Qiu-ling Wu

Effect of betulinic acid on the regulation of Hiwi and cyclin B1 in human gastric adenocarcinoma AGS cells

AbstractAim:To investigate the effect of betulinic acid (BA) on the proliferation, apoptosis and cell cycle of gastric adenocarcinoma cell AGS in vitro and the underlying mechanism.Methods:The effect of BA on the proliferation of AGS cells was measured by using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium (MTT) assay. Apoptosis was analyzed by using Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) double-labeled flow cytometry (FCM) and Hoechst 33258 staining. The influence of BA on cell cycle of AGS cells was tested by PI staining. Both FCM and reverse transcription-PCR (RT-PCR) technologies were applied to detect the expression of Hiwi and Cyclin B1.Results:BA exhibited significant cell proliferation inhibition, as well as its potency of inducing apoptosis in AGS cells in vitro in a time- and dose-dependent manner. The IC50 value for 24 h was 18.25 μg/mL (95% confidence interval: 15.16 to 27.31 μg/mL). Cells treated with BA showed increased cell population in G2/M phase, with decreased S phase population. The expression of Hiwi and Cyclin B1 was down-regulated in BA-treated AGS cells in a dose-dependent manner.Conclusion:BA exerted potent effect on growth inhibition, G2/M cell cycle arrest and induction of apoptosis in AGS cells in vitro, possibly associated with the down-regulation of Hiwi and its downstream target Cyclin B1 expression. The potent antitumor capacity of BA suggested that it could be a promising new experimental anticancer agent in human gastric adenocarcinoma treatment.

Regulating expressions of cyclin D1, pRb, and anti-cancer effects of deguelin on human Burkitt's lymphoma Daudi cells in vitro

AbstractAim:To investigate anticancer effects and molecular mechanism of deguelin on human Burkitts lymphoma Daudi cells in vitro and compare the cytotoxicities of deguelin on Daudi cells and human peripheral blood monocular cells (PBMC).Methods: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 were dectected through Hoechst 33258 staining and Annexin V/PI double-labeled cytometry. The effect of deguelin on the cell cycle of Daudi cells were studied by a propidium iodide method. The expressions of cyclin D1 and pRb were checked by Western blot.Results:The proliferation of Daudi cells were decreased in deguelin-treated group with a 24-h IC50 value of 51.55 nmol/L. Deguelin induced Daudi cells apoptosis was in a time- and dose-dependent manner. G0/G1 phase increased and S phase decreased in Daudi cells treated with deguelin. With deguelin 0, 5, 10, 20, and 40 nmol/L treatment for 24 h, G0/G1 phase increased from 37.34% to 56.56%, whereas S phase decreased from 37.72% to 21.36%. PBMC was less sensitive to the cytotoxic effect of deguelin than Daudi cells. The expression of cyclin D1 and pRb protein were decreased sharply in Daudi cells treated with deguelin.Conclusion:Deguelin is able to inhibit the proliferation of Daudi cells by regulating the cell cycle that arrested cells at G0/G1 phase and inducing the cell apoptosis. Moreover, deguelin selectively induced apoptosis of Daudi cells with low toxicity in PBMC. The antitumor effects of deguelin were related to down-regulating the expression of cyclin D1 and pRb protein.

Gambogic acid induces death inducer-obliterator 1-mediated apoptosis in Jurkat T cells

AbstractAim:To explore the anticancer effects and the molecular mechanisms of gambogic acid (GA) on Jurkat cells.Methods:Cell viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Annexin V-fluorescein-isothiocyanate/propidium iodide, DNA defragmentation, and comet assay were used to detect apoptosis. Western blotting was used to study the expression of death inducer-obliterator-1 (DIO-1), Bcl-2, NF-κB, and procaspase 3, as well as 2 activated subunits: p17 and p20. The subcellular localization of DIO-1 was examined by immunofluorescence and Hoechst33258 staining.Results:GA inhibited the proliferation of Jurkat cells with 50% inhibitory concentration values of 1.51±0.09 (24 h), 0.98±0.13 (48 h), and 0.67±0.12 μmol/L (72 h). GA was able to induce apoptosis of Jurkat cells. Treated by GA, the expression of DIO-1 was upregulated, and that of Bcl-2 and NF-κB was downregulated, leading to the activation of pro-caspase 3. GA induced the translocation of DIO-1 to the nucleus.Conclusion:GA suppressed the proliferation of Jurkat cells by apoptosis induction. DIO-1 triggered early-stage cell death in GA-treated Jurkat cells.

Effects of betulinic acid on proliferation and apoptosis in Jurkat cells and its in vitro mechanism.

The anti-cancer effects of betulinic acid (BA) on Jurkat cells and its in vitro mechanism were examined by using MTT assay. Apoptosis was detected by using Hoechst33258 staining and annexin-V/PI double-labeled cytometry. The effects of betulinic acid on the cell cycle of Jurkat cells were studied by propidium iodide method. RT-PCR and Western blotting were used to analyze the changes of cyclin D3, bcl-xl mRNA and protein levels in Jurkat cells after treatment with betulinic acid. Our results showed the proliferation of Jurkat cells was decreased in betulinic acid-treated group with a 24-h IC50 value being 70.00 μmol/L. Betulinic acid induced apoptosis of Jurkat cells in a time- and dose-dependent manner. The number of Jurkat cells treated with betulinic acid showed an increase in G0/G1 phase and decrease in S phase. After treatment with 0, 20, 60, 100 μmol/L betulinic acid for 24 h, the number of Jurkat cells was increased from (31.00±1.25)% to (58.84±0.32)% in G0/G1 phase, whereas it was decreased from (61.45±1.04)% to (35.82±1.95)% in S phase. PBMCs were less sensitive to the cytotoxicity of betulinic acid than Jurkat cells. The expressions of cyclin D3, bcl-xl mRNA and protein were decreased sharply in Jurkat cells treated with betulinic acid. It is concluded that betulinic acid is able to inhibit the proliferation of Jurkat cells by regulating the cell cycle, arrest cells at G0/G1 phase and induce the cell apoptosis. The anti-tumor effects of betulinic acid are related to the down-regulated expression of cyclin D3 and bcl-xl.SummaryThe anti-cancer effects of betulinic acid (BA) on Jurkat cells and its in vitro mechanism were examined by using MTT assay. Apoptosis was detected by using Hoechst33258 staining and annexin-V/PI double-labeled cytometry. The effects of betulinic acid on the cell cycle of Jurkat cells were studied by propidium iodide method. RT-PCR and Western blotting were used to analyze the changes of cyclin D3, bcl-xl mRNA and protein levels in Jurkat cells after treatment with betulinic acid. Our results showed the proliferation of Jurkat cells was decreased in betulinic acid-treated group with a 24-h IC50 value being 70.00 μmol/L. Betulinic acid induced apoptosis of Jurkat cells in a time- and dose-dependent manner. The number of Jurkat cells treated with betulinic acid showed an increase in G0/G1 phase and decrease in S phase. After treatment with 0, 20, 60, 100 μmol/L betulinic acid for 24 h, the number of Jurkat cells was increased from (31.00±1.25)% to (58.84±0.32)% in G0/G1 phase, whereas it was decreased from (61.45±1.04)% to (35.82±1.95)% in S phase. PBMCs were less sensitive to the cytotoxicity of betulinic acid than Jurkat cells. The expressions of cyclin D3, bcl-xl mRNA and protein were decreased sharply in Jurkat cells treated with betulinic acid. It is concluded that betulinic acid is able to inhibit the proliferation of Jurkat cells by regulating the cell cycle, arrest cells at G0/G1 phase and induce the cell apoptosis. The anti-tumor effects of betulinic acid are related to the down-regulated expression of cyclin D3 and bcl-xl.

Deguelin regulates nuclear pore complex proteins Nup98 and Nup88 in U937 cells in vitro

AbstractAim:To investigate the anticancer effects and the molecular mechanisms of deguelin on human U937 leukemia cells, and to explore the underlying mechanism regulating nucleoporin 98 (Nup98) and nucleoporin 88 (Nup88) in vitro.Methods:The effects of deguelin on the growth of U937 cells were studied by MTT assay. The effect of deguelin on the cell cycle of U937 cells was studied by using a propidium iodide method. The localization of the nuclear pore complex proteins Nup98 and Nup88 was investigated by using immunofluorescence and immunoelectron microscopy. The expression of Nup98 and Nup88 in U937 cells was investigated by using flow cytometry and Western blot.Results:The proliferation of U937 cells was inhibited in the deguelin-treated group, with a 24-h IC50 value of 21.61 nmol/L and a 36-h IC50 value of 17.07 nmol/L. U937 cells treated with deguelin had reduced percentages of cells in the G0/G1 phase, whereas cells accumulated in the S and G2/M phases. Nup88 and Nup98 were found on both the nuclear and cytoplasmic sides of the U937 cells by using immunofluorescence and immunoelectron microscopy. The expression of Nup98 was upregulated and that of the Nup88 protein was downregulated in U937 cells treated with deguelin.Conclusion:Deguelin is able to inhibit the proliferation of U937 cells by regulating the cell cycle such that cells are arrested at the S and G2/M phases, so that the proportion of cells in the G0/G1 phase decreases. The antitumor effects of deguelin are related to upregulating the expression of Nup98 and downregulating the expression of Nup88 protein in U937 cells.

Endothelial microparticles carrying hedgehog-interacting protein induce continuous endothelial damage in the pathogenesis of acute graft-versus-host disease

Accumulating evidence suggests that endothelial microparticles (EMPs), a marker of endothelial damage, are elevated in acute graft-versus-host disease (aGVHD), and that endothelial damage is implicated in the pathogenesis of aGVHD, but the mechanisms remain elusive. In this study, we detected the plasma EMP levels and endothelial damage in patients and mice with aGVHD in vivo and then examined the effects of EMPs derived from injured endothelial cells (ECs) on endothelial damage and the role of hedgehog-interacting protein (HHIP) carried by EMPs in these effects in vitro. Our results showed that EMPs were persistently increased in the early posttransplantation phase in patients and mice with aGVHD. Meanwhile, endothelial damage was continuous in aGVHD mice, but was temporary in non-aGVHD mice after transplantation. In vitro, EMPs induced endothelial damage, including increased EC apoptosis, enhanced reactive oxygen species, decreased nitric oxide production and impaired angiogenic activity. Enhanced expression of HHIP, an antagonist for the Sonic hedgehog (SHH) signaling pathway, was observed in patients and mice with aGVHD and EMPs from injured ECs. The endothelial damage induced by EMPs was reversed when the HHIP incorporated into EMPs was silenced with an HHIP small interfering RNA or inhibited with the SHH pathway agonist, Smoothened agonist. This work supports a feasible vicious cycle in which EMPs generated during endothelial injury, in turn, aggravate endothelial damage by carrying HHIP into target ECs, contributing to the continuously deteriorating endothelial damage in the development of aGVHD. EMPs harboring HHIP would represent a potential therapeutic target for aGVHD.

Expression of histone H2AX phosphorylation and its potential to modulate adriamycin resistance in K562/A02 cell line

SummaryDNA repair processes play a role in the development of drug resistance which represents a huge obstacle to leukemia chemotherapy. Histone H2AX phosphorylation (ser139) (γH2AX) occurs rapidly at the onset of DNA double strand break (DSB) and is critical to the regulation of DSB repair. If DNA repair is successful, cells exposed to anti-neoplastic drugs will keep entering the cycle and develop resistance to the drugs. In this study, we investigated whether γH2AX can be used as an indicator of tumor chemosensitivity and a potential target for enhancing chemotherapy. K562 and multi-drug resistant cell line K562/A02 were exposed to adriamycin (ADR) and γH2AX formed. Flow cytometry revealed that percentage of cells expressing γH2AX was increased in a dose-dependent manner and the percentage of K562/A02 cells was lower than that of K562 cells when treated with the same concentration of ADR. In order to test the potential of γH2AX to reverse drug resistance, K562/A02 cells were treated with PI3K inhibitor LY294002. It was found that LY249002 decreased ADR-induced γH2AX expression and increased the sensitivity of K562/A02 cells to ADR. Additionally, the single-cell gel electrophoresis assay and the Western blotting showed that LY249002 enhanced DSBs and decreased the expression of repair factor BRCA1. These results illustrate chemosensitivity can partly be measured by detecting γH2AX and drug resistance can be reversed by inhibiting γH2AX.DNA repair processes play a role in the development of drug resistance which represents a huge obstacle to leukemia chemotherapy. Histone H2AX phosphorylation (ser139) (γH2AX) occurs rapidly at the onset of DNA double strand break (DSB) and is critical to the regulation of DSB repair. If DNA repair is successful, cells exposed to anti-neoplastic drugs will keep entering the cycle and develop resistance to the drugs. In this study, we investigated whether γH2AX can be used as an indicator of tumor chemosensitivity and a potential target for enhancing chemotherapy. K562 and multi-drug resistant cell line K562/A02 were exposed to adriamycin (ADR) and γH2AX formed. Flow cytometry revealed that percentage of cells expressing γH2AX was increased in a dose-dependent manner and the percentage of K562/A02 cells was lower than that of K562 cells when treated with the same concentration of ADR. In order to test the potential of γH2AX to reverse drug resistance, K562/A02 cells were treated with PI3K inhibitor LY294002. It was found that LY249002 decreased ADR-induced γH2AX expression and increased the sensitivity of K562/A02 cells to ADR. Additionally, the single-cell gel electrophoresis assay and the Western blotting showed that LY249002 enhanced DSBs and decreased the expression of repair factor BRCA1. These results illustrate chemosensitivity can partly be measured by detecting γH2AX and drug resistance can be reversed by inhibiting γH2AX.

Comparison of outcomes of idarubicin intensified TBI–CY and traditional TBI–CY conditioning regimen for high-risk acute lymphoblastic leukemia undergoing allogeneic hematopoietic stem cell transplantation: A single center experience

High-risk acute lymphoblastic leukemia (ALL) carries a very poor prognosis, even after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Exploring novel conditioning regimen to more effectively eliminate leukemic clone while not alter transplant-related mortality (TRM) has become focus of attention. We retrospectively evaluated outcomes of 87 high-risk ALL patients undergoing allo-HSCT: 47 patients received idarubicin (IDA) intensified TBI-CY and 40 patients received traditional TBI-CY regimen. In IDA intensified group, patients received TBI (8Gy) on day-8, IDA of 15mg/m2/d from day-6 to -5, followed by CY (60mg/kg/d) on day-3 to -2. The cumulative incidence of relapse was significantly lower in IDA intensified group compared with TBI-CY group (P=0.018). Oropharyngeal mucositis was observed more frequent in IDA intensified group (P=0.013), while not followed by increased TRM. Very high-risk ALL patients benefit from IDA intensified regimen with only two of eight patients in no remission (NR) pre-transplantation and two of twelve ph+ALL patients relapsed after transplantation. After a median follow-up for all survivors of 21 months (range, 12-53 months), 2-year estimated OS and DFS was 66.2% vs 45.3% (P=0.031) and 62.5% vs 43.5% (P=0.044), respectively. In conclusion, IDA intensified TBI-CY regimen may reduce relapse while not increasing TRM, providing better survival for high-risk ALL patients undergoing allo-HSCT.

Angiogenic factors are associated with development of acute graft-versus-host disease after allogeneic hematopoietic stem cell transplantation

SummaryAcute 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.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.

Anti-cancer Effects of Deguelin on Human Leukemia K562 and K562/ADM Cells In Vitro *

In order to investigate the anti-cancer effects of deguelin and on K562 and K562/ADM cells in vitro and the underlying molecular mechanism and compare the cytotoxicity of deguelin on K562, K562/ADM cells and human peripheral blood mononuclear cells (PBMCs). The effects of deguelin on cell proliferation were assessed by MTT assay. Apoptosis were detected by Annexin V/PI double-labeled cytometry. The effects of deguelin on the cell cycle were studied by a propidium iodide method. Our study showed that deguelin inhibited the proliferation of K562 cell and K562/ADM cell in a time- and dose-dependent manner and had minimal effects on normal human peripheral blood mononuclear cells. The ratio of IC50 value of deguelin of 24 h on K562/ADM cells to K562 cells was only 1.27, which was significantly lower than the ratio of IC50 value of ADM (higher than 20). Deguelin could induce apoptosis of K562 cells and K562/ADM cells. K562 cells were arrested at G2/M phase while K562/ADM cells were arrested at G0/G1 phase. Our results suggested that deguelin was a novel anti-leukemia agents with high efficacy and low toxicity and it is also a promising agent for reversing drug resistance.