This study was aimed to explore the effect of homoharringtonine in combination with AG490 on JAK2-STAT5 associated signal pathway in HEL cells, and analyze its mechanism so as to provide theoretical basis for therapy of chronic myeloproliferative neoplasma by new program. The cell survival rates were tested by MTT, apoptosis was tested by flow cytometry after HEL cells were treated by 20 ng/ml HHT, 100 µmol/L AG490 and 20 ng/ml HHT in combination with 100 µmol/L AG490, while the signal proteins such as P-JAK2, P-STAT5 and BCL-xL activated by abnormal activated JAK2 were tested by Western blot. The results showed that both HHT and AG490 could inhabit the HEL cell proliferation after being treated for 24 hours, and Annexin V-PI double staining confirmed early apoptosis while HHT effect was more obvious, Western blot showed that the expressions of P-JAK2 and P-STAT5 were down-regulated, while the total protein levels of JAK2 and STAT5 were stable. It is concluded that HHT combined with AG490 can obviously inhibit the proliferation and induce early apoptosis of HEL cells, and there is synergistic effect between the two drugs. HHT possibly acts as a broad-spectrum PTK inhibitor and synergistically with AG490 inhibits the phosphorylation of signal proteins caused by JAK2V617F, thus down-regulating the transcription of STAT5.
Cell Line, Tumor ; Harringtonines ; pharmacology ; Humans ; Janus Kinase 2 ; metabolism ; STAT5 Transcription Factor ; metabolism ; Signal Transduction ; drug effects ; Tyrphostins ; pharmacology

OBJECTIVETo study the effect of matrine and oxymatrine on proliferation and the expression of Stat3, Stat5 mRNA in SMMC-7721 cell line.

METHODTreated with matrine and oxymatrine, the inhibitory effect on SMMC-7721 cell proliferation was detected by MTT, double fluorescence labeling was applied to measure the apotosis ratios of SMMC-7721cells, the expression of Stat3 and Stat5 mRNA in SMMC-7721 cell line were assessed with RT-PCR.

RESULTMatrine and oxymatrine could inhibit the proliferation and induce the apoptosis of SMMC-7721 cells and it was time and dose dependent, the expression of Stat3 and Stat5 mRNA in SMMC-7721 cell with matrine and oxymatrine were significantly lower than those in control group (P<0.05 or P<0.01). Compared with the same dose of matrine and oxymatrine, matrine showed stronger effect on cell proliferation, apoptosis, and Stat3 and Stat5 mRNA (P<0.05 or P<0.01).

CONCLUSIONMatrine and oxymatrine inhibited the proliferation and induced the of SMMC-7721 cells significantly, the mechanism of which might be related to the down-regulation of stat3 and stat5 mRNA and inhibition of the signaling transduction pathway.

Alkaloids ; pharmacology ; Apoptosis ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Drugs, Chinese Herbal ; pharmacology ; Gene Expression Regulation ; drug effects ; Humans ; Quinolizines ; pharmacology ; Reverse Transcriptase Polymerase Chain Reaction ; STAT3 Transcription Factor ; genetics ; STAT5 Transcription Factor ; genetics

OBJECTIVETo investigate the effects of Sinopodophyllum hexundrum on apoptosis in K562 cells.

METHODSK562 cells were treated with Sinopodophyllum hexundrum at different concentrations and for different lengths of time to determine the optimal conditions of SinoPodophyllum hexandrum treatment for K562 cells using CCK8 assay. The cell apoptotic rate was detected by flow cytometry, and the cell morphology and nuclear morphology of K562 cells were observed with Wright staining and DPAI staining, respectively. The protein expressions of BCR/ABL, p-BCR/ABL, STAT5, p-STAT5 and the apoptosis-related proteins PARP, caspase-3 and cleaved-caspase-3 were determined with Western blotting.

RESULTSThe cell proliferation was inhibited in a concentration-and time-dependent manner by 1, 2, and 3 µg/mL Sinopodophyllum hexundrum. The treatment was optimal with a Sinopodophyllum hexundrum concentration of 2 µg/mL a treatment time of 48 h, and the cell apoptotic rate increased in a time-dependent manner and significantly increased at 48 h (P<0.001). The expression of apoptosis-related proteins PARP, caspase-3 and cleaved-caspase-3 were also activated in a time-dependent manner. The cells showed typical apoptotic changes after treatment with 2 µg/mL Sinopodophyllum hexundrum for 48 h with significantly reduced expressions of BCR/ABL, p-BCR/ABL, STAT5, AND p-STAT5.

CONCLUSIONSinopodophyllum hexundrum promotes K562 cell apoptosis possibly by inhibiting BCR/ABL-STAT5 survival signal pathways and activating the mitochondrion-associated apoptotic pathways.

Apoptosis ; drug effects ; Caspase 3 ; metabolism ; Cell Proliferation ; Drugs, Chinese Herbal ; pharmacology ; Fusion Proteins, bcr-abl ; metabolism ; Humans ; K562 Cells ; Mitochondria ; metabolism ; STAT5 Transcription Factor ; metabolism ; Signal Transduction

OBJECTIVE: To investigate the effects of paclitaxel, quizartinib and their combination on proliferation, apoptosis and FLT3/STAT5 pathway of human leukemia cell line MV4-11 (FLT3-ITD+). METHODS: MV4-11 cells were treated with paclitaxel and quizartinib at different concentrations for 24 h, 48 h and 72 h, respectively, and then the two drugs were combined at 48 h to compare the inhibition of proliferation, the apoptosis rate was detected by flow cytometry, the expression of FLT3 and STAT5 mRNA was determined by fluorescence quantitative PCR, and the protein expression of FLT3, p-FLT3, STAT5 and p-STAT5 was determined by Western blot. RESULTS: Different combination groups of paclitaxel and quizartinib had synergistic inhibitory effect. The cell survival rate in the combination group was significantly lower than that in the single drug group (P<0.05). The cell apoptosis rate in the combination group was significantly higher than that in the single drug group (P<0.001). The expression of FLT3 mRNA in combination group was significantly higher than that in two single drugs (P<0.01). The expression of STAT5 mRNA in combination group was significantly higher than that in quizartinib group (P<0.001); increased compared with paclitaxel group, but there was no statistical significance. The expression level of p-FLT3、p-STAT5 protein in the combination group was significantly lower than that in the single drug group (P<0.05, P<0.05). CONCLUSION Paclitaxel combined with quizartinib can synergistically inhibit the proliferation of MV4-11 cell line and promote the apoptosis of MV4-11 cell line by inhibiting the activity of FLT3/STAT5 pathway.
Apoptosis ; Benzothiazoles ; Cell Line, Tumor ; Humans ; Leukemia, Myeloid, Acute/genetics* ; Paclitaxel/therapeutic use* ; Phenylurea Compounds ; RNA, Messenger ; STAT5 Transcription Factor/pharmacology* ; Signal Transduction ; fms-Like Tyrosine Kinase 3

OBJECTIVETo investigate the effect of curcumin on STAT 5 signaling molecule in K562 cells and its molecular mechanism of antileukemia.

METHODSCell proliferation was studied by tetrazolium dye assay. The expressions of STAT5 mRNA and protein were assayed by in situ hybridization, and Western blotting respectively.

RESULTSCurcumin could inhibit K562 cell proliferation in a time- and dose-dependent manner. The percentage of STAT5-positive cells was 19% in curcunin group, significantly less than 31% of that in K562 cell group (P < 0.01). The A value of the expression level of STAT5 protein in curcumin group was 15 266 +/- 769, significantly less than 25 781 +/- 1240 of that in K562 cell group (P < 0.01).

CONCLUSIONThe expressions of STAT5 mRNA and protein in K562 cells were inhibited by curcumin and curcumin could inhibit K562 cell proliferation.

Cell Proliferation ; drug effects ; Curcumin ; pharmacology ; Gene Expression Regulation, Leukemic ; drug effects ; Humans ; K562 Cells ; Leukemia ; genetics ; metabolism ; physiopathology ; STAT5 Transcription Factor ; genetics ; metabolism ; Signal Transduction ; drug effects

OBJECTIVETo study the expression of signal transducer and activator of transcription 5b (STAT5b) mRNA and interleukin-4 (IL-4) mRNA in blood mononuclearcells in a rat model of asthma and the effect of montelukast (MK) and BCG-polysaccharide and nucleic acid injection (BCG-PSN) on STAT5b mRNA and IL-4 mRNA expression.

METHODSFifty-two male Sprague-Dawley rats (weight:140-200 g) were randomly divided into four groups: asthma, MK treated and BCG-PSN-treated and control groups. Rat model of asthma was prepared by ovalbumin (OVA) sensitization. The rats were sacrificed 24 hrs after the last sensitization. Blood eosinophils (EOS) were counted. Plasma contens of IL-4 and interferon-gamma (IFN-gamma) were measured using ELISA. Expression of STAT5b mRNA and IL-4 mRNA in blood mononuclearcells was detected with SYBR GREEN I fluorescent quantitation PCR method.

RESULTSBlood contents of STAT5b mRNA and IL-4 mRNA in the untreated asthma group were significantly higher than those in the other three groups (<0.01). Blood EOS count and plasma IL-4 contents in the untreated asthma group significantly increased, while plasma IFN-gamma contents significantly decreased compared with the other three groups (<0.01). There were no significant differences in the parameters measured among the MK-treated, the BCG-PSNjtreated and the control groups. STAT5b mRNA expression was positively correlated to IL-4 mRNA expression, IL-4 content and EOS count (r=0.730,0.650, 0.664, respectively; <0.01), but negatively correlated to IFN-gamma content (r=-0.798; <0.01).

CONCLUSIONSSTAT5b mRNA and IL-4 mRNA were strongly expressed in blood mononuclearcells in rats with asthma, and there was a positive correlation between them. MK and BCG-PSN had inhibitory effects on the expression of STAT5b mRNA and IL-4 mRNA, which might be contributed to suppression of airway inflammation in asthma.

Acetates ; pharmacology ; Animals ; Asthma ; blood ; drug therapy ; pathology ; BCG Vaccine ; pharmacology ; Interferon-gamma ; biosynthesis ; Interleukin-4 ; genetics ; Leukocytes, Mononuclear ; metabolism ; Male ; Quinolines ; pharmacology ; RNA, Messenger ; blood ; Rats ; Rats, Sprague-Dawley ; STAT5 Transcription Factor ; genetics

Mechano-growth factor (MGF) is one of IGF-1 isoforms. MGF is mechanosensitive and has important functions in muscle hypertrophy, regeneration and nerve injury recovery. In this study, MGF cDNA (330 bp) was cloned from stretched osteoblasts by RT-PCR. In order to avoid prolin residue inhibiting enterokinase cleavage, 9bp of MGF cDNA 5' end sequence was truncated by primer, then the obtained truncated MGF (des(1-3)MGF) cDNA (321 bp) was subcloned in pET32a(+) vector to construct a prokaryotic recombination expression plasmid. Trx/des(1-3)MGF fusion protein, existing in forms of solution, was expressed in transformed Escherichia coli strain BL21(DE3) by IPTG induction at 30 degres C. The supernatant of cell lysates was subjected to ion exchange chromatography and Ni2+ metal affinity chromatography, and the fusion protein was obtained with the purity over 95%. After the fusion protein was cleaved by enterokinase, Trx and des(1-3)MGF was isolated by reverse-phase HPLC. Through these procedures, des(1-3) MGF was obtained with the purity of 98%. The protein molecular mass was conformity to the theoretical value by SDS-PAGE and mass spectrometry analysis. The purified des(1-3)MGF was incubated with MC3T3-E1 for cell proliferation and migration assays. The results show that des(1-3)MGF exhibited more facilitative effects on proliferation and migration of MC3T3-E1 than that of des(1-3)IGF-1.
Cloning, Molecular ; DNA, Complementary ; genetics ; Escherichia coli ; genetics ; metabolism ; Humans ; Insulin-Like Growth Factor I ; Osteoblasts ; metabolism ; Protein Isoforms ; biosynthesis ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; pharmacology ; STAT5 Transcription Factor ; biosynthesis ; genetics ; Tumor Suppressor Proteins ; biosynthesis ; genetics

OBJECTIVETo investgate the signal transduction and regulation in erythropoiesis by angelica polysaccharides (APS) to clarify the mechanism for APS promoting hematopoiesis.

METHODThe mononuclear cells were isolated from foetus umbilic cord blood (mononuclear cells, MNCs), after MNCs were incubated in the presence of APS group (APS 200 mg x L(-1)) and control group for 24 h, the cells were stimulated with Epo (5 U x mL(-1)) for 0, 2, 5, 30 min, respectively. STAT5 was measured by ICC and laser confocal scanning microscope. JAK2, STAT5 in nucleus and cytoplasm were measured by western-blotting-ECL.

RESULTAngelica polysaccharide cooperated with Epo has significant impact on the expression of STAT5. The expression of STAT5 has significant difference between APS group and the control group at 4 time points. JAK2, STAT5 expressed in cytoplasm and nuclear of APS group significantly increased as compared to those of control group, and they expressed the strongest at 5 min.

CONCLUSIONJAK2, STAT5 signal transduction molecule plays an important role in the effect of APS cooperated with Epo on promoting hematopoiesis.

Angelica ; chemistry ; Blotting, Western ; Cells, Cultured ; Erythropoietin ; pharmacology ; Gene Expression ; drug effects ; Hematopoiesis ; drug effects ; Humans ; Janus Kinase 2 ; metabolism ; Microscopy, Confocal ; Polysaccharides ; pharmacology ; STAT5 Transcription Factor ; metabolism ; Signal Transduction ; drug effects

OBJECTIVETo investigate the activation pattern of signal transducers and activators of transcription (STAT) induced by vascular endothelial growth factor (VEGF) in CD34+ hematopoietic progenitor cells, and gain an insight into the molecular mechanism and signal transduction pathway of VEGF that has an effect on CD34+ hematopoietic progenitor cells.

METHODSAfter isolated from umbilical cord blood by using a high-gradient magnetically activated cell sorting system (MACS), CD34+ cells were stimulated by VEGF (50 ng/ml) for different time (0, 15, 30, 45, 60, 90 min) to detect the tyrosine phosphorylation and nuclear translocation of STAT-3 and STAT-5 with Western blot and immunocytochemistry methods. The expression of VEGF receptor-2 (VEGFR2) on the membrane of CD34+ progenitor cells was examined by immunocytochemistry. ATWLPPR, an effective peptide screened from phage epitope library by affinity for membrane-expressed VEGFR2 and blocking the binding of VEGF to VEGFR2, was used to determine whether the activation of STAT pathway induced by VEGF was blocked.

RESULTSTyrosine phosphorylation of STAT-3 and STAT-5 was undetectable in unstimulated CD34+ cells, but was evident at 15 min in response to VEGF stimulation. VEGF resulted in a rapid and transient tyrosine phosphorylation of STAT-3 and STAT-5. The maximal tyrosine phosphorylation was catched at 30 and 45 min, respectively (P = 0.0001), and returned to basal levels at 90 min. Immunocytochemistry confirmed that increased phosphorylated STAT-3 was translocated into the nuclei at 30 min (P = 0.0001), and mainly in cytoplasms again at 90 min after stimulation with VEGF. However, compared with unstimulated CD34+ cells, there was only increased phosphorylation of STAT-5 appeared mainly in cytoplasms, but no significant nuclear translocation was found after stimulation with VEGF (P > 0.05). The presence of VEGFR2 was confirmed using anti-VEGFR2 antibody staining by immunocytochemistry, moreover, the phosphorylation of STAT-3 and STAT-5 failed to be activated by the co-culture with ATWLPPR and VEGF, suggesting that activation of the STAT pathway be specifically mediated by VEGFR2 in CD34+ progenitor cells.

CONCLUSIONSSTAT signaling pathway participates in the signal transduction of VEGF via VEGFR2 in CD34+ hemopoietic progenitor cells.

Adult ; Antigens, CD34 ; metabolism ; DNA-Binding Proteins ; Endothelium, Vascular ; drug effects ; metabolism ; Female ; Fetal Blood ; cytology ; Hematopoietic Stem Cells ; metabolism ; physiology ; Humans ; Milk Proteins ; Phosphorylation ; Pregnancy ; Receptors, Vascular Endothelial Growth Factor ; metabolism ; STAT3 Transcription Factor ; STAT5 Transcription Factor ; Signal Transduction ; Trans-Activators ; metabolism ; Transcription, Genetic ; Tyrosine ; metabolism ; Vascular Endothelial Growth Factor A ; pharmacology

OBJECTIVETo explore the mechanism of anti-proliferative effect of indomethacin (IN) on chronic myelogenous leukemia (CML) cells.

METHODSMTT was applied to assay CML cells viability under IN intervention. STAT1, STAT5 proteins were analyzed by Western blot, the expressions of phosphorylated STAT1 or STAT5 by immunoprecipitation combined with Western blot, the cellular localization of p-STATs proteins by indirect immunofluorescence technique, and the detection of Bcl-X(L) and COX-2 protein by Western blot.

RESULTSIN could significantly inhibit the viability of CML cells. 0 approximately 400 micromol/L of IN could down-regulate the expression of p-STAT1 or p-STAT5 in a dose-response manner, p-STATs were distributed mainly in the nucleus as scattering spots. The expression of COX-2 protein could be detected in K562 cells. Both Bcl-X(L) and COX-2 proteins could be inhibited by IN in a dose-dependent manner.

CONCLUSIONSIN could significantly inhibit the proliferation of CML cells, the mechanism of which might be related to the suppression of STATs/Bcl-X(L) signal transduction pathway. There exists COX-2 protein expression in K562 cells, the anti-leukemia effect of IN was possibly dependent on COX-2 pathway.

Blotting, Western ; Cell Proliferation ; drug effects ; Cyclooxygenase 2 ; metabolism ; Cyclooxygenase Inhibitors ; pharmacology ; Down-Regulation ; drug effects ; Humans ; Indomethacin ; pharmacology ; K562 Cells ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; metabolism ; pathology ; Microscopy, Fluorescence ; Phosphorylation ; drug effects ; STAT1 Transcription Factor ; metabolism ; STAT5 Transcription Factor ; metabolism ; bcl-X Protein ; metabolism