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1.
Braz. j. med. biol. res ; 54(2): e9173, 2021. tab, graf
Article in English | ColecionaSUS, LILACS, ColecionaSUS | ID: biblio-1142586

ABSTRACT

This study aimed to explore the correlation of kinesin family member 2A (KIF2A) expression with disease risk, clinical characteristics, and prognosis of acute myeloid leukemia (AML), and investigate the effect of KIF2A knockdown on AML cell activities in vitro. Bone marrow samples were collected from 176 AML patients and 40 healthy donors, and KIF2A expression was measured by real-time quantitative polymerase chain reaction. Treatment response, event-free survival (EFS), and overall survival (OS) were assessed in AML patients. In vitro, KIF2A expression in AML cell lines and CD34+ cells (from healthy donors) was measured, and the effect of KIF2A knockdown on AML cell proliferation and apoptosis in HL-60 and KG-1 cells was detected. KIF2A expression was greater in AML patients compared to healthy donors, and receiver operating characteristic curve indicated that KIF2A expression predicted increased AML risk (area under curve: 0.793 (95%CI: 0.724-0.826)). In AML patients, KIF2A expression positively correlated with white blood cells, monosomal karyotype, and high risk stratification. Furthermore, no correlation of KIF2A expression with complete remission or hematopoietic stem cell transplantation was found. Kaplan-Meier curves showed that KIF2A expression was negatively correlated with EFS and OS. In vitro experiments showed that KIF2A was overexpressed in AML cell lines (KG-1, HL-60, ME-1, and HT-93) compared to CD34+ cells, moreover, cell proliferation was reduced but apoptosis was increased by KIF2A knockdown in HL-60 and KG-1 cells. In conclusion, KIF2A showed potential to be a biomarker and treatment target in AML.


Subject(s)
Humans , Male , Female , Adult , Middle Aged , Leukemia, Myeloid, Acute/diagnosis , Leukemia, Myeloid, Acute/genetics , Kinesin/genetics , Biomarkers, Tumor/genetics , Survival Rate , Risk Factors , Apoptosis , HL-60 Cells , Cell Proliferation , Gene Knockdown Techniques
2.
Braz. j. med. biol. res ; 54(2): e9869, 2021. tab, graf
Article in English | ColecionaSUS, LILACS, ColecionaSUS | ID: biblio-1142585

ABSTRACT

Severe blockage in myeloid differentiation is the hallmark of acute myeloid leukemia (AML). Trdmt1 plays an important role in hematopoiesis. However, little is known about the function of Trdmt1 in AML cell differentiation. In the present study, Trdmt1 was up-regulated and miR-181a was down-regulated significantly during human leukemia HL-60 cell differentiation after TAT-CT3 fusion protein treatment. Accordingly, miR-181a overexpression in HL-60 cells inhibited granulocytic maturation. In addition, our "rescue" assay demonstrated that Trdmt1 3′-untranslated region promoted myeloid differentiation of HL-60 cells by sequestering miR-181a and up-regulating C/EBPα (a critical factor for normal myelopoiesis) via its competing endogenous RNA (ceRNA) activity on miR-181a. These findings revealed an unrecognized role of Trdmt1 as a potential ceRNA for therapeutic targets in AML.


Subject(s)
Humans , Leukemia, Myeloid, Acute/genetics , MicroRNAs/genetics , DNA (Cytosine-5-)-Methyltransferases/genetics , Cell Differentiation , HL-60 Cells
3.
Journal of Experimental Hematology ; (6): 1775-1779, 2021.
Article in Chinese | WPRIM | ID: wpr-922333

ABSTRACT

OBJECTIVE@#To explore the regulatory function of RNA binding motif protein 38 (RBM38) in human acute myeloid leukemia cells HL-60 and its mechanism.@*METHODS@#The lentivirus carriers of overexpressed and knockdown RBM38 were constructed. After HL-60 cells were transfected, Western blot was used to analyze the expression level of RBM38 in HL-60 cells. The cell proliferation and cycle of HL-60 were detected by CCK-8 assay and flow cytometry assay, respectively. RNA immunoprecipitation coupled real-time PCR (RIP-qPCR) was used to detect the combination of RBM38 with mRNAs. Actinomycin D treatment followed by real-time PCR (AcD-qPCR) was used to detect the effect of RBM38 on the stability of target mRNAs.@*RESULTS@#RBM38 in HL-60 cells was overexpressed or inhibited by lentivirus transduction. Overexpressed RBM38 promoted the cell cycle and proliferation of HL-60, while RBM38 knockdown repressed the two processes. RBM38 showed an interaction with FZD1 mRNA and enhancement of its stability.@*CONCLUSION@#RBM38 can regulate cell proliferation of HL-60 by improving the stability of FZD1 mRNA.


Subject(s)
Cell Proliferation , Frizzled Receptors , HL-60 Cells , Humans , Leukemia, Myeloid, Acute , RNA Stability , RNA-Binding Proteins/metabolism
4.
Journal of Experimental Hematology ; (6): 1019-1027, 2021.
Article in Chinese | WPRIM | ID: wpr-888513

ABSTRACT

OBJECTIVE@#To detect the expression of different transcripts of lactamase β(LACTB) gene in leukemic cell lines.@*METHODS@#NCBI website and DNAstar software were used to detect the Bioinformatics analysis of LACTB. The expression of different transcripts of LACTB gene in leukemic cell lines (THP-1, HL60, K562, U937, Jurkat and Raji) was detected by reverse transcription PCR (RT-PCR), DNA and clone sequencing; the expression of different transcripts of LACTB gene in leukemic cell lines was detected by Quantitative Real-time PCR.@*RESULTS@#There were a variety of splicing isomers in LACTB, and it could produce a variety of protein isomers with conserved N-terminal and different C-terminal, moreover, there were many splice isoforms of LACTB in leukemia cell lines, and there were different expression patterns in different cell lines, including XR1, V1, V2 and V3. The expression of total LACTB showed high in HL60 cells, while low in Raji cells, and the difference was statistically significant (P<0.05). The V1 was high expression in U937 cells but low in Raji cells, and the difference was statistically significant (P<0.05). V2 was high expression in HL60 cells but lowly in Raji cells, and the difference was statistically significant (P<0.05). The expression of V3 was low in THP-1 cells, which was significantly different as compared with that in normal bone marrow (P<0.05).@*CONCLUSION@#The reaserch found that there are many splice isomers of LACTB in leukemic cell lines, and there are different expression patterns in different cell lines.


Subject(s)
Alternative Splicing , HL-60 Cells , Humans , Leukemia/genetics , Membrane Proteins/genetics , Mitochondrial Proteins/genetics , RNA Splicing , U937 Cells , beta-Lactamases/genetics
5.
Article in Chinese | WPRIM | ID: wpr-880133

ABSTRACT

OBJECTIVE@#To investigate the effect of V-9302 (an antagonist of transmembrane glutamine flux) on the proliferation and apoptosis of acute myeloid leukemia cells HL-60 and KG-1.@*METHODS@#HL-60 and KG-1 cells at logarithmic phase were treated by different concentrations of V-9302. CCK-8 assay was used to detect the proliferation of the cells. Annexin V-FITC / PI double staining flow cytometry was used to detect the apoptosis of HL-60 and KG-1 cells. The expressions of BAX, BCL-2 and Caspase3 were detected by RT-qPCR and Western blot.@*RESULTS@#V-9302 could significantly inhibit the growth of HL-60 and KG-1 cells. The concentration of V-9302 at 10, 20 μmol/L could significantly promote the apoptosis of HL-60 and KG-1 cells(P<0.05). The results of apoptosis related gene detection showed that when V-9302 was applied to HL-60 and KG-1 cell lines at 10 and 20 μmol/L, the expression levels of Pro-apoptotic protein genes BAX and Caspase3 in HL-60 and KG-1 were significantly higher than those in control group (P<0.05), while the expression level of anti-apoptotic protein gene BCL-2 was significantly lower than that in the control group (P<0.05). The results of Western blot were basically consistent with that of RT-qPCR.@*CONCLUSION@#Competitive antagonist of transmembrane glutamine flux V-9302 can significantly promote the apoptosis of acute myeloid leukemia cell lines HL-60 and KG-1.


Subject(s)
Apoptosis , Cell Proliferation , Glutamine , HL-60 Cells , Humans , Leukemia, Myeloid, Acute
6.
Article in Chinese | WPRIM | ID: wpr-880094

ABSTRACT

OBJECTIVE@#To analyze the relationship of the expression of transcription factor MYB targeted regulation by miR-96 to cell invasion and apoptosis in pediatric acute myeloid leukemia (AML).@*METHODS@#A total of 65 children with AML in The 928 Hospital of PLA Joint Logistics Support Forces from January 2017 to November 2019 were selected, including 35 cases diagnosed as primary AML and 30 cases as complete remission AML. Thirty children with immune thrombocytopenia were selected as control group. The clinical characteristics were analyzed and compared between the two groups. The levels of miR-96 and MYB in peripheral blood samples were detected by qRT-PCR and compared between the two groups. The miR-96 mimics and its negative control (NC), inhibitor-miR-96 and its NC transfected HL60 cells induced by liposome (Lipofectamine 2000), respectively, Then the expression levels of MYB were detected with Western blot and compared among four HL60 cell groups. The invasion ability of four HL60 cell groups were detected with Transwell assay. The cell proliferation ability of four HL60 cell groups were detected with MTT at 24 h, 48 h, and 72 h, respectively. The apoptosis rates of four HL60 cell groups were detected with flow cytometry.@*RESULTS@#Compared with control group, the level of miR-96 in AML children were higher, but MYB lower (P0.05). The promotion of over-expression level of miR-96 on the invasion ability of HL 60 cells was confirmed by Transwell assay. MTT assay showed that miR-96 could promote the proliferation of HL60 cells, inhibit the apoptosis of HL60 cells, and the effect was time-dependent manner (r=0.804). The inhibition of miR-96 on HL60 cells apoptosis was also confirmed with flow cytometry.@*CONCLUSION@#MiR-96 has significant negative effect on invasion and apoptosis of AML cells by targeting regulation MYB, and it might be a potential novel strategy for pediatric AML treatment.


Subject(s)
Apoptosis , Cell Line, Tumor , Cell Proliferation , Child , HL-60 Cells , Humans , Leukemia, Myeloid, Acute/genetics , MicroRNAs/genetics , Proto-Oncogene Proteins c-myb
7.
Article in Chinese | WPRIM | ID: wpr-880089

ABSTRACT

OBJECTIVE@#To investigate the influence of GPT2(glutamic pyruvate transaminase 2)to biological characteristics of human acute myeloid leukemia cell line HL-60.@*METHODS@#The expression of GPT2 in hematological tumor and AML cell was detected. The lentvirus-mediated of short-hairpin RNA (shRNA) was constricted, and the knock-down efficiency of HL-60 in AML cell after infected by lentvirus-mediated was detected by Western blot and Q-PCR. CCK-8 assay and soft agar colony formation assay were used to detect the effect of GPT2 gene deletion to the cell proliferation potential. Fluorescence activated cell sorting(FACS) was used to analyze the effect of gene deletion to the cell cycle and Caspase 3/7 Activity Assay Kit was used to analyze the effect of GPT2 gene deletion to the cell apoptosis.@*RESULTS@#GPT2 showed mRNA high expression in AML patients. CCK-8, soft agar assay, and Caspase 3/7 Activity Assay Kit results showed that compared with shCtrl group, the cells in shGPT2-1、shGPT2-2、shGPT2-3 group showed the slowing down on proliferation, decreasing on colony ability, and the apoptosis of the cells was increasing significantly. FACS showed that GPT2 gene was related to the cycle of HL-60 cell.@*CONCLUSION@#GPT2 appears to involve the proliferation, cycle distribution and apoptosis of AML cell HL-60. The deletion of GPT gene can lead to the inhibitation of cells proliferation and increase apoptosis.


Subject(s)
Apoptosis , Cell Proliferation , HL-60 Cells , Humans , Leukemia, Myeloid, Acute , Pyruvates , Transaminases
8.
Article in Chinese | WPRIM | ID: wpr-880032

ABSTRACT

OBJECTIVE@#To explore the effects and mechanisms of PKC412 inhibitor on proliferation and apoptosis of HL-60 cell line.@*METHODS@#CCK-8 assay was used to detect the effect of PKC412 on the proliferation of HL-60 cells at different concentrations; Wright-Giemsa staining was used to estimated the effect of PKC412 on the apoptosis of HL-60 cells; the mRNA expression of BCL-2 and P53 genes was detected by qRT-PCR, the expression of BCL-2 and P53 proteins was detected by Western blot. HL-60 cells were injected into mouse caudal vein to construct acute myeloid leukemia model, PKC412 was administered to tail vein for 31.25 nmol/kg, normal saline was injected into the same site of the mice as control group, and the inhibitory effect of PKC412 on HL-60 cells in mice was observed. ELISA assay was used to detect the effect of PKC412 on the inflammatory factors of TNF-α and TGF-β in tumor mice.@*RESULTS@#PKC412 could inhibit the proliferation of HL-60 cell, which was in a dose dependent manner(r=0.9973) (IC50 was 0.31 μmol/L), and induce apoptosis of HL-60 cells. After HL-60 cell was treated by PKC412 for 48 h the expression of BCL-2 gene was down regulated(0.417±0.044 vs 0.933±0.033, t=9.347, P0.05) as compared with control group. And the expression of BCL-2 protein was decreased, while the expression of P53 protein was increased. PKC412 could inhibited the growth of HL-60 tumor cells in vivo, the survival rate of mice after administration was 50% and the weight was increased as compared with that in control group(18.02±0.403 g vs 16.44±0.562 g, t=2.272, P=0.0356). The secretion of TNF-α and TGF-β cytokine in serum and spleen cells in PKC412 group was significantly lower than that in control group (P<0.05).@*CONCLUSION@#PKC412 can induce apoptosis of HL-60 cells by inhibiting the expression level of BCL-2 gene, PKC412 administration in vivo can inhibit the growth of the tumors.


Subject(s)
Animals , Apoptosis , Cell Proliferation , HL-60 Cells , Humans , Leukemia, Myeloid, Acute , Mice , Proto-Oncogene Proteins c-bcl-2 , Staurosporine/analogs & derivatives
9.
Article in Chinese | WPRIM | ID: wpr-880027

ABSTRACT

OBJECTIVE@#To investigate the effect of norcantharidin (NCTD) to proliferation of leukemia cells through disrupting key regulators of sonic Hedgehog (SHH) pathway and its downstream transcription factor SOX2.@*METHODS@#CCK8 was used to detected the HL60 and NB4 cells after inhibited by NCTD, SMO and GLI1 inhibitor for 24 hours. Expression level of SMO, GLI1 and SOX2 in HL60 cells with NCTD treatment was detected by immunoblot. HL60 cells were transfected with pcDNA3.1 plasmid expressing GLI1 or SOX2. Empty vector and pcDNA3. 1-EGFP were divided into negative and positive control group, respectively. The expression of exogenous GLI1 or SOX2 in HL60 cells was confirmed by immunoblot, and growth curve of HL60 cell was checked by CCK8. Proliferation of genetic modified HL60 cells treated by various dose of NCTD was detected.@*RESULTS@#NCTD, SMO/GLI1 inhibitors could inhibit the proliferation of NB4 and HL60 cells in a dose-dependent manner. Compared with solvent (DMSO)-treated control group, NCTD remarkably decreased protein level of SMO, GLI1 and SOX2. GLI1 and SOX2 were overexpressed in HL60 cells as compared with pcDNA3.1 empty vector-transfected group. Growth curve demonstrated significant proliferative advantage of GLI1/SOX2-transfected cells. CCK8 assay indicated that GLI1/SOX2-overexpressed HL60 cells were more resistant to NCTD treatment.@*CONCLUSION@#NCTD attenuates HL60 proliferation via targeting the Hedgehog/SOX2 axis.


Subject(s)
Bridged Bicyclo Compounds, Heterocyclic , Cell Proliferation , HL-60 Cells , Hedgehog Proteins , Humans , Leukemia, Myeloid, Acute , SOXB1 Transcription Factors , Zinc Finger Protein GLI1
10.
Article in Chinese | WPRIM | ID: wpr-829038

ABSTRACT

OBJECTIVE@#To explore the molecular mechanism by which miR-218 targeting Bmi-1 inhibits the proliferation of acute promyelocytic leukemia (APL) cells.@*METHODS@#APL cell line HL-60 was transfected by miR-218 and RNA-negative control sequences, respectively. The expression of miR-218 in cells was detected by real-time fluorescence quantitative PCR. The effect of transfected miR-218 on the proliferation of APL cells was detected by MTT assay. Cell apoptosis was detected by flow cytometry. The regulation effect of miR-218 on Bmi-1 expression was determined by Western blot. The correlation of miR-218 expressions with Bmi-1 was analyzed by Spearman test. The targeted relationship between miR-218 and Bmi-1 was verified by luciferase assay.@*RESULTS@#MTT assay showed that the proliferation of HL-60 cells in vitro was inhibited by high expression miR-218 significantly. Flow cytometry showed that the G1 and G2 phase cells increased while the S phase cells decreased after transfected by miR-218. Western blot showed that the level of Bmi-1 protein in HL-60 cells decreased significantly after transfection of miR-218 (P<0.05). Spearman correlation analysis showed that the mRNA level of miR-218 negatively correlated with the protein content of Bmi-1 (r=-0.326, P<0.01). Luciferase assay indicated that Bmi-1 could targeted on miR-218 directly.@*CONCLUSION@#miR-218 can inhibit the proliferation, metastasis and invasion of APL cells, which can be related with the down-regulated of Bmi-1.


Subject(s)
Apoptosis , Cell Proliferation , Gene Expression Regulation, Neoplastic , HL-60 Cells , Humans , Leukemia, Promyelocytic, Acute , Genetics , MicroRNAs , Genetics , Polycomb Repressive Complex 1 , Genetics
11.
Article in Chinese | WPRIM | ID: wpr-829035

ABSTRACT

OBJECTIVE@#To investigate the effects of high dose vitamin C on proliferation and apoptosis of acute myeloid leukemia (AML) cell lines including HL-60, U937 and primary CD34 leukemia cells in AML.@*METHODS@#CD34 cells were sorted by using immunomagnetic cell sorting system, then the primary CD34 leukemia cells, including HL-60 and U937 cell lines were cultured in vitro. Cells in each group were treated with different concentrations of vitamin C, the survival rate of cells was determined by MTT assay, the apoptosis rate of cells was evaluated by Annexin V/PI double staining, the expression of apoptotic proteins-including cleaved caspase 3, cleaved caspase-9 and cleaved PARP were detected by Western blot.@*RESULTS@#The proliferation of HL-60 and U937 cells could be inhibited by high dose vitamin C, which showed a concentration-dependent manner (r=-0.9664; r=-0.9796). HL-60 and U937 cells were treated with different concentrations of vitamin C (8 and 20 mmol/L) for 24 hours, respectively, it was found that with the increasing of vitamin C concentration, cell apoptosis rate was significantly increased (r=0.9905; r=0.9971), and the expression of apoptosis related proteins including cleaved caspase 3, cleaved caspase-9 and cleaved PARP was aslo significantly increased with the increasing of concentration. In addition, it was found that with or without the mutation of TET2, high dose vitamin C could inhibit the proliferation (r=-0.9719; r=-0.9699) and promote the apoptosis (r=0.9998; r=0.9901) of primary CD34 leukemia cells in AML, which showed a dose-dependent manner, but it showed no effect on the proliferation (r=-0.2032) and apoptosis (r=0.1912) of normal CD34 cells.@*CONCLUSION@#High dose vitamin C can inhibit the proliferation and promote the apoptosis of acute myeloid leukemia cells, and selectively kill primary CD34 leukemia cells in AML.


Subject(s)
Apoptosis , Ascorbic Acid , Cell Proliferation , HL-60 Cells , Humans , Leukemia, Myeloid, Acute , U937 Cells
12.
Journal of Experimental Hematology ; (6): 1826-1830, 2020.
Article in Chinese | WPRIM | ID: wpr-879978

ABSTRACT

OBJECTIVE@#To investigate the regulatory effects of Olaparib on natural killer cell activating receptor (NKG2D) ligands expression on human acute myeloid leukemia (AML) cell line HL-60, and to explore the molecular mechanism of Olaparib on HL-60 cells.@*METHODS@#After HL-60 cells in logarithmic growth phase were treated with Olaparib at different concentrations for different times (24, 48 h), the expression of NKG2D ligand on the surface of HL-60 cells was detected by flow cytometry. Western blot was used to dectect the expression of ERK expression in HL-60 cells. The killing effect of NK cells to HL-60 cells was detected by CFSE/PI method.@*RESULTS@#10 μmol/L Olaparib could upregulate the expression of NKG2D ligand on the surface of HL-60 cell at 24 and 48 hours, while 5 μmol/L Olaparib could induce up-regulation of the expression of ULBP-2 and ULBP-3 at 48 hours. Western blot analysis showed that ERK phosphorylation of HL-60 cells was enhanced after treating with Olaparib. The killing effect of NK cells to HL-60 cells could be enhanced by Olaparib, however, ERK inhibitor could suppress the killing effect of NK cells to HL-60 cells.@*CONCLUSION@#Olaparib can upregulate NKG2D ligands expression on the surface of HL-60 cells and enhance the cytotoxicity of NK cell to HL-60 cells. The mechanism may be related to Olaparib promoting ERK phosphorylation expression.


Subject(s)
Cell Line, Tumor , Cytotoxicity, Immunologic , HL-60 Cells , Histocompatibility Antigens Class I , Humans , Ligands , NK Cell Lectin-Like Receptor Subfamily K , Phthalazines , Piperazines , Poly(ADP-ribose) Polymerase Inhibitors
13.
Electron. j. biotechnol ; 39: 1-7, may. 2019. graf
Article in English | LILACS | ID: biblio-1051553

ABSTRACT

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.


Subject(s)
Apoptosis , HL-60 Cells/metabolism , Juglans/chemistry , Polyphenols/metabolism , Antioxidants/metabolism , Cell Survival , Chromatography, High Pressure Liquid , Cell Culture Techniques , Membrane Potential, Mitochondrial
14.
Journal of Experimental Hematology ; (6): 1736-1741, 2019.
Article in Chinese | WPRIM | ID: wpr-781404

ABSTRACT

OBJECTIVE@#To explore the role of bone marrow niche in the chemotherapy resistance of patient with acute myeloid leukemia (AML), and to investigate the effects of the MSCs on the apoptosis of HL-60 cell and its underlying mechanisms.@*METHODS@#MSCs were derived from the bone marrow of newly diagnosed AML patients (AML-MSCs) and health donors(MSCs) were co-cultured with HL-60 cells respectively. The apoptosis of HL-60 cells in the presence/absence of MSCs and/or Daunorubicin were determined by flow cytometry with Annexin V/PI double staining. In addition, the morphological features of HL-60 cells were observed by Wright-Giemsa staining, and the ratio of blasts and differentiated cells were counted. Furthermore, the expressions of apoptosis-related factors including Caspase-3, Caspase-8,Caspase-9 and Survivin were detected by Western blot.@*RESULTS@#The flow cytometry showed that there was no significant change in apoptosis of HL-60 cells co-cultured with MSC derived from healthy donors or AML patients. After adding Daunorubicin into different cultural systems, the apoptotic rates of HL-60, HL-60 co-cultured with normal MSCs and HL-60 co-cultured with AML-MSCs were (49.57±7.44)%, (30.72±4.05)% and (22.99±4.08)%, respectively, which showed that normal MSCs and AML-MSCs could remarkably supress Daunorubicin-induced HL-60 apoptosis, however, there was no statistically significant difference of apoptosis between HL-60 co-cultured with normal MSCs and HL-60 co-cultured with AML-MSCs. Wright-Giemsa staining showed that most of the HL-60 cells co-cultured with AML-MSCs were primitive, and cell differentiation was unusual. In AML-MSCs co-cultured group, the cell apoptosis and differentiation caused by DNR was significant decreased, and most of HL-60 cells were initial. Western blot showed that the cleavage activity of Caspase-3 of HL-60 in AML-MSCs and normal MSCs co-cultured group was decreased, compared with HL-60 in single cultured group, moreover, the decrease was significantly in AML-MSC group. Additionally, the expression of survivin in AML-MSCs and normal MSCs co-cultured group was increased, compared with that in single cultured group, and increase was significant in AML-MSCs group.@*CONCLUSION@#MSCs can suppress Daunorubicin-induced HL-60 apoptosis via inhibiting Caspase-3 and maintaining survivin level.


Subject(s)
Apoptosis , Bone Marrow Cells , Caspase 3 , Cell Proliferation , Daunorubicin , HL-60 Cells , Humans , Leukemia, Myeloid, Acute , Mesenchymal Stem Cells , Survivin
15.
Journal of Experimental Hematology ; (6): 1083-1087, 2019.
Article in Chinese | WPRIM | ID: wpr-775760

ABSTRACT

OBJECTIVE@#To investigate the differentiation of acute promyelocytic leukemia (APL) cells induced by adenosine targeting Prx III.@*METHODS@#HL-60 cells were divided into four groups: control group, all-trans retinoic acid (ATRA) group, adenanthin group and ATRA+adenanthin group. Cell morphologic changes were observed under optical microscope. The influence of adenanthin on the differentiation of HL-60 was observed by nitro blue tetrazolium chloride (NBT) test. Cell surface differentiation antigens CD11b expression was measured by flow cytometry. The protein expression of Prx III was detected by immunohistochemical assay.@*RESULTS@#Adenanthin could induce the differentiation of HL-60 cells; the NBT reduction positive rate in ATRA+adenanthin group was significantly higher than that in ATRA group and adenanthin group (P<0.05). The percentage of CD11b positive cells in ATRA+adenanthin group (43.62%±1.38%) was higher than that in adenanthin group (28.15%±1.78%), ATRA group (36.72%±1.33%) and control group (7.99%±1.78%) (P<0. 05). The content of Prx Ⅲ protein in adenanthin group was significantly higher than that in control group and ATRA group (P<0.05).@*CONCLUSION@#Adenanthin and ATRA have a synergistic effect on the differentiation and maturation of HL-60 cells, and its mechanism may be related with regulation of Prx III expression.


Subject(s)
Cell Differentiation , Diterpenes, Kaurane , HL-60 Cells , Humans , Leukemia, Promyelocytic, Acute , Peroxiredoxin III , Tretinoin
16.
Journal of Experimental Hematology ; (6): 1259-1264, 2019.
Article in Chinese | WPRIM | ID: wpr-775731

ABSTRACT

OBJECTIVE@#To explore the role of bone marrow microenvironment(niche) in the development of acute myeloid leukemia (AML) and the effect of AML patients-derived MSC on the proliferation, cell cycle and immuno-phenotypes of HL-60 cells.@*METHODS@#The MSC derived from bone marrow of patients with newly diagnosed AML were isolated and co-cultured with HL-60 cells. The effect of MSC on proliferation of HL-60 cells was detected by using 3H-TdR incorporation method, the cell cycle and immunophenotypes of HL-60 cells were detected by flow cytometry.@*RESULTS@#The results of 3H-TdR incorporation assay showed that both AML-MSCs and normal MSCs remarkably suppressed the HL-60 cell proliferation in a time- and dose-dependent manner. The results of cell cycle analysis demonstrated that AML MSCs and normal MSCs induced arrest of the HL-60 cells in G/G phase. The results of immunophenotyping revealed that MSCs suppressed the expression of CD11a and CD154 on the surface of HL-60 cells. Moreover, AML MSCs exhibited increased inhibitory effects than that of normal MSCs. However, no remarkable effect of MSCs on CD54 expressions of HL-60 cells was observed in the current study.@*CONCLUSION@#AML-MSCs possess effects on HL-60 cell proliferation, cell cycle and immunophenotypes similiar to normal MSCs, but exhibited increased suppressive capacity on the expression of CD11a and CD154.


Subject(s)
Bone Marrow Cells , Cell Cycle , Cell Proliferation , HL-60 Cells , Humans , Immunophenotyping , Leukemia, Myeloid, Acute , Mesenchymal Stem Cells , Tumor Microenvironment
17.
Journal of Experimental Hematology ; (6): 1424-1430, 2019.
Article in Chinese | WPRIM | ID: wpr-775704

ABSTRACT

OBJECTIVE@#To investigate the relationship of miR-140 expression level with the therapeutic effect of decitabine, and to explore whether the molecular mechanism is dependent on the regulation of TLR4 expression.@*METHODS@#Forty-seven patients with acute myeloid leukaemia (AML) were enrolled in our study and divided into decitabine combination treatment group (22 cases) and traditional treatment group (25 cases). The clinical efficacy was compared between these two groups. Real-time PCR was used to determine the plasma level of miR-140 in AML patients. Decitabine, miR-140 mimic and miR140 inhibitor were used to treat AML HL-60 cells in vitro, the real-time PCR and Western blot were used to detect the expressions of miR-140, TLR4 and NF-κB at both mRNA and protein levels.@*RESULTS@#Compared with traditional treatment group, decitabine combination treatment group showed more significant clinical efficacy. Plasma miR-140 level in both 2 treatment groups both decreased, but the plasma miR-140 level was higher in decitabine combination treatment group as compared with traditional treatment group. Experiment in vitro showed that 0.3 μmol/L decitabine significantly inhibited the HL-60 cell proliferation accompanied by up-regulation of miR-140 expression and down-regulation of expression of TLR4 and NF-κB. These effects induced by decitabine were partly reversed by pretreating the cells with 200 nmol/L miR-140 inhibitor.@*CONCLUSION@#Decitabine-induced up-regulation of miR-140 expression may be related with its chemotherapeutic effects, and miR-140/TLR4/NF-κB pathway may partly mediate the pharmacologic action of decitabine.


Subject(s)
Decitabine , Therapeutic Uses , Down-Regulation , HL-60 Cells , Humans , Leukemia, Myeloid, Acute , Drug Therapy , MicroRNAs , NF-kappa B
18.
Article in Chinese | WPRIM | ID: wpr-774302

ABSTRACT

OBJECTIVE@#To study the effect of traditional chinese medicine (TCM) compound on myeloid leukemia cells and to explore its anti-leukemic mechanism.@*METHODS@#Myeloid leukemia cell lines were cultured in vitro and treated with TCM compound. The proliferation of the leukemia cells was measured by CCK8 method. The differentiation of the leukemia cells was evaluated by using Wright's staining method and by light microscopy, and the expression of differentiation-related surface antigens such as CD11B was measured and by flow cytometry, the apoptosis of the leukemia cells was detected by flow cytometry with using Annexin V staining.@*RESULTS@#Compared with untreated 4 leukemia cell lines HL-60, MOLM-13, MV4-11, AML-M5, the proliferations of 4 leukemia cells treated with different concentrations of TCM compound decreased (P<0.05), and their proliferation inhibition were in a dose-dependent manner (r=0.9236; r=0.7488; r=0.8889; r=0.8119); compared with HL-60 and AML-M5 leukemia cells, the drug-treated 2 leukemia cells displayed obvious differentiated changes; compared with untreated HL-60 leukemia cell line, the expression of surface antigen CD11B increased by 85%±7.13% in HL-60 cells treated IC50 concentration of drug; compared with untreated AML-M5 leukemia cell line, the apoptotic rate of AML-M5 treated with 1.5 and 2 μl doses of TCM compound increased. (P<0.05).@*CONCLUSION@#The traditional chinese medicine compound may inhibit the proliferation of leukemia cell lines mainly by inducing leukemia cell differentiation and apoptosis.


Subject(s)
Apoptosis , Cell Differentiation , Cell Proliferation , HL-60 Cells , Humans , Leukemia, Myeloid, Acute
19.
Article in Chinese | WPRIM | ID: wpr-774301

ABSTRACT

OBJECTIVE@#To investigate the effect of targeting the silent information regulator 2 hemolog 2 (SIRT2) expression on the apoptosis of drug-resistant AML cell line HL-60/A and its mechanism.@*METHODS@#The expression of SIRT2 and antophagy-related protein LCT, P62 in HL-60/A and HL-60 was detected by Western blot, the effect of cytorabine on the apoptosis of HL-60/A cells was detected by using Annexin V/PI double staining after targeting inhibition of SIRT2 expression resulting from transfecting HL-60/A cells with SiRNA. The Western blot and transmission electray microscopy were used to detect the cell autophagy. To further clarify the role of autophragy in the regulatory effect of SIRT2 on the drug-resistance of HL-60/A cells, the autophagy-specific agonist, repamycin, was added into the cell culture medium after SIRT2-siRNA transfection. Then, the autophagy and apoptosis of HL-60/A were detected, respectively.@*RESULTS@#The SIRT2 protein expression obviously increased in HL-60/A cells than that in HL-60 cells. Moreover, the expression rate of LC3II/I was higher, but P62 expression was lower in HL-60/A cells. After siRNA successfully transfecting into HL-60/A cells, quantitative PCR and Western blot should that the expression of SIRT2 significantly decreased. Meawhile, Western blot showed that the expression of LC3 II/I decreased, but P62 increased. Meanwhile, By TEM found that the number of autophagosome also decreased, suggesting the autophagy was inhibited after down-regulation of SIRT2. In addition, the drug senstivity of HL-60/A cells to cytarabine in siRNA-transfection group increased, and the apoptotic rate detected by Annexin V/PI double staining significantly increased. However, after co-culture with rapamycin, the suppressed autophagy in siRNA-trasfect HL-60/A cells was activated, leading to the reappearance of drug resistance of cells to cytarabine, and more significantly decrease of apoptotic rate.@*CONCLUSION@#The high expression of SIRT2 in HL-60/A cells activates the protective autophagy mechanism, which closely related with drug resistance.


Subject(s)
Apoptosis , Autophagy , Drug Resistance, Neoplasm , HL-60 Cells , Humans , Sirtuin 2 , Metabolism
20.
Article in Chinese | WPRIM | ID: wpr-771883

ABSTRACT

OBJECTIVE@#To investigate the regulation of miR-34a on HDAC1 expression and its effect on the apoptosis of acute myeloid leukemia (AML) cells.@*METHODS@#miR-34a mimics, miR-34a inhibitor and miR-34a scramble were transfected into HL-60 cells. The effects of miR-34a expression levels on proliferation and apoptosis of HL-60 cell were detected by CCK8 assay and flow cytometry respectively. The expression of HDAC1 protein was assessed by Western blot after regulating miR-34a expression, the 3'UTR of HDAC1 was cloned and ligated to construct a dual luciferase reporter vector, and then the dual luciferase reporter assay was applied to verify the target of miR-34a, the expression vector pcDNA3.1-HDAC1 was constructed, the interaction of miR-34a and HDAC1 was analyzed by reversion test.@*RESULTS@#miR-34a over-expression could inhibit the proliferation of HL-60 cells and induce their apoptosis. Bioinformatics analysis indicated that the HDAC1 was a target gene of miR-34a. Western blot indicated that miR-34a overexpression down-regulated the expression of HDAC1. Dual luciferase reporter assay and reversion test showed that miR-34a could act at the 3-UTR of HDAC1 gene to regulate its expression.@*CONCLUSION@#miR-34a promotes the apoptosis of HL-60 cells via regulating HDAC1 expression.


Subject(s)
Apoptosis , Cell Proliferation , HL-60 Cells , Histone Deacetylase 1 , Metabolism , Humans , Leukemia, Myeloid, Acute , Genetics , MicroRNAs , Genetics
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