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1.
Chinese Journal of Biotechnology ; (12): 2413-2423, 2020.
Article in Chinese | WPRIM | ID: wpr-878497

ABSTRACT

Human acute leukemia (AL) is a clonal malignancy with abnormal hematopoietic stem cells. Clinically, AL is very difficult to cure due to its sudden onset and short course of disease progression. Previous studies have shown that eukaryotic initiation factor 4B (eIF4B) plays a critical role in the development of chronic leukemia. However, the involvement of eIF4B in human acute leukemia is still largely unknown. Therefore, we studied eIF4B function and its regulatory mechanism in human acute leukemia. We found that phosphorylation levels of eIF4B in acute leukemia cells were significantly reduced in response to treatment with either LY294002 (PI3K inhibitor), AKTi (AKT inhibitor) or SMI-4A (Pim inhibitor). Co-treatment with inhibitors targeting JAK/STAT5/Pim and PI3K/AKT/mTOR signaling dramatically promoted apoptosis of acute leukemia cells by downregulating eIF4B phosphorylation. Furthermore, in vitro and in vivo functional experiments showed that eIF4B played an important anti-apoptosis role in the acute leukemia cells by regulating the expression of anti-apoptotic proteins Bcl-2 and Bcl-XL. In contrast, silencing eIF4B inhibited the growth of acute leukemia cells as engrafted tumors in nude mice. Taken together, our results indicate the synergistic role of JAK/STAT5/Pim and PI3K/AKT/mTOR signaling pathways in regulating eIF4B phosphorylation in acute leukemia, and highlight eIF4B as a candidate therapeutic target for treatment of acute leukemia.


Subject(s)
Animals , Apoptosis , Cell Line, Tumor , Leukemia , Mice , Mice, Nude , Phosphatidylinositol 3-Kinases/metabolism , Proto-Oncogene Proteins c-akt/metabolism , STAT5 Transcription Factor/metabolism
2.
Journal of Experimental Hematology ; (6): 1088-1093, 2019.
Article in Chinese | WPRIM | ID: wpr-775759

ABSTRACT

OBJECTIVE@#To investigate the effects of inhibiting proliferation and inducing apoptosis of low-dose triptolide and sorafenib alone or in combination on FLT3-ITD acute myeloid leukemia cell line MV4-11 and STAT5 pathway.@*METHODS@#The MV4-11 cells were treated with low dose triptolide(IC) and sorafenib(IC) alone or in combination for 48 hours. The cell proliferation and inhibition were detected by using CCK-8 kit, the cell apoptosis was detected by flow cytometry, the expression of FLT3,STAT5 in mRNA and protein levels was detected by RT-PCR and Western blot respectively.@*RESULTS@#The treatment of MV4-11 cells with low dose triptolide and sorafenib alone and in combination for 48 hours could inhibit cell proliferation and induce cell apoptosis, moreover the inhibitory rate and apoptotic rate of MV4-11 cells in drug-combination group both were higher than those in single drug group. The mRNA expression and protein expression of FLT3,STAT5 signaling pathway in drug combination group were significantly lower than those in single drug group.@*CONCLUSION@#Low-dose triptolide combined with sorafenib can synergistically inhibit the proliferation and induce the apoptosis of MV4-11 cells, which may be related with the inhibition of FLT3 and STAT5 pathway.


Subject(s)
Apoptosis , Cell Line, Tumor , Diterpenes , Epoxy Compounds , Humans , Leukemia, Myeloid, Acute , Phenanthrenes , STAT5 Transcription Factor , Sorafenib , fms-Like Tyrosine Kinase 3
3.
Rev. chil. endocrinol. diabetes ; 11(3): 97-102, jul. 2018. ilus
Article in Spanish | LILACS | ID: biblio-915180

ABSTRACT

Abstract: Sex hormones play a major role during pubertal growth. Estradiol (E2) and testosterone (T) levels progressively increase during puberty and in the presence of growth hormone (GH), growth velocity increases. Understanding the interactions between sex hormones and GH, may optimize the treatment of pubertal children with growth disorders. The aim of our study was to investigate possible molecular mechanisms which might potentiate longitudinal growth during puberty due to E 2or T combined with GH. We evaluated the GH/JAK2/STAT5 signaling pathway in the human hepatoma cell line HEPG2. Our results suggest that sex hormones potentiate the GH signaling pathway in a dose dependent fashion. Relatively low concentrations of E 2associated with GH induce a substantial activation of the GH pathway, whereas relatively high concentrations of T associated with GH produce a similar effect. These findings are concordant with the physiology of the pubertal growth spurt, which is an early event in girls (when E 2 circulating levels are low), and a late event in boys (when T circulating levels are high).


Resumen: Las hormonas sexuales, modulan el crecimiento durante la pubertad. Los niveles de estradiol (E2) y testosterona (T) aumentan progresivamente durante la pubertad y en combinación con la hormona de crecimiento (GH), producen un incremento en la velocidad de crecimiento en este período conocido como el "estirón puberal". El estudio de la interacción entre las hormonas sexuales y la GH, es de gran importancia para optimizar el tratamiento de niños(as) con alteraciones del crecimiento durante la pubertad. El objetivo de nuestro estudio fue investigar los posibles mecanismos que podrían potenciar el crecimiento longitudinal durante la pubertad, en especial las interacciones entre E 2o T en combinación con GH. Se evaluó la activación de la vía de señalización GH/JAK2/STAT5 frente al estímulo combinado con estas hormonas en cultivos celulares de hepatoma humana HEPG2. Nuestros resultados sugieren que existe un efecto potenciador de las hormonas sexuales sobre la vía de señalización de GH. Observamos que concentraciones relativamente bajas de E2 junto con GH producen una clara activación de la vía de señalización para GH, mientras que concentraciones relativamente altas de T junto con GH producen una activación similar. Estos hallazgos son concordantes con la fisiología del estirón puberal, que es más precoz en niñas (cuando los niveles circulantes de E2 son bajos), y más tardíos en varones (cuando los niveles circulantes de T son altos).


Subject(s)
Humans , Testosterone/physiology , Growth Hormone/physiology , Estradiol/physiology , STAT5 Transcription Factor/physiology , Janus Kinase 2/physiology , Puberty
4.
Journal of Experimental Hematology ; (6): 1726-1730, 2018.
Article in Chinese | WPRIM | ID: wpr-773029

ABSTRACT

OBJECTIVE@#To study the effects of iron metabolism abnormality on EPO-STAT5 signaling pathway in anemia patients.@*METHODS@#According to diseases, the patients were divided into 3 groups: lower risk myelodysplastic syndrome (MDS) group (30 cases) including 14 cases of non-iron over load and 16 cases of iron over load, 12 cases of them were treated by iron chelation therapy; anemia of chronic disease (ACD) group (12 cases) and iron deficiency anemia (IDA) group (12 cases). In addition, the healthy control group was selected. The iron metaloslism index (SF, SI, TIBC), serum level of EPO, plasma level of P-STAT5 and STAT-5 mRNA expression in peripheral blood cells were detected and compared in different groups. Moreover, the effects of iron metabolism abnormality on the expression of EPO and STAT5 in anemia patients were analyzed.@*RESULTS@#compared with non-iron over load group, the EPO level in iron over load group significantly increased (P<0.05), the expression of STAT5 mRNA and P-STAT5 significantly decreased (P<0.05). After iron chelation therapy, the EPO level in serum significantly decreased (P<0.05), the expression of STAT5 mRNA and P-STAT5 was up-regulated significantly (P<0.05). Compared with healthy control group, the expression of EPO in ACD group was down-regulated significantly, while the expression of STAT5 mRNA was not different, but the P-STAT5 expression was down-regulated significantly (P<0.05). Compared with the healtly control group, the EPO expression in IDA group was enhanced significantly (P<0.05), the expression of STAT5 mRNA and P-STAT5 were also significantly enhanced (P<0.05).@*CONCLUSION@#The excessive iron load or chronic inflammation may inhibit the activation of EPO-STAT5 signaling pathway and aggravate the anemia.


Subject(s)
Anemia , Anemia, Iron-Deficiency , Erythropoietin , Humans , Iron , STAT5 Transcription Factor , Signal Transduction
5.
Article in Chinese | WPRIM | ID: wpr-273783

ABSTRACT

<p><b>OBJECTIVE</b>To investigate the effects of Sinopodophyllum hexundrum on apoptosis in K562 cells.</p><p><b>METHODS</b>K562 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.</p><p><b>RESULTS</b>The 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.</p><p><b>CONCLUSION</b>Sinopodophyllum hexundrum promotes K562 cell apoptosis possibly by inhibiting BCR/ABL-STAT5 survival signal pathways and activating the mitochondrion-associated apoptotic pathways.</p>


Subject(s)
Apoptosis , 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
6.
Article in English | WPRIM | ID: wpr-7401

ABSTRACT

Entamoeba histolytica is a tissue-invasive protozoan parasite causing dysentery in humans. During infection of colonic tissues, amoebic trophozoites are able to kill host cells via apoptosis or necrosis, both of which trigger IL-8-mediated acute inflammatory responses. However, the signaling pathways involved in host cell death induced by E. histolytica have not yet been fully defined. In this study, we examined whether calpain plays a role in the cleavage of pro-survival transcription factors during cell death of colonic epithelial cells, induced by live E. histolytica trophozoites. Incubation with amoebic trophozoites induced activation of m-calpain in a time- and dose-dependent manner. Moreover, incubation with amoebae resulted in marked degradation of STAT proteins (STAT3 and STAT5) and NF-kappaB (p65) in Caco-2 cells. However, IkappaB, an inhibitor of NF-kappaB, was not cleaved in Caco-2 cells following adherence of E. histolytica. Entamoeba-induced cleavage of STAT proteins and NF-kappaB was partially inhibited by pretreatment of cells with a cell-permeable calpain inhibitor, calpeptin. In contrast, E. histolytica did not induce cleavage of caspase-3 in Caco-2 cells. Furthermore, pretreatment of Caco-2 cells with a calpain inhibitor, calpeptin (but not the pan-caspase inhibitor, z-VAD-fmk) or m-calpain siRNA partially reduced Entamoeba-induced DNA fragmentation in Caco-2 cells. These results suggest that calpain plays an important role in E. histolytica-induced degradation of NF-kappaB and STATs in colonic epithelial cells, which ultimately accelerates cell death.


Subject(s)
Caco-2 Cells , Calcium-Binding Proteins , Calpain/genetics , Caspase 3/genetics , Caspases , Cell Death , Colon/cytology , Entamoeba histolytica/physiology , Epithelial Cells/cytology , Humans , I-kappa B Proteins/metabolism , Intestinal Mucosa/cytology , NF-kappa B/genetics , RNA Interference , RNA, Small Interfering , STAT3 Transcription Factor/genetics , STAT5 Transcription Factor/genetics , Signal Transduction
7.
Article in Chinese | WPRIM | ID: wpr-749364

ABSTRACT

OBJECTIVE@#To study the expression of Interleukin2 (IL-2) signaling pathway related factors and Treg cell in nasal tissue of nasal polyps, so that to investigate the possible mechanism of IL-2 signaling pathway in the progress of nasal polyps and the correlation between IL-2 pathway and Treg cell.@*METHOD@#Thirty patients were enrolled for study, including the patients with nasal polyps and those with only deviated nasal septum as normal control. The nasal polyps tissue and the turbinate mucosa of the patients were collected during surgery. The expression levels of IL-2 and IL-2R were measured by means of ELISA. The level of pSTAT5 was evaluated by Western blot. We measured the level of Foxp3 mRNA in the tissue by real-time PCR, and the proportion of Treg cells was evaluated by flow cytometry. Finally. we analyzed the correlation between IL-2 pathway related factors and Treg cells in nasal polyps.@*RESULT@#The expression levels of IL-2. IL-2R and pSTAT5 were significantly decreased in the nasal polyps compared with normal control (P < 0.05), and the level of Foxp3 mRNA and proportion of Treg cells in patients with nasal polyps were significantly lower than in normal control (P < 0.05). Moreover, there was positive correlation between the levels of IL-2 pathway related factors and the levels of Foxp3 mRNA and Treg cells proportion in nasal polyps.@*CONCLUSION@#The activated state of IL-2 signaling pathway got changed in nasal polyps tissue, the level of which was positively correlated with the expression of Treg cells, indicating that the IL-2 signaling pathway may play a crucial role in the development of nasal polyps, and the decreased level of Treg cells in nasal polyps may result from the downregulation of IL-2 signaling pathway.


Subject(s)
Adult , Female , Forkhead Transcription Factors , Metabolism , Humans , Interleukin-2 , Metabolism , Male , Middle Aged , Nasal Polyps , Metabolism , Receptors, Interleukin-2 , Metabolism , STAT5 Transcription Factor , Metabolism , Signal Transduction , T-Lymphocytes, Regulatory , Cell Biology
8.
Chinese Journal of Pediatrics ; (12): 930-933, 2013.
Article in Chinese | WPRIM | ID: wpr-288810

ABSTRACT

<p><b>OBJECTIVE</b>To analyze clinical manifestations and gene mutations in a child with severe short stature, explore its molecular mechanism and further clarify the diagnostic procedure for short stature.</p><p><b>METHOD</b>We observed clinical characteristics of a patient with short stature and did diagnostic examinations, assessed the function of GH-IGF-1 axis, and surveyed its family members.Genomic DNA was extracted from peripheral blood, GHR, IGFALS, STAT5b and GH1 gene were amplified by PCR for sequencing, including exons and splicing areas.</p><p><b>RESULT</b>The patient presented symmetrical short stature (height -8.2 SDS) and facial features, and other congenital abnormalities.It displayed non-growth hormone deficiency. The baseline value of GH was 21 µg/L, and the peak was 57.9 µg/L. The value of IGF-1 was less than 25 µg/L, and the IGFBP-3 less than 50 µg/L. And IGF-1 generation test showed no response. There was no similar patients in the family members.Sequencing of GHR in the patient revealed a homozygous point mutation (c.Ivs6+1G>A), and her father and mother had the same heterozygous mutation. The same mutation was not identified for her sister.No other candidate gene was found.</p><p><b>CONCLUSION</b>As the result of combined clinical characteristics and lab examinations, as well as gene detection, the case was diagnosed with Laron syndrome and GHR gene mutation is the molecular mechanism.We should explicit the etiological diagnosis for short stature, and avoid missed diagnosis and misdiagnosis.</p>


Subject(s)
Base Sequence , Body Height , Child , DNA Mutational Analysis , Exons , Growth Disorders , Blood , Genetics , Pathology , Human Growth Hormone , Blood , Humans , Insulin-Like Growth Factor Binding Protein 3 , Blood , Insulin-Like Growth Factor I , Laron Syndrome , Blood , Genetics , Pathology , Male , Molecular Sequence Data , Mutation , Pedigree , Receptors, Somatotropin , Genetics , STAT5 Transcription Factor , Genetics
9.
Journal of Experimental Hematology ; (6): 1398-1404, 2012.
Article in Chinese | WPRIM | ID: wpr-325251

ABSTRACT

This study was aimed to explore the JAK2V617F mutation and p-STAT5 expression in patients with myeloproliferative neoplasm (MPN), and investigate their relations with clinical characteristics so as to provide theoretical basis for clinical practice and target therapy. Forty-five confirmed BCR-ABL-negative MPN patients and 15 healthy adults were enrolled in this study. Real-time fluorescent quantitative PCR and Western blot were respectively used to detect JAK2V617F mutation proportion and p-STAT5 expression level. In addition, their relations with clinical characteristics of MPN were analyzed. The results showed that the positive rate of JAK2V617F mutation in MPN patients was 73.3% (33/45), including 83.3% in polycythemia vera (PV) patients (20/24), 68.8% in essential thrombocythemia (ET) patients (11/16) and 40.0% in idiopathic myelofibrosis (IMF) patients (2/5). Mutation proportions of JAK2V617F in PV, ET and IMF patients were 0.472 ± 0.245, 0.216 ± 0.162, 0.435 ± 0.239 respectively; gray values of p-STAT5 protein in PV, ET and IMF patients were 1.396 ± 0.758, 0.760 ± 0.623, 0.792 ± 0.612 respectively. JAK2V617F mutation proportion and p-STAT5 protein expression level showed a linear correlation (P < 0.05). PV patients with higher JAK2V617F mutation proportion had higher white blood cell count, hemoglobin level and hematocrit, but lower platelet count; ET patients with higher mutation proportion showed older and higher white blood cell count, hemoglobin level and hematocrit, there was no significant difference between platelet count; IMF patients with higher JAK2V617F mutation proportion showed lower white blood cell count, platelet count, hemoglobin level and hematocrit. Patients with JAK2V617F positive mutation were more likely complicated by splenomegaly, bleeding and thrombotic events. It is concluded that the incidence rate of JAK2V617F mutation is high in patients with MPN. Higher mutation proportion always connected with higher expression of p-STAT5, and easily complicates by splenomegaly and thrombotic events.


Subject(s)
Adult , Aged , Case-Control Studies , Female , Humans , Janus Kinase 2 , Genetics , Male , Middle Aged , Mutation , Myeloproliferative Disorders , Blood , Genetics , STAT5 Transcription Factor , Blood , Young Adult
10.
Journal of Experimental Hematology ; (6): 1496-1500, 2012.
Article in Chinese | WPRIM | ID: wpr-325231

ABSTRACT

Signal transducer and activator of transcription 5 (STAT5) is an important transcription factor existing in the cytoplasm of various types of cells. Once activated, STAT5 dimers translocate into nucleus and bind to the corresponding DNA sequence to regulate the transcription of its target genes. There are two isoforms of STAT5: STAT5A and STAT5B with 96% sequence homology and are encoded by two closely related but different genes. Studies have shown that STAT5 can regulate the survival, proliferation, differentiation and death of hematopoietic cells. Furthermore, elevated activation of STAT5 was found in many malignant hematologic diseases and therefore raised the possibility that STAT5 may be used as a new therapeutic target for blood related diseases. This review discusses the regulatory role of STAT5 in hematopoietic cells and its effect on the occurrence and development of blood diseases.


Subject(s)
Animals , Hematologic Diseases , Genetics , Metabolism , Hematopoietic System , Metabolism , Humans , STAT5 Transcription Factor , Genetics , Metabolism
11.
Chinese Journal of Hematology ; (12): 480-483, 2012.
Article in Chinese | WPRIM | ID: wpr-359452

ABSTRACT

<p><b>OBJECTIVE</b>To investigate the expressions of STAT5 phosphorylation in CD34(+)CD38(-)CD123(+) bone marrow cells of the patients with myelodysplastic syndromes (MDS), and then evaluate the level of activation of STAT5 associated with cell proliferation in MDS clone cells.</p><p><b>METHODS</b>The bone marrow mononuclear cells (BMMNC) were extracted from 36 MDS patients and 14 normal controls. The mean fluorescence intensities (MFI) of phosphorylated STAT5(P-STAT5) in CD34(+)CD38(-)CD123(+) and CD34(+)CD38(-)CD123(-)cells, with or without the stimulation of 10 U/ml EPO, were examined by flow cytometry (FCM).</p><p><b>RESULTS</b>Without stimulation, the P-STAT5 MFI in CD34(+)CD38(-)CD123(+) cells of low/high risk MDS patients was 113.71 ± 67.22/173.05 ± 102.78, which was significantly higher than that of CD34(+)CD38(-)CD123(-) cells (58.84 ± 27.51/68.99 ± 50.42, P < 0.01, P < 0.05) and the normal controls CD34(+)CD38(-)CD123(-) cells (63.06 ± 21.06, P < 0.05), there was no significant difference between the CD34(+)CD38(-)CD123(-) cells of MDS patients and the normal control CD34(+)CD38(-)CD123(-) cells; With the EPO stimulation, the P-STAT5 MFI in CD34(+)CD38(-)CD123(+) cells of low/high risk MDS patients was 144.04 ± 58.11/239.45 ± 152.05, which was significantly higher than that of CD34(+)CD38(-)CD123(-) cells (68.41 ± 25, 10/64.21 ± 23.43, P < 0.01) and the normal controls CD34(+)CD38(-)CD123(-) cells (75.21 ± 27.02, P < 0.01), there was no significant difference between the CD34(+)CD38(-)CD123(-) cells of MDS patients and the normal control CD34(+)CD38(-)CD123(-) cells; The P-STAT5 MFI in the CD34(+)CD38(-)CD123(+) cells of low/high risk MDS patients with or without EPO stimulation were 21.80/28.86, which was significantly higher than that of CD34(+)CD38(-)CD123(-) cells (7.42/5.50, P < 0.01, P < 0.05) and the normal controls CD34(+)CD38(-)CD123(-) cells (6.39, P < 0.05), there was no significant difference between the CD34(+)CD38(-)CD123(-) cells of MDS patients and the normal controls CD34(+)CD38(-)CD123(-) cells; There was no significant difference of P-STAT5 MFI with or without EPO stimulation and the increased P-STAT5 MFI between the CD34(+)CD38(-)CD123(+) cells of low and high risk MDS.</p><p><b>CONCLUSION</b>STAT5 associated with cell proliferation was activated in CD34(+)CD38(-)CD123(+) bone marrow cells in MDS, which had more significant reactions to EPO than CD34(+)CD38(-)CD123(-) cells, indicating that CD34(+)CD38(-)CD123(+) bone marrow cells might be the real malignant MDS clone cells in MDS.</p>


Subject(s)
Adult , Aged , Aged, 80 and over , Antigens, CD34 , Metabolism , Bone Marrow Cells , Cell Biology , Metabolism , Cell Proliferation , Cells, Cultured , Female , Flow Cytometry , Humans , Male , Middle Aged , Myelodysplastic Syndromes , Metabolism , Pathology , Phosphorylation , STAT5 Transcription Factor , Metabolism
12.
Article in Chinese | WPRIM | ID: wpr-336765

ABSTRACT

<p><b>OBJECTIVE</b>To investigate synergistically killing effect of histone deacetylase (HDAC) inhibitor suberoylanilide hydroxamic acid (SAHA) combined with imatinib on human chronic myeloid leukemia (CML) cell line.</p><p><b>METHODS</b>K562 cells were co-treated with SAHA and imatinib. Cell growth was measured by MTT assay. Apoptosis was determined using Hoechst staining apoptosis detection kit and flow cytometric analysis. Activation of Caspase pathway, expression of Bcr-Abl and its downstream target genes, and expression of anti-apoptotic proteins were detected by Western blot.</p><p><b>RESULTS</b>SAHA synergized the cytotoxicity of imatinib against leukemia K562 cells, concomitantly with increased apoptosis and enhanced activation of Caspase-3, -8 and PRAP. The combination therapy resulted in significantly lower levels of Bcr-Abl,phosphorylated Bcr-Abl compared to treatment with either SAHA or imatinib alone. Furthermore,the co-treatment resulted in down-regulation of anti-apoptotic protein Mcl-1 expression. Also,marked down-regulated expression of JAK2,STAT5,and phosphorylated STAT5 was detected in the combination therapy.</p><p><b>CONCLUSION</b>Combining HDAC inhibitor SAHA with imatinib can kill CML cells synergistically by inhibiting cell growth and inducing apoptosis, which is associated with activation of Caspase pathway and regulation of anti-apoptotic proteins.</p>


Subject(s)
Antineoplastic Agents , Pharmacology , Apoptosis , Benzamides , Caspase 3 , Metabolism , Caspase 8 , Metabolism , Drug Synergism , Fusion Proteins, bcr-abl , Metabolism , Histone Deacetylase Inhibitors , Pharmacology , Humans , Hydroxamic Acids , Pharmacology , Imatinib Mesylate , Intracellular Signaling Peptides and Proteins , Metabolism , Janus Kinase 2 , Metabolism , K562 Cells , Piperazines , Pharmacology , Pyrimidines , Pharmacology , STAT5 Transcription Factor , Metabolism
13.
Chinese Journal of Hematology ; (12): 378-382, 2011.
Article in Chinese | WPRIM | ID: wpr-251946

ABSTRACT

<p><b>OBJECTIVE</b>To study the effects of hermap gene on kinases in erythroid signal transduction pathway and investigate the mechanism of hermap on erythroid differentiation.</p><p><b>METHODS</b>The K562 cells expressing hermap and hermap-siRNA respectively were established for up- and down-regulating the expression of hermap gene. These K562 cells were then induced by Ara-C to erythroid differentiation and analyzed at 0, 24, 48, 72 and 96 h, respectively, for cell morphology and biphenylamine staining positive cells, determination of CD235a, CD36, kinases p-STAT5, p-Akt, p-MAPK and p-c-JUN by FCM; and quantification of hermap gene and γ (Aγ,Gγ) globin gene by FQ-PCR.</p><p><b>RESULTS</b>With up-regulating hermap gene and inducing by Ara-C, K562 cells were changing to low ratio of nucleus to cytoplasm, cytoplasm colour from basophilic to pinkish or amethyst tinge, increase of number of biphenylamine positive cells and expression of CD235a, CD36, γ (Aγ,Gγ) globin gene, hermap gene and p-STAT5 from 0 to 96 h. At 0, 24, 48, 72 and 96 h of culture, the positive rates of p-STAT5 cells were detected of 0.46%, 4.54%, 20.01%, 23.65% and 33.08%, respectively. This results demonstrated that there was a positive correlation between expression of p-STAT5 and hermap gene expression (P < 0.05).</p><p><b>CONCLUSION</b>hermap gene can stimulate erythroid differentiation of Ara-C induced K562 cells mainly through JAK/STAT5 signal transduction pathway.</p>


Subject(s)
Cell Differentiation , Erythrocyte Membrane , Erythrocytes , Cell Biology , Erythropoiesis , Gene Expression , Humans , K562 Cells , Receptors, Erythropoietin , Genetics , STAT5 Transcription Factor , Metabolism , Signal Transduction
14.
Journal of Experimental Hematology ; (6): 1117-1120, 2011.
Article in Chinese | WPRIM | ID: wpr-261918

ABSTRACT

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.


Subject(s)
Cell Line, Tumor , Harringtonines , Pharmacology , Humans , Janus Kinase 2 , Metabolism , STAT5 Transcription Factor , Metabolism , Signal Transduction , Tyrphostins , Pharmacology
15.
Chinese Journal of Cancer ; (12): 969-979, 2010.
Article in English | WPRIM | ID: wpr-296329

ABSTRACT

<p><b>BACKGROUND AND OBJECTIVE</b>Leukemic microenvironment has a major role in the progression of leukemia. Leukemic cells can induce reversible changes in microenvironmental components, especially the stromal function which results in improved growth conditions for maintaining the malignant leukemic cells. This study aimed to investigate the survival advantage of leukemic cells over normal hematopoietic cells in stromal microenvironment in long term.</p><p><b>METHODS</b>The mice were injected intraperitoneally with N-N' ethylnitrosourea (ENU) to induce leukemia; the mice received injection of normal saline were used as control. At 180 days after ENU induction, the mice were killed and the bone marrows were cultured for 19 days. Colony-forming assays were used to analyze the formation of various cell colonies. The expression of Sca-1, CD146, VEGFR2, CD95, pStat3, pStat5, and Bcl-xL in marrow cells were detected by flow cytometry.</p><p><b>RESULTS</b>Long-term leukemic bone marrow culture showed abnormal elongated stromal fibroblasts with almost absence of normal hematopoietic cells. Adherent cell colonies were increased, but CFU-F and other hematopoietic cell colonies were significantly decreased in leukemia group (P<0.001). Primitive progenitor-specific Sca-1 receptor expression was decreased with subsequent increased expression of CD146 and VEGFR-2 in leukemic bone marrow cells. Decreased Fas antigen expression with increased intracellular pStat3, pStat5 and Bcl-xL proteins were observed in leukemic bone marrow cells.</p><p><b>CONCLUSIONS</b>Stromal microenvironment shows altered morphology and decreased maturation in leukemia. Effective progenitor cells are decreased in leukemia with increased leukemia-specific cell population. Leukemic microenvironment plays a role in promoting and maintaining the leukemic cell proliferation and survivability in long term.</p>


Subject(s)
Animals , Antigens, Ly , Metabolism , Bone Marrow Cells , Metabolism , Pathology , CD146 Antigen , Metabolism , Cell Count , Cells, Cultured , Colony-Forming Units Assay , Erythroid Precursor Cells , Metabolism , Pathology , Ethylnitrosourea , Female , Fibroblasts , Metabolism , Pathology , Granulocyte-Macrophage Progenitor Cells , Metabolism , Pathology , Granulocytes , Metabolism , Pathology , Hematopoiesis , Hematopoietic Stem Cells , Metabolism , Pathology , Leukemia , Metabolism , Pathology , Male , Membrane Proteins , Metabolism , Mice , Myeloid Progenitor Cells , Metabolism , Pathology , Phenotype , STAT3 Transcription Factor , Metabolism , STAT5 Transcription Factor , Metabolism , Tumor Microenvironment , Physiology , Vascular Endothelial Growth Factor Receptor-2 , Metabolism , bcl-X Protein , Metabolism , fas Receptor , Metabolism
16.
Chinese Medical Journal ; (24): 566-570, 2009.
Article in English | WPRIM | ID: wpr-311822

ABSTRACT

<p><b>BACKGROUND</b>Tumor necrosis factor a receptor 1 (TNFalphaR1) plays an important role in the signal pathway of apoptosis. The objective of this study was to investigate the effects of TNFalphaR1 knockout on the up-regulation of erythropoietin receptor (Epo-R) and the coordinated anti-apoptosis functions during myocardial ischemia-reperfusion injury in mice.</p><p><b>METHODS</b>The ischemia-reperfusion injury model for cardiomyocytes was performed by ligating the left circumflex branch artery of TNFalphaR1 knockout (P55(-/-)) C17 B6 mice, as well as wild-type (P55(+/+)) C17 B6 mice. Triphenyltetrazolium chloride (TTC) staining was performed to observe the damaged area of the heart. TUNEL staining and DNA fragmentation were used to identify apoptosis. Mitochondrial Bcl-2 and Bax as well as expression of Epo-R and its downstream genes (Jak-2, stat-5, Akt, IkB-alpha, HIF-1alpha) were measured by Western blotting. The gene knockout mice were assigned into those undergoing the apoptosis surgical model group (KO group), and those subjected to sham operation (KOs group). Similarly, wild-type mice were either exposed to the surgical model (WT group) or subject to a sham operation (WTs group).</p><p><b>RESULTS</b>The myocardial damage ratio of the wild-type group after the operation was significantly higher than that of the knockout group, (50.5 +/- 6.4)% vs (36.9 +/- 6.9)%, P < 0.01. Similarly, TUNEL positive ratio of the wild-type group was significantly higher than that of the knockout group, (63.1 +/- 5.6)% vs (42.1 +/- 4.7)%, P < 0.01. The gray value ratios of Epo-R, Jak-2, stat-5, Akt, IkB-alpha, HIF-1 and mitochondrial Bcl-2 in the KO group were significantly higher than those of the WT group, P < 0.05; however, mitochondrial Bax was significantly lower than that of the WT group significantly (P < 0.05).</p><p><b>CONCLUSIONS</b>Using the ischemia-reperfusion injury model in mice, cardiomyocytes of TNFalphaR1 knockouts exhibited anti-apoptotic characteristics. This information could be used to coordinate the prevention of myocardial apoptosis by up-regulating and activating the Epo-R pathway.</p>


Subject(s)
Animals , Apoptosis , Blotting, Western , Disease Models, Animal , I-kappa B Proteins , Metabolism , In Situ Nick-End Labeling , In Vitro Techniques , Janus Kinase 2 , Metabolism , Male , Mice , Mice, Knockout , Myocardial Reperfusion Injury , Genetics , Metabolism , Pathology , Myocytes, Cardiac , Metabolism , Pathology , NF-KappaB Inhibitor alpha , Oncogene Protein v-akt , Metabolism , Proto-Oncogene Proteins c-bcl-2 , Metabolism , Receptors, Erythropoietin , Metabolism , Receptors, Tumor Necrosis Factor , Genetics , Metabolism , STAT5 Transcription Factor , Metabolism , Up-Regulation , bcl-2-Associated X Protein , Metabolism
17.
Chinese Journal of Hematology ; (12): 394-398, 2009.
Article in Chinese | WPRIM | ID: wpr-314473

ABSTRACT

<p><b>OBJECTIVE</b>To investigate the frequency of JAK2 V617F mutation in 145 myeloproliferative disorders (MPDs) patients, analyze the correlation between JAK2 V617F mutation and clinical features.</p><p><b>METHODS</b>The JAK2 V617F mutation was detected by direct DNA sequencing of PCR product and allele-specific PCR respectively. The expression of JAK2, phospho-JAK2 and phospho-STAT5 proteins was determined by Western blot. The clinical data of MPDs patients with or without JAK2 V617F mutation was collected and analyzed for evaluating the clinical significance of JAK2 V617F mutation.</p><p><b>RESULTS</b>1) The frequency of JAK2 V617F mutation for PV, IMF, ET was 92%, 58%, 50% respectively. Compared with conventional DNA sequencing (PV 84%, IMF 44%, ET 39%, respectively), allele-specific PCR exhibited a higher sensitivity in JAK2 V617F mutation detection. 2) The expression levels of phospho-JAK2 and phospho-STAT5 in peripheral blood mononuclear cells (PBMNCs) were upregulated significantly in JAK2 V617F-positive patients than in JAK2 V617F negative patients. 3) Compared with the patients with no JAK2 V617F mutation, the JAK2 V 617F-positive patients' features were as follows: older age of onset, higher mean leukocyte counts, lower platelet counts and smaller spleen volume. Frequency of thrombosis events in PT, ET, IMF was 17%, 32%, 16% respectively for JAK2 V617F positive group, and 0% (PV), 16% (ET), 5% (IMF) for JAK2 V617F negative group.</p><p><b>CONCLUSIONS</b>MPDs patients display higher frequency of JAK2 V617F mutation. JAK2 V617F mutation positive patients predispose to a thrombosis tendency.</p>


Subject(s)
Female , Humans , Janus Kinase 2 , Genetics , Metabolism , Male , Mutation , Myeloproliferative Disorders , Genetics , Metabolism , Phosphorylation , STAT5 Transcription Factor , Metabolism , Sequence Analysis, DNA
18.
Article in Chinese | WPRIM | ID: wpr-347983

ABSTRACT

<p><b>OBJECTIVE</b>To 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.</p><p><b>METHODS</b>Fifty-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.</p><p><b>RESULTS</b>Blood 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).</p><p><b>CONCLUSIONS</b>STAT5b 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.</p>


Subject(s)
Acetates , Pharmacology , Animals , Asthma , Blood , Drug Therapy , Pathology , BCG Vaccine , Pharmacology , Interferon-gamma , Interleukin-4 , Genetics , Leukocytes, Mononuclear , Metabolism , Male , Quinolines , Pharmacology , RNA, Messenger , Blood , Rats , Rats, Sprague-Dawley , STAT5 Transcription Factor , Genetics
19.
Article in Chinese | WPRIM | ID: wpr-281075

ABSTRACT

<p><b>OBJECTIVE</b>To investgate the signal transduction and regulation in erythropoiesis by angelica polysaccharides (APS) to clarify the mechanism for APS promoting hematopoiesis.</p><p><b>METHOD</b>The 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.</p><p><b>RESULT</b>Angelica 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.</p><p><b>CONCLUSION</b>JAK2, STAT5 signal transduction molecule plays an important role in the effect of APS cooperated with Epo on promoting hematopoiesis.</p>


Subject(s)
Angelica , Chemistry , Blotting, Western , Cells, Cultured , Erythropoietin , Pharmacology , Gene Expression , Hematopoiesis , Humans , Janus Kinase 2 , Metabolism , Microscopy, Confocal , Polysaccharides , Pharmacology , STAT5 Transcription Factor , Metabolism , Signal Transduction
20.
Chinese Journal of Hematology ; (12): 815-818, 2008.
Article in Chinese | WPRIM | ID: wpr-239954

ABSTRACT

<p><b>OBJECTIVE</b>To investigate the effect of meisoindigo on bcr-abl signaling pathway and to explore the mechanism of meisoindigo inducing apoptosis in K562 cells.</p><p><b>METHODS</b>Apoptosis and mitochondria membrane potential (MMP) were evaluated by flow cytometry. In K562 cells, the expression level of Bcl-2 family members, cleaved caspase members, bcr-abl, STAT5 and CRKL were determined by Western blot and bcr-abl mRNA expression level was measured by RT-PCR before and after meisoindigo treatment. The DNA binding potential of STAT3 and STAT5 was checked by electronic mobility shift assay (EMSA).</p><p><b>RESULTS</b>Down-regulation of total and phosphorylated bcr-abl protein level in K562 cells was observed when treated with 20 micromol/L meisoindigo, but its mRNA level was not changed. The expression level of phosphorylated STAT5 and CRKL was decreased and the DNA binding potential of STAT3 and STAT5 were inhibited in K562 cell after exposure to meisoindigo. Exposure to 5 - 20 micromol/L meisoindigo induced apoptosis accompanied with activating of caspase 3, 8, 9 and decreasing of MMP in K562 cells in a dose-dependent manner. The apoptosis was blocked by 50 micromol/L z-DEVD-fmk, z-IETD-cho, z-LETD-fmk, the specific inhibitors of caspase 3, 8, 9, respectively. No change in Bcl-2, Bax and Bid protein expression levels were observed before and after meisoindigo inducing apoptosis.</p><p><b>CONCLUSION</b>Meisoindigo can inhibit the proliferation of K562 cells by affecting the bcr-abl signaling transduction pathway. Meisoindigo induces K562 cell apoptosis through a novel caspase dependent pathway in addition to the contribution of mitochondria. The Bcl-2 family members are not involved in the apoptosis induction by meisoindigo in K562 cells.</p>


Subject(s)
Apoptosis , Caspases , Metabolism , Fusion Proteins, bcr-abl , Genetics , Metabolism , Humans , Indoles , Pharmacology , K562 Cells , Proto-Oncogene Proteins c-bcl-2 , Metabolism , RNA, Messenger , Genetics , STAT3 Transcription Factor , Metabolism , STAT5 Transcription Factor , Metabolism , Signal Transduction , bcl-2-Associated X Protein , Metabolism
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