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OBJECTIVE To explore whether diterpenoid 12-deoxyphorbol-13-palmitate （DP） from Euphorbia fischeriana can exert anti-leukemia effects through the phosphatidylinositol 3-kinase （PI3K）/protein kinase B （Akt） signal pathway， and to provide experimental evidence for developing it into a new anti-leukemia drug. METHODS Using LY294002 （PI3K specific inhibitor） as tool drug， the effects of 24 h DP treatment on the proliferation and apoptosis of human myeloid leukemia HL60 cells were detected by MTT method， Annexin Ⅴ-FITC/PI staining and AO-EB staining. ELISA method was used to detect lactic dehydrogenase （LDH） release and the activities of cysteinyl aspartate specific proteinase 3 （caspase-3） and caspase-9. The transcriptional level of caspase-3， caspase-9， forkhead box O3a （FoxO3a） and B cell lymphoma 2 interacting mediator of cell death （Bim） mRNA were detected by real-time quantitative polymerase chain reaction （qRT-PCR）. The protein expression of phosphorylated FoxO3a （p- FoxO3a） and phosphorylated Akt （p-Akt） were detected by Western blot method. The nuclear translocation of FoxO3a protein was detected by immunostaining combined with laser confocal microscopy. RESULTS 10 μmol/L DP and 10 μmol/L DP+LY294002 could inhibit the proliferation and induce the apoptosis of HL60 cells （P＜0.01）. After treatment of 5， 10， 20 μmol/L DP， HL60 cells showed typical morphological characteristics of apoptosis； DP could significantly increase the levels of LDH release and the activities of caspase-3 and caspase-9 （P＜0.05 or P＜0.01）， in dose-dependent manner. After treatment of 10 μmol/L DP and 10 μmol/L DP+LY294002， the transcriptional levels of caspase-3， caspase-9 and Bim mRNA were increased significantly （P＜0.05 or P＜0.01）， and transcriptional level of FoxO3a mRNA and protein expressions of p-FoxO3a and p-Akt were decreased significantly （P＜0.05 or P＜0.01）. Nuclear translocation changes were observed in FoxO3a protein in 10 μmol/L DP+LY294002 group， and the change was more significant than that of LY294002 group. CONCLUSIONS DP can inhibit the proliferation and induce the apoptosis of HL60 cells via inhibiting PI3K/Akt signaling pathway.
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Background : Acute promyelocytic leukemia (APL) affects both kids and adults, however it is more prevalent in younger population. Although APL has a favorable prognostic, patients that relapse often do not respond positively to additional chemotherapy. Therefore, there is a need to further identify ways to overcome these challenges. Hypothesis: In this study, we examined antileukemic effects of xanthohumol (XN), a prenylated flav onoid derived from hops ( Humulus lupulus L ), on human promyelocytic HL - 60 cells. Materials and Methods : HL - 60 cells were exposed to different concentrations of XN (?M) for 24 h. Cell viability, cell morphology, chromatin condensation, cPARP - 1 level, and caspase - 3 activation, and the expression of p21 WAF1/Cip1 were analyzed. Results : XN reduced HL - 60 cell viability in a dose - dependent manner. XN induced a dose - dependent morphological changes including cell shrinkage and b lebbing , and significantly increased the number of cells with condensed chromatin. XN significantly increased the level of cPARP - 1, active caspase - 3, and the expression of p21WAF/CIP mRNA. Conclusion : These data indicate that XN induces HL - 60 cell death by regula ting cell cycle progression and apoptosis. This study suggests that XN may have antileukemic preventive effects.
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BACKGROUND: Juglone is a naphthoquinone currently obtained by chemical synthesis with biological activities including antitumor activity. Additionally, juglone is present in the green husk of walnut, which suggests evaluating the effect of GH extracts on carcinogenic cell lines. RESULTS: Walnut green husk ethanolic extract was obtained as 169.1 mg juglone/100 g Green Husk and antioxidant activity (ORAC) of 44,920 µmol Trolox Equivalent/100 g DW Green Husk. At 1 µM juglone in HL-60 cell culture, green husk extract showed an antiproliferative effect, but pure juglone did not; under these conditions, normal fibroblast cells were not affected. A dose-dependent effect on mitochondrial membrane potential loss was observed. Apoptosis of HL-60 was detected at 10 µM juglone. Despite high ORAC values, neither purified juglone nor the extract showed protective effects on HL-60 cells under oxidative conditions. CONCLUSIONS: Green husk extract generates an antiproliferative effect in HL-60 cells, which is related to an induction of the early stages of apoptosis and a loss of mitochondrial membrane potential. The normal cells were not affected when juglone is present at concentrations of 1 µM, while at higher concentrations, there is loss of viability of both cancerous and healthy cells.
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Apoptosis , HL-60 Cells/metabolism , Juglans/chemistry , Polyphenols/metabolism , Antioxidants/metabolism , Cell Survival , Chromatography, High Pressure Liquid , Cell Culture Techniques , Membrane Potential, MitochondrialABSTRACT
Objective The inhibitory effect of the PARP inhibitor olaparib on human acute myeloid leukemia HL-60 cells was studied. Methods The HL-60 cells in logarithmic growth phase were treated with different concentrations(1. 25,2. 5,5 and 10 μmol/L) of olaparib for different time. The CCK-8 assay was used to detect the inhibitory effect of olaparib on HL-60 cells. The apoptotic level of HL-60 cells was detected by Annexin-V/PI double staining method,and the expression of related signal proteins ( PARP-1 and caspase-3)in HL-60 cells was detected by Western blot. Results HL-60 cells were inhibited by olaparib at dif-ferent concentrations(1. 25,2. 5,5 and 10 μmol/L) for 48 h,and the inhibition rate gradually increased with the prolongation of the action time;at the same time,the apoptotic rate was increased in HL-60 cells after olaparib treatment for 48 h,showing a dose-de-pendent manner;the PARP activity was inhibited and caspase-3 was activated in HL-60 cells treated with olaparib. Conclusion The PARP inhibitor olaparib not only inhibits proliferation of HL-60 cells,but it also promotes apoptosis of HL-60 cells by inhibi-ting PARP activity and activating caspase-3.
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Objective@#To evaluate the effect of down-regulating SALL4 on the radiosensitivity of leukemia cells, aiming to provide new ideas for improving radiosensitivity of leukemia patients.@*Methods@#Human acute myeloid leukemia cell line HL-60 infected with shRNA SALL4 and shRNA control lentivirus was classified into the Lv-shSALL4 group and Lv-shNC group. The levels of SALL4 mRNA and protein in cells were detected by RT-PCR and Western blot. The infected cells treated with 8 Gy dose irradiation were assigned into the Lv-shSALL4+ radiation and Lv-shNC+ radiation groups. The cell apoptosis was detected by flow cytometry. The levels of cleaved Caspase-3, cleaved Caspase-9 and Bax proteins in cells were determined by Western blot. The cells in the Lv-shSALL4 and Lv-shNC groups were exposed to 0, 2, 4, 6 and 8 Gy irradiation. The radiosensitivity ratio was determined by cell clone test.@*Results@#The level of SALL4 in the Lv-shSALL4 group was significantly lower than that in the Lv-shNC group (P<0.05). The cell apoptosis rate was significantly increased, the levels of cleaved Caspase-3, cleaved Caspase-9 and Bax proteins were remarkably up-regulated in cells compared with those in the Lv-shNC group (all P<0.05). The cell proliferation ability in the Lv-shSALL4+ radiation was significantly reduced, the cell apoptosis rate was considerably increased, the levels of cleaved Caspase-3, cleaved Caspase-9 and Bax proteins were significantly up-regulated compared with those in the Lv-shSALL4 and Lv-shNC+ radiation groups (all P<0.05). The cell radiosensitization ratio in the Lv-shSALL4 group was 1.323.@*Conclusion@#Down-regulating SALL4 can increase the radiosensitivity of leukemic cells, inhibit the cell proliferation and induce the apoptosis of leukemic cells.
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OBJECTIVE: To study the algorithm of four-parameter logistic regression and apply the model in the potency calibration of bacterial endotoxin standard. METHODS: The potency of endotoxin reference standard was estimated by a novel monocyte activation test,HL60-IL6 assay.The endotoxin national standard was set as standard, while the endotoxin working standard was set as test. The four-parameter logistic model was fitted by the method of nonlinear least squares when observations of standard or sample solutions were plotted against endotoxin concentration. The algorithm was performed by C Language, the RESULTS: of which were compared by those of softmax, R Language as well as Microsoft Office Excel. RESULTS The estimated parameters as well as estimated potency were parallel between the four statistical tools. The goodness-of-fit were all over 0.99.The variance was within 0.1%. CONCLUSION: The four-parameter logistic model can be established by the four statistical tools. Researchers can select a suitable tool according to demands.
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Objective To evaluate the effect of down-regulating SALL4 on the radiosensitivity of leukemia cells,aiming to provide new ideas for improving radiosensitivity of leukemia patients.Methods Human acute myeloid leukemia cell line HL-60 infected with shRNA SALL4 and shRNA control lentivirus was classified into the Lv-shSALL4 group and Lv-shNC group.The levels of SALL4 mRNA and protein in cells were detected by RT-PCR and Western blot.The infected cells treated with 8 Gy dose irradiation were assigned into the Lv-shSALL4 + radiation and Lv-shNC+radiation groups.The cell apoptosis was detected by flow cytometry.The levels of cleaved Caspase-3,cleaved Caspase-9 and Bax proteins in cells were determined by Western blot.The cells in the Lv-shSALL4 and Lv-shNC groups were exposed to 0,2,4,6 and 8 Gy irradiation.The radiosensitivity ratio was determined by cell clone test.Results The level of SALL4 in the Lv-shSALL4 group was significantly lower than that in the Lv-shNC group (P＜0.05).The cell apoptosis rate was significantly increased,the levels of cleaved Caspase-3,cleaved Caspase-9 and Bax proteins were remarkably up-regulated in cells compared with those in the Lv-shNC group (all P＜ 0.05).The cell proliferation ability in the Lv-shSALL4 + radiation was significantly reduced,the cell apoptosis rate was considerably increased,the levels of cleaved Caspase-3,cleaved Caspase-9 and Bax proteins were significantly up-regulated compared with those in the Lv-shSALL4 and Lv-shNC+ radiation groups (all P＜0.05).The cell radiosensitization ratio in the Lv-shSALL4 group was 1.323.Conclusion Down-regulating SALL4 can increase the radiosensitivity of leukemic cells,inhibit the cell proliferation and induce the apoptosis of leukemic cells.
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OBJECTIVE: To investigate the effects of 12-deoxyphorbol-13-palmitate (DP) on the proliferative inhibition and induction apoptosis of human acute myelocytic leukemia cell line HL60. METHODS: HL60 cells were treated with different concentrations of DP. The morphological changes were observed with optical microscope. The effect of DP in cell proliferation was tested using cell counting kit-8(CCK-8) assay, to analyze the cell cloning capacity by methyl cellulose colone-forming experiment. DNA agarose electrophoresis method was used to detect apoptosis. The expression of pro-and anti-apoptotic genes Bcl-2 and Bax were examined by reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: CCK-8 METHODS and methyl cellulose colone-forming experiment indicated DP had an certainly inhibitory effect on HL60 cells(P<0.05, P<0.01). The characteristics of apoptosis was presented under optical microscope. DNA agarose electrophoresis showed the obvious DNA 1adder. In accordance with RT-PCR experiments, compared with the control group(0 mg·L-1), DP could down-regulate the gene expression of Bcl-2 and up-regulate the gene expression of Bax in HL60 cells(P<0.01). CONCLUSION: DP can inhibit the proliferation and induce the apoptosis of cultured HL60 cells in a dose-effect and time-effect relationship in vitro, and its mechanism may be related to down-regulating gene expression of Bcl-2, up-regulating gene expression of Bax, rising Bax/Bcl-2 value.
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Objective To investigate the characteristics and mechanism of spironolactone ent-kaurane diterpene type diterpene rabdosin B (RB) induced HL-60 differentiation into mature granulocytes. Methods The effects of RB on proliferation, cell cycle, NBT reducing power, phagocytosis, and cell surface antigen CD11b expression of HL-60 were detected by trypan blue staining, Giemsa staining, NBT reducing power assay, and flow cytometry, respectively. Results RB inhibited the proliferation of HL-60 cells at the concentration of 1.0, 3.0, and 5.0 μmol/L, which resulted in the arrest of G0/G1 phase, increased the ratio of renal nucleated and lobate nucleus, and markedly enhanced the NBT reducing power and phagocytic capacity and the significant increase of CD11b positive cell rate. Further tests showed that the active oxygen scavenger NAC (300 μmol/L), NADPH oxidase inhibitor APO (100 μmol/L), and DPI (0.1 μmol/L) not only significantly inhibited the intracellular ROS upregulation induced by RB, but also inhibited various features of RB induced HL-60 cell differentiation. Conclusion RB can increase the activity of NADPH oxidase in HL-60 cells, increase the intracellular ROS concentration, and induce the differentiation of HL-60 cells to mature granulocytes.
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Objective To evaluate the effects of Treponema pallidum (T.pallidum) on the expression of chemokine ligands (CXCL) in human brain microvascular endothelial cells (HBMECs).Methods HBMECs were randomly divided into 4 groups,which were treated with viable T.pallidum suspension (T.pallidum group),heat-inactivated T.pallidum suspension (inactivated T.pallidum group),200 μg/L lipopolysaccharide (LPS group) and cell culture medium (blank control group),respectively,for 6,12 and 24 hours.Fluorescence-based quantitative PCR and enzyme-linked immunosorbent assay (ELISA) were performed to determine the mRNA and protein expression of CXCL6,CXCL8 and CXCL10 in HBMECs in the above groups respectively.Transwell migration assay was conducted to evaluate the effects of T.pallidum-stimulated HBMECs on the chemotaxis of human promyelocytic HL-60 leukemia cells (HL-60 cells).Results At 6,12 and 24 hours,the T.pallidum group showed significantly higher mRNA expression of CXCL6,CXCL8 and CXCL10 in HBMECs compared with the blank control group and inactivated T.pallidum group (all P ＜ 0.05),while there were no significant differences between the blank control group and inactivated T.pallidum group (all P ＞ 0.05).Compared with the LPS group,the T.pallidum group showed significantly decreased mRNA expression of CXCL6 and CXCL8 (P ＜ 0.05),but similar mRNA expression of CXCL10(P ＞ 0.05)at 6,12 and 24 hours.At these time points,the levels of CXCL6 and CXCL8 in the culture supernatant of HBMECs were significantly higher in the T.pallidum group than in the blank control group and the inactivated T.pallidum group (all P ＜ 0.05),but no significant differences were observed between the blank control group and the inactivated T.pallidum group (both P ＞ 0.05).Moreover,there were no significant differences in the level of CXCL10 in the culture supernatant of HBMECs among the T.pallidum group,the inactivated T.pallidum group and the blank control group (all P ＞ 0.05).The number of migratory HL-60 cells in the lower Transwell chambers was significantly higher in the T.pallidum group than in the inactivated T.pallidum group and the blank control group (both P ＜ 0.05).Conclusion Viable T.pallidum can up-regulate the gene expression of CXCL6,CXCL8 and CXCL10 in HBMECs,promote the secretion of CXCL6 and CXCL8,and enhance the chemotactic effect of HBMECs on HL-60 cells,which may play a certain role in the occurrence of neurosyphilis.
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AIM:To investigate the effect of suppression of mel18 gene on the differentiation of human acute myeloid leukemia cell line HL 60 induced by cinnamaldehyde ( CA) .METHODS:HL60 cells were treated with low con-centration of CA or all-trans retinoic acid (ATRA).shRNAmel18 vector and shRNALuc control vector were employed to package lentiviruses which were then used to infect HL 60 cells.The virus-infected HL60 cells were treated with low con-centration of CA , and ATRA was used as a positive control of differentiation-inducing agent .The differentiation markers on the cell surface and cell cycle of virus-infected HL60 cells were analyzed by flow cytometry .Western blot was used to deter-mine the expression of MEL18, cyclin D1 and p27, as well as the phosphorylation level of Akt .RESULTS:Low concen-tration of CA and ATRA increased the expression of granulocytic differentiation marker CD 11b on the HL60 cells, with the decreased expression of MEL 18 in the HL60 cells.The expression of MEL18 decreased in shmel18 virus-infected HL60 cells (shmel18-HL60 cells), but did not change in shLuc-HL60 cells.The expression of CD11b on shmel18-HL60 cells increased with G 1-phase arrest , which went even higher after treatment with CA .The phosphorylation level of Akt and the expression of cyclin D1 decreased in shmel18-HL60 cells with the increase in the expression of p27.CONCLUSION:In-hibition of mel18 gene leads HL60 cell granulocytic differentiation .mel18 gene may affect the differentiation of HL 60 cells by regulating cyclin D1 and p27 via PI3K/Akt signaling pathway.PI3K/Akt signaling pathway is also involved in CA-in-duced differentiation of HL60 cells by suppressing mel18 gene expression.
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AIM:To investigate the effect of suppression of mel18 gene on the differentiation of human acute myeloid leukemia cell line HL 60 induced by cinnamaldehyde ( CA) .METHODS:HL60 cells were treated with low con-centration of CA or all-trans retinoic acid (ATRA).shRNAmel18 vector and shRNALuc control vector were employed to package lentiviruses which were then used to infect HL 60 cells.The virus-infected HL60 cells were treated with low con-centration of CA , and ATRA was used as a positive control of differentiation-inducing agent .The differentiation markers on the cell surface and cell cycle of virus-infected HL60 cells were analyzed by flow cytometry .Western blot was used to deter-mine the expression of MEL18, cyclin D1 and p27, as well as the phosphorylation level of Akt .RESULTS:Low concen-tration of CA and ATRA increased the expression of granulocytic differentiation marker CD 11b on the HL60 cells, with the decreased expression of MEL 18 in the HL60 cells.The expression of MEL18 decreased in shmel18 virus-infected HL60 cells (shmel18-HL60 cells), but did not change in shLuc-HL60 cells.The expression of CD11b on shmel18-HL60 cells increased with G 1-phase arrest , which went even higher after treatment with CA .The phosphorylation level of Akt and the expression of cyclin D1 decreased in shmel18-HL60 cells with the increase in the expression of p27.CONCLUSION:In-hibition of mel18 gene leads HL60 cell granulocytic differentiation .mel18 gene may affect the differentiation of HL 60 cells by regulating cyclin D1 and p27 via PI3K/Akt signaling pathway.PI3K/Akt signaling pathway is also involved in CA-in-duced differentiation of HL60 cells by suppressing mel18 gene expression.
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OBJECTIVE: To establish the cell model using human leukemia cell line HL-60 for exposure of coke oven emissions( COE) in vitro and to explore the mechanism of COE-induced acute toxicity in HL-60 cells. METHODS: HL-60 cells were collected in their logarithmic growth phase and cultured in medium that had final concentrations of COE in 2. 5,5. 0,10. 0 and 20. 0 mg / L for 24 hours. Cell survival rate was examined by CCK-8 assay. The cytotoxicity was evaluated using lactate dehydrogenase release assay. Reactive oxygen species( ROS) production was determined by the 2',7'-dichlorofluorescein diacetate and nitroblue tetrazolium method. The activation of nuclear factor-κB( NF-κB) pathway was evaluated by western blot. RESULTS: With the increasing exposure concentrations of COE,the cytotoxicity of HL-60 cells increased( P < 0. 01),the cell survival rate decreased( P < 0. 01),intracellular ROS decreased( P < 0. 01),whereas extracellular ROS increased( P < 0. 01). These changes had a dose-effect relationship. The levels of phospho-nuclear factor-kappa B p65 and phospho-inhibitor of kappa Bα were higher in all the COE-treated cells compared with untreated cells( P < 0. 05),with no dose-effect relationship. CONCLUSION: COE could cause acute toxicity in HL-60 cells in a doseeffect relationship. The mechanism may be related to the COE-induced in-balanced ROS release and removal,leading to the activation of NF-κB pathway. HL-60 cells can be used as a common cell line for COE hematotoxicity analysis.
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Objectives@#To investigate the effect of β-catenin interacting protein 1 (ICAT) silence in Wnt signaling pathway and sterol drug NSC67657 induced cell differentiation of HL60 cell.@*Methods@#HL-60 cells were treated with NSC67657, the cell surface antigen CD14 expression was detected by flow cytometry. Lentivirus LV-ICAT-RNAi vector was constructed and infected HL-60 cells. Then the ICAT gene and protein expression were analyzed using real-time qPCR and Western blot technique. Furthermore, Co-immunoprecipitation assay was used to confirm the interaction of β-catenin/ICAT proteins, and Western blot was employed to compare the expressions of Wnt signaling pathway downstream targets Cyclin D1, TCF-1 and c-Jun between Lentivirus LV-ICAT-RNAi vector infected HL-60 (HL-60i) cells and un-infected HL-60 (HL-60v) cells. The cellular differentiation of HL-60i and HL-60v cells treated with NSC67657 for 24 h was evaluated by Wright’s staining, transmission electron microscopy and flow cytometry analysis.@*Results@#HL-60 cells could be induced to differentiate into monocytes by 10 μmol/L NSC67657. The CD14 positive cells could reach to (92.30±5.14) % after NSC67657 treatment for 5d. The co- immunoprecipitation assay demonstrated that ICAT protein did interact with β-catenin protein, and the absorbance of protein electrophoresis bands increased in differentiated cells. The expressions of Wnt signaling pathway downstream target proteins in HL-60i cells were higher than that in HL-60v cells when they were treated by 10 μmol/L NSC67657, but lower than NSC67657 untreated cells. CD14 positive HL-60i cells were significantly lower than that of HL-60v cells[ (8.33±3.14) % vs (19.08±4.73) %]when treated with NSC67657, but still higher than that of uninfected and untreated HL60 cells[ (0.60±0.03) %] (F=119.24, P=0.010) . The results of cellular morphology and ultrastructure observation were also in accord with that of cell surface antigen analysis.@*Conclusions@#ICAT does participate in HL-60 cells monocytic differentiation induced by NSC67657, and Wnt/β-catenin signaling pathway might play a bridge role.
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Objective To investigate the effect of down-regulation of lysine specific demethylase 1 (LSD1) by shRNA on the apoptosis and cell cycle of human acute myelogenous leukemia cells.Methods The lentiviral vector-mediated LSD1-shRNA was transfected into human acute promyelocytic leukemia HL-60 cells and acute monocytic leukemia SHI-1 cells.The expressions of LSD1 mRNA and protein were examined by real time quantitative PCR and Western blot,respectively.The flow cytometry was applied to detect the apoptosis and cell cycle distribution after AnnexinV-PE/7-AAD and PI dying,respectively.Results The expressions of LSD1 mRNA and protein in HL-60 and SHI-1 LSD1-shRNA group were significantly decreased compared with the blank control group and the negative shRNA group (P ＜ 0.01,respectively).The apoptosis levels of HL-60 and SHI-1 cells were significantly increased after knockdown of LSD1 (P ＜ 0.01).Moreover,the cell cycle distribution in the G0/G1 phases was also significantly increased(P ＜ 0.01).Conclusion LSDI-shRNA promotes cell apoptosis and increases the percentage of cells in the G0/G1 phases of human acute myelogenous leukemia cells.
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Objective:To investigate the potential pro-apoptotic activity of arsenic trioxide (ATO) in human leukemia HL-60 cells,as well as the potential mechanism with focus on mitochondrial pathway.Methods:After treatment with different concentrations of ATO (1 μg/mL,5 μg/mL or 10 μg/mL) for 24 h,apoptotic cell death was detected by flow cytometry,oxidative stress was determined by measuring MDA and GSH levels,the expression of apoptotic factors was detected by western blot,and mitochondrial membrane potential (MMP) was determined by immunofluorescence staining.Results:ATO at the concentrations of 5 μg/mL or 10 μg/mL induces apoptotic cell death and increases oxidative stress in human leukemia HL-60 cells.ATO significantly increases the expression of pro-apoptotic factors (Bax and Caspase-3),whereas decreases the expression of anti-apoptotic factor Bcl-2.Compared with the control group,ATO treatment significantly decreases the MMP level in HL-60 ceils.Conclusions:Arsenic trioxide induces apoptotic cell death through mitochondrial pathway in human leukemia HL-60 cells.
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Objective To construct the ICAT gene interference lentivirus expression vector targeting and to establish stable transfected cell line HL60 .Methods The interference sequence targeted at human ICAT gene was designed and synthesized ,after annealing ,which was connected to PGLV3 interference vector and with PG‐p1‐VSVG ,PG‐p1‐REV ,PG‐p1‐RRE were co‐transfected into 293T cells The lentivirus particles were packaged and generated .The virus titer was detected .HL60 cells were transfected for establishing the stable cell line ;RT‐PCR and Western Blot techniques were used to detect ICAT gene and protein expression in sta‐ble HL60 cells ,then the results were compared with those in the control group .Results The lentivirus expression vector targeted at ICAT was successfully constructed and the virus titer was 2 × 108 U/mL .Stable transfected HL60 cell line was established .The effective interference verification revealed that shICAT could significantly reduce the mRNA and protein level of ICAT ( P<0 .001) .Conclusion The shRNA lentiviral expression vector of ICAT gene is successfully constructed and the HL 60 cell line stably interfering ICAT expression is established .
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Bisfenol A (BPA) é um insumo largamente utilizado na produção de plástico policarbonato e amplamente difundido no meio ambiente, levando o ser humano à exposição crônica desde o período intrauterino. A literatura aponta a possibilidade de BPA aumentar o risco de diversos tipos de câncer, mas são necessários estudos que possibilitem o entendimento de mecanismos pelos quais isso pode ocorrer. Neste trabalho foram investigados os efeitos do BPA ou nitro-BPA em células HL-60, MCF-7 e tecidos de ratos. Células HL-60 foram expostas ao BPA ou nitro-BPA nas concentrações de 25, 100 e 250 µM (0,1 % DMSO v/v) por 2, 24 ou 48 horas na presença ou ausência de H2O2 (40 nmol/5 x 104 células). Células MCF-7 foram expostas da mesma forma, sem o uso de H2O2, mas na presença e ausência de agonista (PCB) de receptor Ah. Ratos Sprague-Dawley machos receberam BPA diariamente ao longo de 4 semanas (50 mg/kg de peso corpóreo) por gavagem, na vigência e ausência de diabetes, com subsequente coleta de urina, fígado, rins, medula óssea e sangue. Nos experimentos com as células, a viabilidade, ciclo celular, fragmentação do DNA e a produção intracelular de espécies reativas de oxigênio (ROs) foram avaliadas por citometria de fluxo, a atividade da cadeia respiratória mitocondrial pelo ensaio do XTT, e a atividade de MPO de células HL-60 por ensaio de fluorescência, bem como a produção de â¢NO. A metilação e hidroximetilação global do DNA e os adutos 8-oxodG, CEdG, 1,N6-εdA, 1,N2-εdG e BPA-Gua no DNA das células, tecidos, meio de cultura e urina foram analisados por HPLC-ESI-MS/MS. O hemograma e mielograma dos animais foram obtidos no Laboratório de Hematologia Experimental da FCF USP. Observou-se que tanto BPA quanto nitro-BPA induziram a geração de ROS em células HL-60 logo após 2h de incubação. BPA levou subsequentemente à perda de atividade da cadeia respiratória mitocondrial, aumento da permeabilidade da membrana plasmática, fragmentação do DNA, parada na fase G2/M do ciclo celular e hipermetilação acompanhada de hipohidroximetilação global do DNA. A citotoxicidade induzida pelas mesmas concentrações de nitro-BPA em células HL-60 foi menos pronunciada, sem perda de atividade da cadeia respiratória mitocondrial, com pouca fragmentação do DNA, mas com parada na fase G0/G1 do ciclo celular e indução de hipohidroximetilação global do DNA na presença de H2O2. Não foi observada a indução de adutos de DNA nas células HL-60 incubadas com BPA, mas sim de CEdG nas células incubadas com nitro-BPA. Os dados obtidos a partir da exposição das células HL-60 a BPA e nitro-BPA nos indicam que as duas moléculas provocam alterações metabólicas distintas nesse tipo celular, independentes da via estrogênica, que levam a alterações predominantemente epigenéticas (BPA) ou genéticas e epigenéticas (nitro-BPA), que podem ter consequências fenotípicas, como progressão maligna, que precisam ser investigadas. Foi observado que as células MCF-7 são mais resistentes que as células HL-60 à citotoxicidade induzida por BPA e nitro-BPA. Como resultado da exposição das células MCF-7 a BPA, houve pequeno aumento da permeabilidade da membrana plasmática (250 µM), indução dos níveis de ROS após 24 h (25 µM) e aumento da população de células em sub G1, ou seja, com DNA fragmentado (100 µM e 250 µM), mas sem alteração do ciclo celular. No caso de nitro-BPA, foi observada parada do ciclo celular em G2/M (25 µM, 100 µM e 250 µM), assim como aumento de permeabilidade da membrana plasmática após 24 h de incubação (25 µM, 250 µM), sem indução de ROS ou aumento de células em sub G1. Entretanto, observou-se aumento dos níveis de CEdG e 8-oxodG no DNA das células incubadas com BPA (100 µM, 250 µM) sem a ativação prévia de receptores Ah. A ativação dos receptores Ah com PCB levou a menor aumento do nível das lesões após as incubações com BPA. A maior resistência das células MCF-7 aos efeitos citotóxicos do BPA está provavelmente relacionada à ação estrogênica desse xenobiótico. A sinalização estrogênica juntamente com o aumento dos níveis de lesões no DNA aumenta a chance de mutações e de transformação maligna. Nas células com ativação do receptor Ah, BPA levou ainda ao aumento da hidroximetilação global, sem alteração da metilação global do DNA. Os animais não diabéticos expostos ao BPA apresentaram quantidades diminuídas de promielócitos, blastos e bastonetes na medula óssea (aplasia medular), sem alteração no hemograma. Houve aumento dos níveis de CEdG no fígado, da metilação e hidroximetilação global do DNA hepático, e não foi observada alteração das marcas epigenéticas e adutos de DNA no rim ou na urina. Os animais diabéticos expostos ao BPA apresentaram aumento do número de eosinófilos e linfócitos na medula óssea, podendo-se sugerir a indução de um estado inflamatório alérgico, e aumento do número total de hemácias circulantes e do hematócrito. Houve aumento dos níveis de CEdG, da metilação e hidroximetilação global do DNA hepático, aumento dos níveis de 8-oxodG no DNA renal, sem alteração das marcas epigenéticas no rim, e não foi observada alteração dos adutos de DNA na urina. Os dados obtidos apontam para a geração de ROS como uma importante via de cito- e genotoxicidade induzidas por BPA. Sua biotransformação para BPA-3,4- quinona nos modelos utilizados parece ter menor importância para os efeitos, uma vez que não foi detectada a lesão BPA-Gua em nenhuma amostra de DNA, meio de cultura das células ou urina dos animais. Alterações metabólicas induzidas por BPA e ROS podem favorecer as alterações das marcas epigenéticas observadas no DNA das células HL-60, MCF-7 e fígado dos animais. Todas essas alterações podem contribuir para a transformação maligna de células expostas ao BPA
Bisphenol A (BPA) is a compound widely used in polycarbonate plastic production and widespread in the environment, humans are chronic exposed to BPA in intrauterine period and entire life. The literature suggests the possibility of BPA increase the risk of developing cancers, but studies are required to enable the understanding of mechanisms by which this can occur. HL -60 cells were exposed to BPA or nitro-BPA at concentrations of 25, 100 and 250 uM (0.1% DMSO v/v) for 2, 24 or 48 hours in presence or absence of H2O2 (40 nmol/5x104 cells), MCF-7 cells followed a similar profile of exposure without the use of H2O2, but in presence or absence of Ah agonist receptor (PCB126). Male Sprague-Dawley rats received BPA daily over 4 weeks (50 mg/kg body weight) by gavage in presence and absence of diabetes, with subsequent collection of urine, liver, kidney, bone marrow and circulating blood. The viability, cell cycle, DNA fragmentation and the intracellular production of reactive oxygen species (ROS) was evaluated by flow cytometry, MPO activity and NO production was evaluated by fluorescence assay for HL- 60 cells, mitochondrial activity by XTT assay, and the global DNA methylation was checked by HPLC-PDA. DNA adducts 8-oxodG, CEdG, 1,N6-εdA, 1,N6-εdG and BPA-Gua were quantified by HPLC- ESI-MS/MS in DNA of cells, culture medium, urine and tissue collected from Sprague-Dawley rats. Blood count and bone marrow examination were obtained in collaboration with Experimental Hematology Laboratory of University of Sao Paulo We observed that both BPA and BPANO2 induced ROS generation in HL- 60 cells after 2 hours of incubation. BPA subsequently led to failure of mitochondrial respiratory chain activity, increased permeability of the plasma membrane, DNA fragmentation, arrest in G2/M phase of cell cycle, DNA hypermethylation with global hipohydroxymethylation. We saw low cytotoxicity in HL-60 cells induced by nitro-bpa n the same concentration, without loss of mitochondrial respiratory chain activity, discrete DNA fragmentation, but leading cell cycle to stopping at G0/G1 phase, and induction of DNA global hypermethylation. No lesions were observed in the DNA of HL-60 cells.The results obtained from the exposure of HL-60 cells to BPA and nitro-BPA indicate that these two molecules induce different metabolic abnormalities in this cell line, independent of estrogen pathway, leading to changes in epigenetic (BPA) or genetic and epigenetic (nitro-BPA) profile, that can induce phenotypic consequences such as malignant progression. It was observed along the study that MCF-7 cells are more resistant than HL-60 cells to cell damage induced by BPA and nitro-BPA. As a result of MCF-7 cells exposure to BPA, we saw a slight increase in membrane permeability (250 mM), ROS generation after 24h (25 mM) and increase in cell population in sub G1, so we had DNA fragmentation (100 uM and 250 uM), but with no effect on cell cycle. However, we observed increased levels of CEdG and 8-oxodG on DNA of cells incubated with BPA (100 uM, 250 mM) without prior activation of Ah receptors. The activation of Ah receptors with PCB took a small increase in the level of DNA lesions after incubations with BPA. MCF-7 cells resistance to the cytotoxic effects of BPA is probably related to estrogen action of this compound. Estrogen signaling in addition with the increased levels of DNA damage increases the chance of mutations and malignant transformation. In cells with Ah receptor activation, BPA also led to increased DNA global hydroxymethylation, without changing the global DNA methylation. Nondiabetic animals exposed to BPA had decreased amounts of promyelocytes, blasts and rods in the bone marrow, with any change in blood count. There were an increase of CEdG levels in liver, methylation and global hydroxymethylation on hepatic DNA, and was observed any alteration on epigenetic markers and DNA adducts in kidney or urine. On the other hand diabetic animals exposed to BPA showed increased numbers of eosinophils and lymphocytes in bone marrow, suggesting the induction of an allergic inflammatory state. There were increased levels of CEdG, methylation and global hydroxymethylation on hepatic DNA, increased 8-oxodG levels on kidney DNA without changing epigenetic markers, was not observed DNA adducts in urine. The data obtained indicate that the generation of ROS could be the major route of cytotoxic and genotoxic induced by BPA exposure. BPA biotransformation to BPA-3,4-quinone used in the models seem to have poor effects, since we was not detected BPA-Gua lesion in any DNA sample, culture medium of cells or urine of the animals. Metabolic changes induced by BPA and ROS can enable changes in epigenetic markers observed in the DNA of HL-60 cells, MCF-7 and liver tissue. All these changes may contribute to malignant transformation of cells that were exposed to BPA
Subject(s)
Animals , Rats , Rats/classification , Bisphenol A-Glycidyl Methacrylate , HL-60 Cells/cytology , Epigenesis, Genetic , MCF-7 Cells/cytology , Polychlorinated Biphenyls/agonists , Diabetes MellitusABSTRACT
@#To determine the effects of celastrol on the proliferation and apoptosis of cell lines HL-60 and Jurkat in human leukemia. Human leukemia cell lines were treated with celastrol at different concentrations. The inhibitory rate of cell proliferation was detected by MTT assay; the apoptosis rate was detected by AnnexinV-FITC/PI double staining; cell cycle was observed by PI staining; cell morphology was observed by transmission electron microscope(TEM). Cell proliferation was inhibited by celastrol, with IC50 of(0. 46±1. 05)μmol/L and(0. 88±1. 13)μmol/L at 24 h. Celastrol induced cell apoptosis in a dose-dependent manner. The cell cycle distribution of G1 phase rate increased, S phase rate decreased(P< 0. 05)with typical cell apoptosis-induced morphological changes. Results showed that celastrol could significantly inhibit cell proliferation and induce apoptosis in human leukemia cell lines of HL-60 and Jurkat.
ABSTRACT
Differentiated HL-60 is an effector cell widely used for the opsonophagocytic-killing assay (OPKA) to measure efficacy of pneumococcal vaccines. We investigated the correlation between phenotypic expression of immunoreceptors and phagocytic ability of HL-60 cells differentiated with N,N-dimethylformamide (DMF), all-trans retinoic acid (ATRA), or 1alpha, 25-dihydroxyvitamin D3 (VitD3) for 5 days. Phenotypic change was examined by flow cytometry with specific antibodies to CD11c, CD14, CD18, CD32, and CD64. Apoptosis was determined by flow cytometry using 7-aminoactinomycin D. Function was evaluated by a standard OPKA against serotype 19F and chemiluminescence-based respiratory burst assay. The expression of CD11c and CD14 gradually increased upon exposure to all three agents, while CD14 expression increased abruptly after VitD3. The expression of CD18, CD32, and CD64 increased during differentiation with all three agents. Apoptosis remained less than 10% until day 3 but increased after differentiation by DMF or ATRA. Differentiation with ATRA or VitD3 increased the respiratory burst after day 4. DMF differentiation showed a high OPKA titer at day 1 which sustained thereafter while ATRA or VitD3-differentiated cells gradually increased. Pearson analysis between the phenotypic changes and OPKA titers suggests that CD11c might be a useful differentiation marker for HL-60 cells for use in pneumococcal OPKA.