摘要
To investigate the role of spinal interleukin-6-Janus kinase 2 (IL-6-JAK2) signaling transduction pathway in regulating astrocytes activation during the maintenance of bone cancer pain (BCP). Methods: NCTC 2472 fibrosarcoma cells were injected into the femur marrow cavity in C3H/HeNCrlVr male mice to establish BCP model and they were replaced by the equal volume of α-MEM in the sham model. The paw withdrawal latency (PWL) was measured after inoculation of tumor cells. The lumbar enlargement of spinal cord (L3-L5) was isolated, and Real-time RT-PCR and Western blot were used to detect the expression of spinal glial fibrillary acidic protein (GFAP) and JAK2 mRNA and protein, respectively. The expression level of spinal GFAP mRNA indirectly reflect astrocytes activation level. Pain behaviors and spinal cord GFAP mRNA and protein expression were observed at the given time points after intrathecal administration of JAK2 antagonist AG-490. Results: The PWL at 10, 14, 21 d after operation in BCP model group were significantly shorter than that in the sham group (P<0.05); the spinal GFAP and JAK2 mRNA and protein levels were higher in the BCP model group in comparison to mice in the sham group (P<0.05); intrathecal injection of JAK2 agonist AG-490 (30 or 90 nmol) significantly alleviated PWL, and downregulated the expression of spinal GFAP mRNA and protein (P<0.05). Conclusion: The IL-6-JAK2 signaling pathway plays an important role in maintaining the BCP by regulating the expression of GFAP in the spinal cord. Intrathecal injection of AG-490 can reduce the BCP, and inhibit the activation of IL-6-JAK2 signaling pathway, which may be one of the mechanisms for spinal astrocyte activation.
Subject(s)
Animals , Male , Mice , Astrocytes , Pathology , Bone Neoplasms , Hyperalgesia , Drug Therapy , Injections, Spinal , Mice, Inbred C3H , Rats, Sprague-Dawley , Spinal Cord , Cell Biology , Pathology , Tyrphostins摘要
To investigate the role of Janus kinase (JAK) inhibitor AG490 in the anti-proliferation and cell cycle in human retinoblastoma HXO-RB44 cell lines in vitro, and to explore its effect on the expression of JAK2/signal transducer and activator of transcription 3 (STAT3). Methods: Cells were divided into an experiment group and a control group, and the experiment group was further divided into 6 sub-groups according to different AG490 concentrations (6.25, 12.50, 25.00, 50.00 or 100.00 μmol/L). Cell proliferation in the different groups was analyzed by cell vitality determination. Cell cycle distribution and apoptosis rate were examined by flow cytometry. The protein levels of STAT3, p-STAT3 and vascular endothelial growth factor (VEGF) were detected by Western blot. Results: After 48 h treatment with AG490, the viability of HXO-RB44 cells was reduced in a concentration-dependent manner. Compared with the control group, there was no significant difference in the experiment groups except the 6.25 μmol/L group (all P>0.05). The apoptosis rates in the experiment groups were significantly increased with increase in concentration of AG490 compared with that in the control group (all P0.05), but there were significant differences in the other experiment groups (all P<0.05). Conclusion: JAK inhibitor AG490 can inhibit proliferation and promote apoptosis of the retinoblastoma HXO-RB44 cells through down-regulation of JAK2/STAT3 signaling pathway.
Subject(s)
Humans , Apoptosis , Cell Line, Tumor , Cell Proliferation , Cell Survival , Enzyme Inhibitors , Pharmacology , Gene Expression Regulation, Neoplastic , Janus Kinase 2 , Genetics , Metabolism , Retinoblastoma , STAT3 Transcription Factor , Genetics , Metabolism , Signal Transduction , Tyrphostins , Pharmacology , Vascular Endothelial Growth Factor A , Metabolism摘要
The aim of the present study was to investigate the mechanism of the inhibitory effect of luteolin on the proliferation of breast cancer cells induced by epidermal growth factor (EGF) in vitro. MTT assay was used to detect the inhibitory effect of luteolin on the proliferation of MCF-7 and MDA-MB-231 cells as well as the effect on the proliferation of MCF-7 cells induced by EGF. Western blotting was used to detect the effects of luteolin on the expression of epidermal growth factor receptor (EGFR), phosphatidylinositol 3-kinase (PI3K)/Akt, mitogen-activated protein kinase (MAPK)/extracellular-signal-regulated kinases (Erk) 1/2 and signal transducers and activators of transcription-3 (STAT3) in MCF-7 cells induced by EGF. The results showed that luteolin could significantly inhibit the proliferation of MCF-7 and MDA-MB-231 cells, and the inhibitory effect on MCF-7 cells was more prominent. Moreover, luteolin could inhibit the proliferation of MCF-7 cells induced by EGF. Western blotting results showed that luteolin and AG1478 (an inhibitor of EGFR signaling) could inhibit the expression of p-EGFR and p-STAT3 in MCF-7 cells induced by EGF. Luteolin, LY294002 (an inhibitor of Akt signaling) and PD98059 (an inhibitor of Erk1/2 signaling) could inhibit the expression of p-Akt and p-Erk1/2 respectively in MCF-7 cells induced by EGF. Our data suggest that luteolin may inhibit EGF-induced activities of EGFR signaling pathway in human breast cancer cell lines, and PI3K/Akt, MAPK/Erk1/2, STAT3 signal pathways may be the major pathways that mediate the inhibitory effect of luteolin on EGFR signaling. Overall, our results may provide a theoretical foundation for the development of luteolin as anti-tumor drug.
Subject(s)
Humans , Breast Neoplasms , Cell Line, Tumor , Cell Proliferation , Chromones , Epidermal Growth Factor , Luteolin , MAP Kinase Signaling System , Mitogen-Activated Protein Kinase 1 , Mitogen-Activated Protein Kinase 3 , Morpholines , Phosphatidylinositol 3-Kinases , Quinazolines , ErbB Receptors , Tyrphostins摘要
To investigate the effect of diosgenin (Dgn) on chondrocytes and its relation to JAK2/STAT3 signaling pathway in mice with osteoarthritis (OA).Fifteen male C57BL/6 mice were randomly divided into three groups:control group, OA group and OA+Dgn group. After 4 weeks of treatment, the histopathological changes of cartilage tissue were observed by toluidine blue staining under light microscopy and the ultrastructure of chondrocytes was observed under electron microscopy. The primarily cultured chondrocytes of OA mice were randomly divided into 4 groups:(1) OA group, (2) Dgn group, (3) Dgn+AG490 group, (4) AG490 group. The expression of p-JAK2, p-STAT3, Bax, succinate dehydrogenase (SDH) and cytochrome c oxidase (COX) were detected by Western blotting, and superoxide dismutase (SOD) was detected using colorimetric method.The morphological observation showed that the chondrocytes of OA group presented considerable pathological changes, while the chondrocytes in OA+Dgn group maintained intact membrane. Electron microscopy observation found obvious injury in cartilage tissues of OA group, while that in OA+Dgn group remained smooth. Compared with OA group, the expressions of p-JAK2 and p-STAT3 in chondrocytes of Dgn group were increased (all<0.05), and the expressions of Bax protein, SDH, COX and SOD were decreased (all<0.05). While compared with Dgn group, the expressions of p-JAK2, p-STAT3, SDH, COX and SOD in chondrocytes of Dgn+AG490 group were decreased (all<0.05), and the expression of Bax protein was increased (<0.05).Diosgenin can inhibit apoptosis and increase mitochondrial oxidative stress capacity of chondrocytes in mice with osteoarthritis, which is closely related to the activation of JAK2/STAT3 signaling pathway.
Subject(s)
Animals , Male , Mice , Apoptosis , Cartilage , Pathology , Chondrocytes , Chemistry , Pathology , Diosgenin , Pharmacology , Electron Transport Complex IV , Metabolism , Janus Kinase 2 , Mice, Inbred C57BL , Mitochondria , Genetics , Osteoarthritis , Genetics , Oxidative Stress , STAT3 Transcription Factor , Signal Transduction , Succinate Dehydrogenase , Metabolism , Superoxide Dismutase , Metabolism , Tyrphostins , Pharmacology , bcl-2-Associated X Protein , Metabolism摘要
OBJECTIVE@#To investigate the effects of combined inhibition of signal transducer and activator of transcription 3 (STAT3) and hypoxia-inducible factor-1α (HIF-1α) in the enhancement of chemosensitivity of the model of human laryngeal squamous cacinoma in nude mice.@*METHOD@#Model nude mice were divided into six groups randomly: control group(A) , cisplatin group(B) , cisplatin and AG490 group(C) , cisplatin and HIF-1α⁻/⁻ group (D), cisplatin combined AG490 and HIF-1α⁻/⁻ group (E), HIF-1α⁻/⁻ group (F) (only in calculating tumor inhibition rate). 3mg/kg cisplatin was administered by peritoneal injection for 3 days. Then cisplatin and 10 mg/kg AG490 were administered every other day for 12 days. The expression of Ki67 and HIF-1α was detected by immunocytochemical method. Western blot was used to detect the expression of p-STAT3.@*RESULT@#The expression of HIF-1α in group C and group D were lower than that in group B, and there were significant difference respectively (t₁ = 2.782, t₂ = 3.873, P 0.05); The expression level of p-STAT3 in group E was significantly lower compared with that in group C and group D respectively (P < 0.01). Tumor inhibition rate of group E was higher than that in group B, group C , as well as group D respectively and there were significant difference respectively (t₁ = 5.509, P < 0.01; t₂ = 3.422, P < 0.05; t₃ = 2.718, P < 0.05 ). Ki67 index of group E was lower than that in group B, group C as well as group D respectively and there were significant difference respectively(t₁ = 8.307, P < 0.01; t₂ = 3.736, P < 0.05; t₃ = 4.524, P < 0.01).@*CONCLUSION@#Combined inhibition of STAT3 and HIF-1α could enhance chemo-sensitivity in the model of human laryngeal squamous cacinoma in nude mice.
Subject(s)
Animals , Mice , Antineoplastic Agents , Pharmacology , Carcinoma, Squamous Cell , Drug Therapy , Metabolism , Cisplatin , Pharmacology , Drug Resistance, Neoplasm , Hypoxia-Inducible Factor 1, alpha Subunit , Genetics , Metabolism , Ki-67 Antigen , Metabolism , Laryngeal Neoplasms , Drug Therapy , Metabolism , Mice, Nude , Neoplasms, Experimental , Drug Therapy , Metabolism , STAT3 Transcription Factor , Genetics , Metabolism , Tyrphostins , Pharmacology摘要
The type 1 insulin-like growth factor receptor (IGF-1R) and its downstream signaling components have been increasingly recognized to drive the development of malignancies, including non-small cell lung cancer (NSCLC). This study aimed to investigate the effects of IGF-1R and its inhibitor, AG1024, on the progression of lung cancer. Tissue microarray and immunohistochemistry were employed to detect the expressions of IGF-1 and IGF-1R in NSCLC tissues (n=198). Western blotting was used to determine the expressions of IGF-1 and phosphorylated IGF-1R (p-IGF-1R) in A549 human lung carcinoma cells, and MTT assay to measure cell proliferation. Additionally, the expressions of IGF-1, p-IGF-1R and IGF-1R in a mouse model of lung cancer were detected by Western blotting and real-time fluorescence quantitative polymerase chain reaction (FQ-PCR), respectively. The results showed that IGF-1 and IGF-1R were overexpressed in NSCLC tissues. The expression levels of IGF-1 and p-IGF-1R were significantly increased in A549 cells treated with IGF-1 as compared to those treated with IGF-1+AG1024 or untreated cells. In the presence of IGF-1, the proliferation of A549 cells was significantly increased. The progression of lung cancer in mice treated with IGF-1 was significantly increased as compared to the group treated with IGF-1+AG1024 or the control group, with the same trend mirrored in IGF-1/p-IGF-1R/IGF-1R at the protein and/or mRNA levels. It was concluded that IGF-1 and IGF inhibitor AG1024 promotes lung cancer progression.
Subject(s)
Adult , Aged , Animals , Female , Humans , Male , Mice , Middle Aged , Carcinoma, Non-Small-Cell Lung , Metabolism , Pathology , Cell Proliferation , Disease Models, Animal , Disease Progression , Insulin-Like Growth Factor I , Metabolism , Lung Neoplasms , Metabolism , Pathology , Receptor, IGF Type 1 , Physiology , Tyrphostins , Pharmacology摘要
<p><b>OBJECTIVE</b>To investigate the role of TGF-beta1 and STAT3 signaling in liver fibrosis using a rat model system and to determine the therapeutic mechanism of AG490 in relation to this signaling pathway.</p><p><b>METHODS</b>Rats were randomly divided into a control group and DENA-induced liver fibrosis model group, and then subdivided into AG490 treatment groups. During fibrosis development, liver tissue samples were collected at different time points (0, 4 and 8 weeks) and evaluated according to the Scheuer scoring system. Expression of STAT3, TGFbeta1, alpha-SMA, E-cadherin, MMP2 and TIMP1 was measured by PCR (mRNA) and immunohistochemistry and western blotting (protein).</p><p><b>RESULTS</b>Increasing degrees of inflammation and fibrosis were observed in liver tissues of DENA-treated rats throughout model establishment. The mRNA expression of TGFbeta1 and STAT3 was significantly increased in DENA-induced rats with advanced fibrosis (AF) compared to those with early fibrosis (EF) (P = 0.034 and P = 0.012 respectively). The protein expression of TGF-beta1, phospho-Smad2, alpha-SMA, E-cadherin, STAT3 and phospho-STAT3 was significantly increased in DENA-induced rats with AF compared to the unmodeled control group (P = 0.048, P = 0.003, P = 0.002, P = 0.028, P = 0.009 and P = 0.039). The protein expression of E-cadherin was lower in the DENA-induced rats with AF than in those with EF (P = 0.026). STAT3 and TGF-beta1 co-expression was detected in AF tissues. DENA-induced AG490-treated rats with AF showed substantially lower protein expression of STAT3, TGF-beta1, MMP2 and TIMP1 compared to DENA-induced untreated rats with AF (P = 0.006, P = 0.018, P = 0.010 and P = 0.005); in addition, the degrees of fibrosis and inflammation were also greatly reduced in the DENA-induced AG490-treated rats with AF compared to DENA-induced untreated rats with AF (P = 0.042 and P = 0.021). Conclusions STAT3 signal transduction may regulate the TGF-beta1 pathway and affect liver fibrosis, especially in the advanced phase. AG490 can inhibit TGFbeta1-STAT3 signaling, resulting in reversal of liver fibrosis.</p>
Subject(s)
Animals , Rats , Disease Models, Animal , Liver Cirrhosis , Metabolism , Rats, Sprague-Dawley , STAT3 Transcription Factor , Metabolism , Signal Transduction , Transforming Growth Factor beta1 , Metabolism , Tyrphostins , Pharmacology摘要
Our previous studies indicate that phosphatidylinositol 4-kinase IIα can promote the growth of multi-malignant tumors via HER-2/PI3K and MAPK pathways. However, the molecular mechanisms of this pathway and its potential for clinical application remain unknown. In this study, we found that PI4KIIα could be an ideal combinatorial target for EGFR treatment via regulating EGFR degradation. Results showed that PI4KIIα knockdown reduced EGFR protein level, and the expression of PI4KIIα shows a strong correlation with EGFR in human breast cancer tissues (r = 0.77, P < 0.01). PI4KIIα knockdown greatly prolonged the effects and decreased the effective dosage of AG-1478, a specific inhibitor of EGFR. In addition, it significantly enhanced AG1478-induced inhibition of tumor cell survival and strengthened the effect of the EGFR-targeting anti-cancer drug Iressa in xenograft tumor models. Mechanistically, we found that PI4KIIα suppression increased EGFR ligand-independent degradation. Quantitative proteomic analysis by stable isotope labeling with amino acids in cell culture (SILAC) and LC-MS/MS suggested that HSP90 mediated the effect of PI4KIIα on EGFR. Furthermore, we found that combined inhibition of PI4KIIα and EGFR suppressed both PI3K/AKT and MAPK/ERK pathways, and resulted in downregulation of multiple oncogenes like PRDX2, FASN, MTA2, ultimately leading to suppression of tumor growth. Therefore, we conclude that combined inhibition of PI4KIIα and EGFR exerts a multiple anti-tumor effect. Dual inhibition of EGFR at protein and activity level via combinatorial blocking of PI4KIIα presents a novel strategy to combat EGFR-dependent tumors.
Subject(s)
Animals , Female , Humans , Male , Mice , Antineoplastic Agents , Pharmacology , Breast Neoplasms , Metabolism , Pathology , Cell Line, Tumor , Cell Survival , ErbB Receptors , Metabolism , HSP90 Heat-Shock Proteins , Metabolism , MCF-7 Cells , Mice, Inbred BALB C , Mice, Nude , Minor Histocompatibility Antigens , Mitogen-Activated Protein Kinases , Metabolism , Phosphatidylinositol 3-Kinases , Metabolism , Phosphotransferases (Alcohol Group Acceptor) , Genetics , Metabolism , Proto-Oncogene Proteins c-akt , Metabolism , Quinazolines , Pharmacology , Transplantation, Heterologous , Tyrphostins , Pharmacology摘要
<p><b>OBJECTIVE</b>To study the relationship of basic fibroblast growth factor (bFGF) and signal transducer and activator of transcription 3(STAT3) in glioma apoptosis and possible mechanisms of its interaction.</p><p><b>METHODS</b>Two glioblastomamultiforme (GBM) cell lines: U87 (wild-type p53) and U251 (mutant p53) were used in this study and divided into normal control group, mock group and experiment group.Small interfering RNA-carried recombinant lentivirus, LV-bFGFsiRNA and LV-STAT3siRNA, targeting bFGF and STAT3 were constructed respectively. After 48 hours of lentivirus transfection, small molecular inhibitors were used to block specific signaling pathways, AG490 20 µmol/L blocking JAK, LY294002 20 µmol/L blocking PI3K/Akt pathways for 24 hours, 48 hours and 72 hours, respectively. Then, apoptosis, changes in apoptosis-related proteins and mitochondrial membrane potential were detected through the methods of flow cytometry, protein chip and confocal microscopy, respectively.Groups were compared using single factor analysis of variance (One-way ANOVA).</p><p><b>RESULTS</b>Western blot results revealed the levels of Tyr705 and Ser727 phosphorylationin reduced in a time dependent manner by blocking JAK and PI3K/Akt pathway respectively. The results of flow cytometry showed that the apoptosis rate in normal control group, mock group, experiment group were 17.97% ± 0.24%, 18.26% ± 0.88%, 46.57% ± 1.63% in U87 cells and 15.94% ± 1.18%, 16.88% ± 0.17%, 39.34% ± 0.87% in U251 cells, respectively. There was no statistically significant change between the normal control group and the mock group (P > 0.05) , while when compared with the experiment group, both group showed statistically significant difference (F = 697.41, 729.58, both P < 0.05). The results of protein chip demonstrated that protein expression of Bad, Caspase3, Cytochrome C, p27 were higher and XIAP was lower in the experiment group compared with the normal control group and mock group. Also, confocal microscopy could detect apoptosis and mitochondrial membrane potential reduced significantly in the experimental group compared with the normal control group and the mock group.</p><p><b>CONCLUSIONS</b>bFGF mainly interacts with STAT3 tyrosine site-pSTAT3(Tyr705) to influence the level of STAT3 phosphorylation;blocking bFGF/STAT3 signaling pathway can induce glioma cell apoptosis through mitochondrial pathway.</p>
Subject(s)
Humans , Apoptosis , Brain Neoplasms , Metabolism , Cell Line, Tumor , Cytochromes c , Fibroblast Growth Factor 2 , Metabolism , Glioma , Metabolism , Mitochondria , Phosphatidylinositol 3-Kinases , Phosphorylation , RNA, Small Interfering , STAT3 Transcription Factor , Metabolism , Signal Transduction , Transfection , Tyrphostins摘要
<p><b>OBJECTIVE</b>To investigate the antitumour molecular mechanisms of WP1066 (STAT-3 inhibitor ) to human tongue squamous cell carcinoma in vitro and in vivo.</p><p><b>METHODS</b>WP1066 was used to inhibit the p-STAT-3 expression in Tscca human tongue squamous cell carcinoma cell line .Real-time PCR was used to detect the microRNA-21 expression after treatment with DMSO and WP1066. Methyl thiazolyl tatrozolium (MTT) assay was employed to determine cell survival. Flow cytometry (FCM) was used to measure apoptosis. The expression level of STAT-3/p-STAT-3, programmed cell death protein 4 (PDCD-4), antigen 67 (Ki-67), B cell lymphoma 2 (Bcl-2) and cleaved cysteinyl aspartate specific proteinase-3 (CCASP-3) was examined by Western blotting. Luciferase reporter gene assay was conducted to verify the regulation of STAT-3 to microRNA-21. Tscca xenograft tumor model was established in BALB/c nude mice and the tumors were divided into control, DMSO and WP1066 treated groups. The tumor tissues were measured by immunohistochemistry stain and terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL) assay.</p><p><b>RESULTS</b>STAT-3/p-STAT-3 protein was suppressed after treatment with WP1066 (STAT-3:F = 15.336, P = 0.004, p-STAT-3: F = 52.837, P = 0.000). MicroRNA-21 relative expression level was down-regulated (F = 8.197, P = 0.019). Cell survival rate was significantly reduced after treatment with WP1066 than control groups (F = 94.388, P = 0.000). Early apoptosis rate increased after treatment with WP1066 (F = 217.080, P = 0.000) . PDCD-4 and cleaved cysteinyl aspartate specific proteinase-3 (CCASP-3) protein expression was increased after treatment with WP1066 (PDCD-4:F = 8.771, P = 0.017; CCASP-3: F = 26.611, P = 0.001) .Ki-67 and Bcl-2 protein was down regulated (Ki-67:F = 5.854, P = 0.039; Bcl-2:F = 125.502, P = 0.000). Luciferase reporter gene assay proved that STAT-3 combined with specific promoter region of microRNA-21.In vivo, the tumor volume after treatment with WP1066 was significantly smaller than control groups by the end of observation (F = 15.390, P = 0.000) .Immunological histological chemistry indicated that PDCD-4 and CCASP-3 protein expression was up-regulated simultaneously while Ki-67 and Bcl-2 protein of tumor tissue was down-regulated after treatment with WP1066 than control groups. TUNEL assay suggested that apoptosis index rose after treatment with WP1066 than control groups (F = 133.368, P = 0.000) .</p><p><b>CONCLUSIONS</b>WP1066 affected Tscca cancer cell proliferation and apoptosis via inhibiting STAT-3/microRNA-21.WP1066 provided new direction and possibility to human tongue squamous cell carcinoma therapy.</p>
Subject(s)
Animals , Humans , Mice , Apoptosis , Apoptosis Regulatory Proteins , Carcinoma, Squamous Cell , Pathology , Cell Line, Tumor , Cell Proliferation , Down-Regulation , Heterografts , In Vitro Techniques , Mice, Nude , Pyridines , Pharmacology , Real-Time Polymerase Chain Reaction , STAT3 Transcription Factor , Signal Transduction , Tongue Neoplasms , Pathology , Tyrphostins , Pharmacology摘要
<p><b>OBJECTIVE</b>To investigate the effect and mechanism of signal transducers and activators of transcription 3 (STAT-3) modulates human tongue squamous cell carcinoma invasion ability via targeting mircoRNA-21.</p><p><b>METHODS</b>Tscca and Tca8113P160 human tongue squamous cell carcinoma cell lines were used.WP1066 (STAT-3 inhibitor) , the small molecule inhibitor of STAT-3 was used to suppress the STAT-3 expression. The half maximal inhibitory concentration (IC50 value) of WP1066 in the two cell lines was determined by methyl thiazolyl tetrazolium (MTT) assay. The expression level of STAT-3 and phosphorylation of STAT-3 (pSTAT-3) was examined by Western blotting. Real-time PCR was used to detect the mircoRNA-21 expression after treated with WP1066. Matrigel matrix and transwell assay were used to determine cancer cell colony formation and invasion ability after treated with WP1066. Tumor invasion related proteins in Tscca and Tca8113P160 cell lines were measured by Western blotting. Luciferase reporter gene assay was conducted to detect the relationship between STAT-3 and mircoRNA-21.</p><p><b>RESULTS</b>The IC50 to WP1066 in Tscca cell was 3.1 and 3.5 µmol/L for Tca8113P160 cell respectively. STAT-3/pSTAT-3 protein level was suppressed significantly (Tscca: STAT-3: F = 887.154, P = 0.000; pSTAT-3: F = 332.212, P = 0.000; Tca8113P160: STAT-3: F = 322.895, P = 0.000; pSTAT-3:F = 788.357, P = 0.000). mircoRNA-21 expression was down-regulated (Tscca:F = 32.157, P = 0.000; Tca8113P160: F = 11.349, P = 0.007). The diameters of culture clone in cell treated with WP1066 were less than control groups (Tscca:F = 15.751, P = 0.004; Tca8113P160: F = 12.964, P = 0.007). The number of tongue cancer cell migrating through the transwell membrane in WP1066 treated group was less than in control groups (Tscca: F = 1688.926, P = 0.000; Tca8113P160: F = 327.528, P = 0.000). In addition, MMP-2/9 protein expression was decreased in both of the cell lines treated with WP1066, while TIMP-3 was up regulated dramatically. STAT-3 could modulate mircoRNA-21 directly.</p><p><b>CONCLUSIONS</b>Reduction of STAT-3 can inhibit tongue cancer cell invasion ability via targeting mircoRNA-21.</p>
Subject(s)
Humans , Carcinoma, Squamous Cell , Genetics , Metabolism , Pathology , Cell Line, Tumor , Cell Movement , Cell Proliferation , Down-Regulation , Matrix Metalloproteinase 2 , Metabolism , Matrix Metalloproteinase 9 , Metabolism , MicroRNAs , Genetics , Metabolism , Phosphorylation , Pyridines , Pharmacology , STAT3 Transcription Factor , Metabolism , Signal Transduction , Tissue Inhibitor of Metalloproteinase-3 , Metabolism , Tongue Neoplasms , Genetics , Metabolism , Pathology , Tyrphostins , Pharmacology摘要
<p><b>BACKGROUND</b>Traumatic brain injury (TBI) is a major cause of death and disability in children and young adults worldwide. Therefore, we investigated the role of AG490 in regulating brain oedema, expression of CD40 and neurological function after TBI.</p><p><b>METHODS</b>Sprague Dawley rats (n = 240) were randomly divided into a sham operation group, TBI+saline group and TBI+AG490 (JAK/STAT inhibitor) group. Members of each group were euthanized at 6, 12, 24 or 72 hours after injury. Neurological severity score (NSS) was used to evaluate the severity of neurological damage. Brain water was quantitated by wet/dry weight method. The expression of CD40 was assessed by flow cytometry.</p><p><b>RESULTS</b>In both the TBI+saline group and the TBI+AG490 group, the brain water content was elevated after TBI, reached a peak at 24-hour and remained high for the rest of the period investigated; the expression of CD40 reached a peak 24 hours after TBI; the NSS was elevated after TBI and then decreased after 6 hours. Elevations in the level of CD40, degree of brain edema and NSS after TBI were significantly reduced in TBI+AG490 group.</p><p><b>CONCLUSION</b>Inhibition of the JAK/STAT signalling pathway reduces brain oedema, decreases the expression of CD40 and exerts neuroprotective effects after TBI.</p>
Subject(s)
Animals , Male , Rats , Brain Edema , Metabolism , Brain Injuries , Drug Therapy , CD40 Antigens , Flow Cytometry , Janus Kinases , Metabolism , Neuroprotective Agents , Therapeutic Uses , Rats, Sprague-Dawley , STAT Transcription Factors , Metabolism , Tyrphostins , Therapeutic Uses摘要
<p><b>OBJECTIVE</b>To detect the expression of phosphorylated-signal transducer and activator of transcription 3 (p-Stat3) and myeloid leukemia-1 (Mcl-1) as well as their correlation, and to investigate the functional role of Stat3 and Mcl-1 in the pathogenesis of esophageal squamous cell carcinoma (ESCC).</p><p><b>METHODS</b>Stat3 activity in ESCC cells was inhibited with JAK/Stat3 inhibitors (AG490 or JSI-124). Specific siRNA was used to inhibit the Stat3 expression. Cell apoptosis was detected by flow cytometry. Expression of Mcl-1 protein was determined by Western blotting. Expression of phospho-Stat3 (Tyr705) and myeloid leukemia-1 (Mcl-1) proteins in ESCC tissues was detected by tissue microarray and immunohistochemistry. The relationship between p-Stat3 or Mcl-1 aberrant expression and clinicopatholohical features of ESCC was analyzed. The correlation of their expression was also analyzed.</p><p><b>RESULTS</b>Suppression of the Stat3 signaling activation in ESCC cells led to marked apoptosis, and dramatic reduction of Mcl-1 protein. The positive rate of phospho-Stat3 (Tyr705) expression was 45.0% in 50/111 of the ESCC tissue samples. The lower the degree of tumor differentiation, the higher the positive rate of phospho-Stat3 (Tyr705), showing a significant difference (P = 0.018). The positive rate of Mcl-1 protein expression was 72.1% (80/111), and the lower the degree of tumor differentiation was, the higher there was the positive rate of Mcl-1, with a significant difference (P = 0.026). There was a positive correlation between the expressions of p-Stat3 and Mcl-1 proteins (P = 0.012).</p><p><b>CONCLUSIONS</b>In a subset of ESCC tissues, p-Stat3 (Tyr705) and Mcl-1 are overexpressed and positively correlated with each other, and both are correlated with tumor differentiation. Persistent activation of Stat3 contributes to apoptotic resistance in ESCC cells, and may be at least partly mediated through upregulation of Mcl-1.</p>
Subject(s)
Humans , Antineoplastic Agents , Pharmacology , Apoptosis , Carcinoma, Squamous Cell , Metabolism , Pathology , Cell Differentiation , Cell Line, Tumor , Cell Survival , Esophageal Neoplasms , Metabolism , Pathology , Myeloid Cell Leukemia Sequence 1 Protein , Metabolism , Neoplasm Grading , Neoplasm Staging , Phosphorylation , RNA, Small Interfering , Genetics , STAT3 Transcription Factor , Genetics , Metabolism , Tyrphostins , Pharmacology摘要
<p><b>OBJECTIVE</b>To explore the effects of EGFR-TKI AG1478 on the expression of FoxMl and FOXO3a genes in non-small cell cancer (NSCLC) cell lines, and explore the effect on cell proliferation and drug sensitivity to AG1478 after down-regulation of FOXMl and FOXO3a expression by RNAi technique.</p><p><b>METHODS</b>Human lung cancer cells were treated with AG1478 at different concentrations. RT-PCR and Western blot were used to examine the expression of P-EGFR, FOXM1, FOXO3a mRNA and protein. After transient transfection of FOXM1 and FOXO3a siRNA, RT-PCR and Western blot were employed to determine the transfection efficiency and expression of the related proteins. CCK-8 assay, colony formation assay and flow cytometry were performed to evaluate the cell proliferation, colony formation ability and the changes in cell cycle distribution.</p><p><b>RESULTS</b>The expressions of FOXM1 mRNA and protein were inhibited by AG1478 in a dose-dependent manner (both P < 0.05). After transfection with FOXM1 siRNA, the expressions of FOXM1 mRNA and protein, and proteins of cyclin B1, c-Myc, and Bcl-2 were significantly down-regulated, and the expressions of p21 and cleaved-PARP proteins were significantly up-regulated (all P < 0.05). The colony number of FOXM1siRNA transfection group was 37.3 ± 8.6, significantly lower than that of the blank control (135.3 ± 7.0) and negative control group (125.3 ± 7.5, P < 0.05). The colony formation inhibition rate was (7.40 ± 0.94)% in the negative control group and (72.4 ± 6.09)% in the FOXM1 siRNA transfection group. FOXM1siRNA transfection induced cell cycle arrest at G2/M phase with a percentage of (55.6 ± 4.83)%, significantly higher than that of the blank control [(24.30 ± 1.95)%] and negative control group [(21.3 ± 2.06)%, P < 0.05]. Additionally, the FOXM1siRNA transfection significantly increased the chemosensitivity of A549 cells to AG1478 (P < 0.05). Besides, AG1478 induced expression and nuclear relocation of FOXO3a. After the FOXO3a siRNA transfection, the expression of FOXM1 protein was significantly up-regulated, and resulted in a reduction of AG1478-induced inhibition of FOXM1.</p><p><b>CONCLUSIONS</b>The expression of FOXM1 is down-regulated by AG1478 via FOXO3a in the NSCLC cell lines, and then increases the chemosensitivity of A549 cells to AG1478. It suggests that FOXM1 could be a potential target for the therapy and drug exploitation for NSCLC.</p>
Subject(s)
Humans , Adenocarcinoma , Metabolism , Pathology , Apoptosis , Cell Cycle , Cell Line, Tumor , Cell Proliferation , Dose-Response Relationship, Drug , Down-Regulation , Forkhead Box Protein M1 , Forkhead Box Protein O3 , Forkhead Transcription Factors , Genetics , Metabolism , Lung Neoplasms , Metabolism , Pathology , Quinazolines , Pharmacology , RNA, Messenger , Metabolism , RNA, Small Interfering , Genetics , ErbB Receptors , Transfection , Tyrphostins , Pharmacology摘要
<p><b>OBJECTIVE</b>To investigate the relationship between EGFR activation and down-regulation of miRNA-145 in lung cancer.</p><p><b>METHODS</b>Normal human lung epithelia cell line (BEAS-2B), human lung adenocarcinoma cell lines with wild-type EGFR (A549 and H292) and human lung adenocarcinoma cell lines with EGFR mutation (H1975 and H1650) were chosen in this study. The levels of miRNA-145 and p-EGFR were determined by quantitative real-time PCR (qRT-PCR) and Western blotting, respectively, and the relationship between p-EGFR and miRNA-145 levels was analyzed. The miRNA-145 levels were determined by qRT-PCR after activating EGFR with EGF or blocking EGFR signal pathway with AG1478. In addition, ERK1/2 inhibitor U0126 was used to inhibit ERK1/2 activation and then the expression of miRNA-145 was detected.</p><p><b>RESULTS</b>The miRNA-145 levels were closely negatively related with p-EGFR in lung cancer cells (r = -0.926, P = 0.024). EGF down-regulated miRNA-145 expression, particularly in BEAS-2B cells (53.0%; t = 30.993, P = 0.001) and A549 cells (42.6%; t = 14.326, P = 0.005).The miRNA-145 was up-regulated after inhibiting p-EGFR with AG1478, and significantly enhanced by 67.5% in H1975 cells when treated with AG1478 (t = 8.269, P = 0.014). The ERK1/2 signal pathway was activated by p-EGFR. U0126 restored the miRNA-145 down-regulation induced by EGFR-activation in lung cancer cells.</p><p><b>CONCLUSION</b>The activation of EGFR down-regulates miRNA-145 expression through ERK1/2 in lung cancer cells.</p>
Subject(s)
Humans , Butadienes , Pharmacology , Carcinoma, Non-Small-Cell Lung , Metabolism , Pathology , Cell Line , Cell Line, Tumor , Down-Regulation , Enzyme Activation , Enzyme Inhibitors , Pharmacology , Epidermal Growth Factor , Pharmacology , Epithelial Cells , Metabolism , Lung , Cell Biology , Lung Neoplasms , Metabolism , Pathology , MAP Kinase Signaling System , MicroRNAs , Metabolism , Mitogen-Activated Protein Kinase 1 , Metabolism , Mitogen-Activated Protein Kinase 3 , Metabolism , Nitriles , Pharmacology , Phosphorylation , Quinazolines , Pharmacology , ErbB Receptors , Metabolism , Tyrphostins , Pharmacology摘要
<p><b>OBJECTIVE</b>To study the molecular mechanism of epidermal growth factor receptor (EGFR) signaling pathway in mediating paclitaxel-resistance and improving paclitaxel sensitivity in human melanoma A375 cells.</p><p><b>METHODS</b>Human melanoma cell line A375 cells were treated with different concentrations of paclitaxel with or without 20 µmol/L AG1478 (EGFR inhibitor), 40 µmol/L PD98059 (extracellular signal conditioning kinase (ERK) 1/2 blockers) or 10 µmol/L LY294002 (PI3K inhibitor). MTT method was used to measure the proliferation of A375 cells. Flow cytometry was used to detect cell cycle and apoptosis in the A375 cells. The expressions of P-EGFR, P-ERK and P-AKT proteins were determined by Western blot analysis.</p><p><b>RESULTS</b>Paclitaxel (0.001 µmol/L to 0.1 µmol/L) inhibited the growth of A375 cells (P < 0.01) and induced apoptosis (P < 0.05) in a dose- and time-dependent manner. AG1478 (20 µmol/L) increased the 0.01 µmol/L paclitaxel-induced inhibition rate from 38.5% to 62.6% at 72 h. Different doses of paclitaxel induced apoptosis in A375 cells by different ways, in which G0/G1 phase cells were decreased and mitotic phase was prolonged at 0.01 µmol/L, and cell cycle arrest at G2/M phase by 0.1 µmol/L paclitaxel. When DNA damage occurred in A375 cells exposed to paclitaxel, expression of P-EGFR, P-ERK and P-AKT proteins was increased. When EGFR signaling pathway was blocked, paclitaxel did not activate MAPK signaling pathway or PI3K/AKT signaling pathway and did not change its effect on cell cycle in vitro. When EGFR was inhibited by 20 µmol/L tyrophostin AG1478, the 0.001 and 0.01 µmol/L paclitaxel-induced early apoptosis rate in A375 cells was increased by 1.73- and 1.80-fold, respectively. When the ERK signaling was blocked by 40 µmol/L PD98059, the 0.001 and 0.01 µmol/L paclitaxel-induced early apoptosis rate in A375 cells was increased by 2.73- and 2.25-fold, respectively. When the AKT signaling was blocked by 10 µmol/L LY294002, the 0.001 and 0.01 µmol/L paclitaxel-induced early apoptosis rate in A375 cells was increased by 2.02- and 1.46-fold, respectively.</p><p><b>CONCLUSIONS</b>Human melanoma A375 cells produce resistance to paclitaxel (0.001 to 0.1 µmol/L) by activating MAPK signaling and PI3K/AKT signaling pathways. Targeting EGFR, ERK and AKT signaling pathways significantly enhances the cytotoxic effect of paclitaxel on human melanoma cells.</p>
Subject(s)
Humans , Antineoplastic Agents, Phytogenic , Pharmacology , Apoptosis , Cell Cycle , Cell Line, Tumor , Cell Proliferation , Chromones , Pharmacology , Dose-Response Relationship, Drug , Drug Resistance, Neoplasm , Extracellular Signal-Regulated MAP Kinases , Metabolism , Flavonoids , Pharmacology , Melanoma , Metabolism , Pathology , Morpholines , Pharmacology , Paclitaxel , Pharmacology , Phosphatidylinositol 3-Kinases , Metabolism , Phosphorylation , Proto-Oncogene Proteins c-akt , Metabolism , Quinazolines , Pharmacology , ErbB Receptors , Metabolism , Signal Transduction , Tyrphostins , Pharmacology摘要
The JAK2/STAT3 signal pathway is an important component of survivor activating factor enhancement (SAFE) pathway. The objective of the present study was to determine whether the JAK2/STAT3 signaling pathway participates in hydrogen sulfide (H2S) postconditioning, protecting isolated rat hearts from ischemic-reperfusion injury. Male Sprague-Dawley rats (230-270 g) were divided into 6 groups (N = 14 per group): time-matched perfusion (Sham) group, ischemia/reperfusion (I/R) group, NaHS postconditioning group, NaHS with AG-490 group, AG-490 (5 µM) group, and dimethyl sulfoxide (DMSO; <0.2%) group. Langendorff-perfused rat hearts, with the exception of the Sham group, were subjected to 30 min of ischemia followed by 90 min of reperfusion after 20 min of equilibrium. Heart rate, left ventricular developed pressure (LVDP), left ventricular end-diastolic pressure (LVEDP), and the maximum rate of increase or decrease of left ventricular pressure (± dp/dt max) were recorded. Infarct size was determined using triphenyltetrazolium chloride (TTC) staining. Myocardial TUNEL staining was used as the in situ cell death detection method and the percentage of TUNEL-positive nuclei to all nuclei counted was used as the apoptotic index. The expression of STAT3, bcl-2 and bax was determined by Western blotting. After reperfusion, compared to the I/R group, H2S significantly improved functional recovery and decreased infarct size (23.3 ± 3.8 vs 41.2 ± 4.7%, P < 0.05) and apoptotic index (22.1 ± 3.6 vs 43.0 ± 4.8%, P < 0.05). However, H2S-mediated protection was abolished by AG-490, the JAK2 inhibitor. In conclusion, H2S postconditioning effectively protects isolated I/R rat hearts via activation of the JAK2/STAT3 signaling pathway.
Subject(s)
Animals , Male , Rats , Hydrogen Sulfide/metabolism , Ischemic Postconditioning , /metabolism , Myocardial Reperfusion Injury/metabolism , /metabolism , Apoptosis , /analysis , Rats, Sprague-Dawley , Signal Transduction , /analysis , Tyrphostins摘要
PURPOSE: There is growing evidence that nasal airway remodeling occurs in allergic rhinitis (AR). Although angiogenesis is an important component of airway remodeling in asthma, its involvement in AR has been little studied. Furthermore, information regarding the role of potent angiogenic factors, such as vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF), in the nasal airway remodeling process is limited. This study was conducted to investigate the role of VEGF and PDGF in nasal airway remodeling, and to assess the preventive effects of anti-angiogenic drugs on this process in a murine AR model. METHODS: Mice were systemically sensitized and subjected to inhalation of ovalbumin (OVA) twice a week for 3 months. Control mice were challenged with phosphate buffered saline, while the treatment group received SU1498, a VEGF receptor inhibitor, and/or AG1296, a PDGF receptor inhibitor, via intraperitoneal injection 4 hours prior to each OVA inhalation. Staining using hematoxylin and eosin, Masson's trichrome, and periodic acid-Schiff were separately performed to assess eosinophil infiltration, subepithelial fibrosis, and goblet cell hyperplasia, respectively, in the nasal airway. Immunohistochemical staining for matrix metalloproteinase-9 (MMP-9) and tissue inhibitor of metalloproteinase-1 (TIMP-1) was also conducted. RESULTS: Repetitive intranasal inhalation of OVA resulted in significant increases in eosinophil infiltration, subepithelial fibrosis, goblet cell count, and MMP-9/TIMP-1 expression. Administration of SU1498 or AG1296 prevented these abnormal responses. CONCLUSIONS: The results of this study suggest that a causal relationship may exist between angiogenic factors and nasal airway remodeling in AR. Inhibition of VEGF or PDGF receptors may, in turn, suppress the remodeling process through the regulation of MMP-9/TIMP-1 expression.
Subject(s)
Animals , Mice , Airway Remodeling , Angiogenesis Inducing Agents , Angiogenesis Inhibitors , Asthma , Cinnamates , Eosine Yellowish-(YS) , Eosinophils , Fibrosis , Goblet Cells , Hematoxylin , Hyperplasia , Inhalation , Injections, Intraperitoneal , Matrix Metalloproteinase 9 , Nose , Ovalbumin , Ovum , Platelet-Derived Growth Factor , Receptors, Platelet-Derived Growth Factor , Receptors, Vascular Endothelial Growth Factor , Rhinitis , Rhinitis, Allergic, Perennial , Tissue Inhibitor of Metalloproteinase-1 , Tyrphostins , Vascular Endothelial Growth Factor A摘要
This study was aimed to investigate the effect of AG490, a JAK2 inhibitor, on expression of PHLPP and p-Akt in K562. K562 cells were treated with different concentrations of AG490. The proliferation of K562 cells was examined by WST-1 assay and apoptosis of K562 cells was detected by flow cytometry with Annexin V-FITC/PI double staining. The expressions of PHLPP, phosphorate-Akt (p-Akt) and total Akt protein were detected by Western blot. The results indicated that AG490 inhibited the proliferation of K562 cells in concentration-and time-dependent manners, with the IC(50) 338.0 µmol/L in 48 h. AG490 100 µmol/L also induced apoptosis of K562 cells in a time-dependent manner. AG490 100 µmol/L time-dependently down-regulated the protein expression of p-Akt and PHLPP, but without significant effect on expression of total Akt. It is concluded that AG490 can inhibit proliferation and induce apoptosis of K562 cells through down-regulation of p-Akt expression, but inhibiting efficacy of AG490 on K562 proliferation also may be limited due to the down-regulation of p-Akt regulatory protein PHLPP expression.
Subject(s)
Humans , Cell Proliferation , Down-Regulation , K562 Cells , Nuclear Proteins , Metabolism , Phosphoprotein Phosphatases , Metabolism , Tyrphostins , Pharmacology摘要
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.