cycle was studied by flow cytometer.The cyclin D3 mRNA expression was checked by Western blotting and RT-PCR techniques.Results BA showed obvious inhibition on proliferation,as well as induction potency of apoptosis on Raji cells in vitro in a time-and dose-dependent manner by Annexin-V/PI double-labeled method.With the IC_(50)value for 24 h being(39.44?0.65)?g/mL,Raji cells treated with BA showed ac- cumulation in G_0/G_1 phase and reduction in the percentage of cells in S phase.The cyclin D3 mRNA ex- pression and protein were sharply decreased in Raji cells treated with BA.Conclusion BA could inhibit the proliferation of Raji cells by regulating the cell cycle that arrests cells at G_0/G_1 phase and induces apop- tosis of Raji cells.The antitumor effects of BA may be related to down-regulation of the expression of cy- clin D3.

Reversing Mechanism of Adriamycin Resisitance of K562/A02 Cell Line by Ciclosporin through H2AX Suppression to Attenuate DNA Damage Repair / 实用儿科临床杂志

Objective To explore the reversing mechanism of multidrug resistance of ciclosporin(CsA) on K562/A02 cell line,attenuating DNA damage repair through H2AX suppression.Methods K563 and K562/A02 respectively co-cultured with adriamycin(ADM) of different concentrations and CsA.MTT assay was employed to determine the inhibitory concentration of 50 percent(IC50),the resistance times and the reversal times.The K562/A02 cells treated with CsA,and reverse transcriptase(RT)-PCR technique was used to examine the mdr1 and H2AX mRNA level.Flow cytometry was used to measure P-glycoprotein(P-gp) and H2AX expression.Neutral comet assay was used to detect the level of DNA double strands break(DSBs) of the K562/A02 treated with ?H2AX antibody.Results 2?10-3g/L CsA could increase the sensitivity of K562/A02 to ADM,and the reversal times was 5.28.Mdr1 mRNA level and H2AX mRNA level were decreased when treated with CsA(P