BACKGROUND: Drug resistance is the main cause of high mortality of lung cancer. This study was conducted to investigate the effect of folic acid (FA) on the resistance of non-small cell lung cancer (NSCLC) cells to Osimertinib (OSM) by regulating the methylation of dual specificity phosphatase 1 (DUSP1). METHODS: The OSM resistant NSCLC cell line PC9R was establishd by gradually escalation of OSM concentration in PC9 cells. PC9R cells were randomly grouped into Control group, OSM group (5 μmol/L OSM), FA group (600 nmol/L FA), methylation inhibitor decitabine (DAC) group (10 μmol/L DAC), FA+OSM group (600 nmol/L FA+5 μmol/L OSM), and FA+OSM+DAC group (600 nmol/L FA+5 μmol/L OSM+10 μmol/L DAC). CCK-8 method was applied to detect cell proliferation ability. Scratch test was applied to test the ability of cell migration. Transwell assay was applied to detect cell invasion ability. Flow cytometry was applied to measure and analyze the apoptosis rate of cells in each group. Real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) method was applied to detect the expression level of DUSP1 mRNA in cells. Methylation specific PCR (MSP) was applied to detect the methylation status of the DUSP1 promoter region in each group. Western blot was applied to analyze the expression levels of DUSP1 protein and key proteins in the DUSP1 downstream mitogen-activated protein kinase (MAPK) signaling pathway in each group. RESULTS: Compared with the Control group, the cell OD450 values (48 h, 72 h), scratch healing rate, number of cell invasions, and expression of DUSP1 in the OSM group were obviously decreased (P<0.05); the apoptosis rate, the methylation level of DUSP1, the expression of p38 MAPK protein, and the phosphorylation level of extracellular regulated protein kinases (ERK) were obviously increased (P<0.05); the cell OD450 values (48, 72 h), scratch healing rate, number of cell invasions, and expression of DUSP1 in the DAC group were obviously increased (P<0.05); the apoptosis rate, the expression of p38 MAPK protein, the phosphorylation level of ERK, and the methylation level of DUSP1 were obviously reduced (P<0.05). Compared with the OSM group, the cell OD450 values (48, 72 h), scratch healing rate, number of cell invasions, and expression of DUSP1 in the FA+OSM group were obviously decreased (P<0.05); the apoptosis rate, the methylation level of DUSP1, the expression of p38 MAPK protein, and the phosphorylation level of ERK were obviously increased (P<0.05). Compared with the FA+OSM group, the cell OD450 values (48, 72 h), scratch healing rate, number of cell invasions, and expression of DUSP1 in the FA+OSM+DAC group were obviously increased; the apoptosis rate, the methylation level of DUSP1, the expression of p38 MAPK protein, and the phosphorylation level of ERK were obviously reduced (P<0.05). CONCLUSIONS FA may inhibit DUSP1 expression by enhancing DUSP1 methylation, regulate downstream MAPK signal pathway, then promote apoptosis, inhibit cell invasion and metastasis, and ultimately reduce OSM resistance in NSCLC cells.
Humans ; Carcinoma, Non-Small-Cell Lung/genetics* ; Lung Neoplasms/genetics* ; Dual Specificity Phosphatase 1/pharmacology* ; Cell Proliferation ; p38 Mitogen-Activated Protein Kinases/pharmacology* ; Methylation ; Apoptosis ; Cell Line, Tumor

OBJECTIVETo investigate the effects of evodiamine (Evo), a component of Evodiaminedia rutaecarpa (Juss.) Benth, on cardiomyocyte hypertrophy induced by angiotensin II (Ang II) and further explore the potential mechanisms.

METHODSCardiomyocytes from neonatal Sprague Dawley rats were isolated and characterized, and then the cadiomyocyte cultures were randomly divided into control, model (Ang II 0.1 μmol/L), and Evo (0.03, 0.3, 3 μmol/L) groups. The cardiomyocyte surface area, protein level, intracellular free calcium ([Ca]) concentration, activity of nitric oxide synthase (NOS) and content of nitric oxide (NO) were measured, respectively. The mRNA expressions of atrial natriuretic factor (ANF), calcineurin (CaN), extracellular signal-regulated kinase-2 (ERK-2), and endothelial nitric oxide synthase (eNOS) of cardiomyocytes were analyzed by real-time reverse transcriptionpolymerase chain reaction. The protein expressions of calcineurin catalytic subunit (CnA) and mitogen-activated protein kinase phosphatase-1 (MKP-1) were detected by Western blot analysis.

RESULTSCompared with the control group, Ang II induced cardiomyocytes hypertrophy, as evidenced by increased cardiomyocyte surface area, protein content, and ANF mRNA expression; increased intracellular free calcium ([Ca]) concentration and expressions of CaN mRNA, CnA protein, and ERK-2 mRNA, but decreased MKP-1 protein expression (P<0.05 or P<0.01). Compared with Ang II, Evo (0.3, 3 μmol/L) significantly attenuated Ang II-induced cardiomyocyte hypertrophy, decreased the [Ca] concentration and expressions of CaN mRNA, CnA protein, and ERK-2 mRNA, but increased MKP-1 protein expression (P<0.05 or P<0.01). Most interestingly, Evo increased the NOS activity and NO production, and upregulated the eNOS mRNA expression (P<0.05).

CONCLUSIONEvo signifificantly attenuated Ang II-induced cardiomyocyte hypertrophy, and this effect was partly due to promotion of NO production, reduction of [Ca]i concentration, and inhibition of CaN and ERK-2 signal transduction pathways.

Angiotensin II ; Animals ; Atrial Natriuretic Factor ; metabolism ; Calcineurin ; genetics ; metabolism ; Calcium ; metabolism ; Dual Specificity Phosphatase 1 ; genetics ; metabolism ; Extracellular Signal-Regulated MAP Kinases ; genetics ; metabolism ; Hypertrophy ; Myocytes, Cardiac ; drug effects ; metabolism ; pathology ; Nitric Oxide ; metabolism ; Nitric Oxide Synthase Type III ; metabolism ; Quinazolines ; pharmacology ; RNA, Messenger ; genetics ; metabolism ; Rats, Sprague-Dawley

Background: Glucocorticoid (GC) is the first-line therapy for asthma, but some asthmatics are insensitive to it. Glucocorticoid-induced transcript 1 gene (GLCCI1) is reported to be associated with GCs efficiency in asthmatics, while its exact mechanism remains unknown. Methods: A total of 30 asthmatic patients received fluticasone propionate for 12 weeks. Forced expiratory volume in 1 s (FEV) and GLCCI1 expression were detected. Asthma model was constructed in wild-type and GLCCI1 knockout (GLCCI1) mice. Glucocorticoid receptor (GR) and mitogen-activated protein kinase phosphatase 1 (MKP-1) expression were detected by polymerase chain reaction and Western blotting (WB). The phosphorylation of p38 mitogen-activated protein kinase (MAPK) was also detected by WB. Results: In asthmatic patients, the change of FEV was well positively correlated with change of GLCCI1 expression (r = 0.430, P = 0.022). In animal experiment, GR and MKP-1 mRNA levels were significantly decreased in asthmatic mice than in control mice (wild-type: GR: 0.769 vs. 1.000, P = 0.022; MKP-1: 0.493 vs. 1.000, P < 0.001. GLCCI1: GR: 0.629 vs. 1.645, P < 0.001; MKP-1: 0.377 vs. 2.146, P < 0.001). Hydroprednisone treatment significantly increased GR and MKP-1 mRNA expression levels than in asthmatic groups; however, GLCCI1 asthmatic mice had less improvement (wild-type: GR: 1.517 vs. 0.769, P = 0.023; MKP-1: 1.036 vs. 0.493, P = 0.003. GLCCI1: GR: 0.846 vs. 0.629, P = 0.116; MKP-1: 0.475 vs. 0.377, P = 0.388). GLCCI1 asthmatic mice had more obvious phosphorylation of p38 MAPK than wild-type asthmatic mice (9.060 vs. 3.484, P < 0.001). It was still higher even though after hydroprednisone treatment (6.440 vs. 2.630, P < 0.001). Conclusions: GLCCI1 deficiency in asthmatic mice inhibits the activation of GR and MKP-1 and leads to more obvious phosphorylation of p38 MAPK, leading to a decremental sensitivity to GCs. Trial Registration ChiCTR.org.cn, ChiCTR-RCC-13003634; http://www.chictr.org.cn/showproj.aspx?proj=5926.
Animals ; Asthma ; drug therapy ; metabolism ; Dual Specificity Phosphatase 1 ; genetics ; metabolism ; Forced Expiratory Volume ; genetics ; physiology ; Glucocorticoids ; therapeutic use ; Mice ; Mice, Knockout ; Phosphorylation ; genetics ; physiology ; Receptors, Glucocorticoid ; deficiency ; genetics ; metabolism ; p38 Mitogen-Activated Protein Kinases ; genetics ; metabolism

The opening of mitochondrial permeability transition pore (MPTP) plays a critical role in platelet activation. However, the potential trigger of the MPTP opening in platelet activation remains unknown. Inflammation is the crucial trigger of platelet activation. In this study, we aimed to explore whether and how the important inflammatory cytokine IL-17 is associated with MPTP opening in platelets activation by using MPTP inhibitor cyclosporine-A (CsA). The mitochondrial membrane potential (ΔΨm) was detected to reflect MPTP opening levels. And the platelet aggregation, activation, and the primary signaling pathway were also tested. The results showed that the MPTP opening levels were increased and Δψm reduced in platelets administrated with IL-17. Moreover, the levels of aggregation, CD62P, PAC-1, P53 and the phosphorylation of ERK2 were enhanced along with the MPTP opening in platelets pre-stimulated with IL-17. However, CsA attenuated these effects triggered by IL-17. It was suggested that IL-17 could induce MPTP opening through ERK2 and P53 signaling pathway in platelet activation and aggregation.
Blood Platelets ; cytology ; drug effects ; metabolism ; Cell Separation ; Cyclosporine ; pharmacology ; Dual Specificity Phosphatase 2 ; genetics ; metabolism ; Gene Expression Regulation ; Humans ; Interleukin-17 ; metabolism ; pharmacology ; Membrane Potential, Mitochondrial ; drug effects ; Mitochondria ; drug effects ; metabolism ; Mitochondrial Membrane Transport Proteins ; agonists ; antagonists & inhibitors ; genetics ; metabolism ; Mitogen-Activated Protein Kinase 1 ; genetics ; metabolism ; P-Selectin ; genetics ; metabolism ; Phosphorylation ; drug effects ; Platelet Activation ; drug effects ; Platelet Aggregation ; drug effects ; Primary Cell Culture ; Signal Transduction ; Tumor Suppressor Protein p53 ; genetics ; metabolism

The purpose of this research is to explore the distribution and expression of MAP kinase phosphatase-1 (MKP-1) in cerebrospinal fluid (CSF)-contacting nucleus in depression, and provide experimental evidence to reveal the biological function and regulatory mechanisms of CSF-contacting nucleus in depression. Depression model was produced by chronic forced swimming stress (CFSS) in Sprague-Dawley (SD) rats. Intracerebroventricular injection of cholera toxin subunit B (CTb) labeled with horseradish peroxidase (CB-HRP) was used to specifically mark distal CSF-contacting nucleus. The rate of animal growth and behavioral tests including sucrose preference test (SPT) and open field test (OFT) were used to validate the model of depression. The expressions of MKP-1 and fos proteins in CSF-contacting nucleus were detected by immunofluorescence. Software Image-Pro Plus version 6.0 was used to count the positive neurons. The results showed that, the distributions of MKP-1 were found in the CSF-contacting nucleus. After 28 days of swimming, the rats in stress group had a lower growth rate, a less consumption of sucrose and lower scores of OFT compared to control group. The number of neurons double labeled with CB-HRP/fos or CB-HRP/MKP-1 in stress group was significantly higher than that in control group (P < 0.01). These results suggest that the CSF-contacting nucleus may be involved in the process of depression via the MKP-1.
Animals ; Cerebrospinal Fluid ; Depression ; enzymology ; Dual Specificity Phosphatase 1 ; physiology ; Neurons ; physiology ; Rats ; Rats, Sprague-Dawley ; Stress, Physiological

OBJECTIVETo investigate the post-transcriptional regulation of dual-specificity phosphatase-1 (DUSP1) by the RNA- binding protein HuR in heat shock.

METHODSThe recombinant plasmids carrying wild-type (WT) HuR or its mutants at threonine 118 were constructed and transiently transfected into NIH 3T3 cells via liposome, and the changes in the expressions of DUSP1 mRNA and protein were detected by quantitative real-time PCR and Western blotting, respectively.

RESULTSHeat shock caused significantly enhanced phosphorylation of HuR at the residue T118. In 3T3 cells transfected with the plasmids carrying wild-type HuR for its over-expression showed significantly up-regulated DUSP1 mRNA and protein expressions at 24 h after transfection. Over-expression of HuR(T118A) down-regulated DUSP1 mRNA and protein expressions in cells challenged with heat shock, while HuR(T118E) over-expression significantly increased DISP1 expression at both mRNA and protein levels. After heat shock, HuR(WT) translocated from the cell nucleus to the cytoplasm to form particles. HuR(T118E) was diffusely distributed in the cytoplasm before heat shock and formed particles after heat shock. HuR(T118A) did not undergo such translocation in response to heat shock challenge.

CONCLUSIONHuR regulates DUSP1 mRNA and protein expression at the post-transcriptional level to increase its expression after heat shock by enhancing the phosphorylation HuR T118.

Animals ; Cell Nucleus ; Cytoplasm ; Dual Specificity Phosphatase 1 ; genetics ; metabolism ; ELAV Proteins ; metabolism ; Gene Expression Regulation ; Heat-Shock Response ; Hot Temperature ; Mice ; NIH 3T3 Cells ; Phosphorylation ; RNA, Messenger ; Real-Time Polymerase Chain Reaction ; Transfection ; Up-Regulation

The aim of this study is to investigate the apoptotic inhibition and its molecular mechanism of dexamethasone (DEX) acting on cisplatin (CDDP)-induced apoptosis of human lung adenocarcinoma cell SPC-A1; SPC-A1 cells were pre-cultured in vitro for 24 hours with DEX in different concentrations and then CDDP was added in different concentrations for culturing for further 48 hours. The survival rates of the cells were determined by MTT. The expression of serum/glucocorticoid-induced kinase (SGK-1) and mitogen-activated protein kinase phosphatase-1 (MKP-1) in SPC-A1 cells after being cultured by 1 micromol/l DEX at different time was detected by semi-quantitative RT-PCR technology. The expression of glucocorticoid receptor (GR) in SPC-A1 cells was measured by immunohistochemistry (IHC) with biotin-labeled anti-GR. The results of MTT showed that SPC-A1 cells had resistance to CDDP-induced apoptosis with pre-cultured DEX and the resistance intensity presented DEX concentration-dependent. The expressing quantity of SGK-1 in SPC-A1 cells stimulated by DEX could be elevated and increased with intention of time, but the express of MKP-1 was not detected. Up-regulated expression of GR in SPC-A1 cells stimulated by DEX was detected by IHC. The number of cells expressing GR in SPC-A1 cells was significantly higher than that in the control group. The results showed that DEX inhibited apoptosis of SPC-A1 cells induced by CDDP. The possible molecular mechanism is that elevated expression of GR induced by DEX up-regulates the expression of SGK-1 which locates at the downstream of anti-apoptosis pathway. The apoptosis resistance of SPC-A1 cells may account for all above the factors.
Adenocarcinoma ; pathology ; Apoptosis ; drug effects ; Cell Line, Tumor ; Cisplatin ; pharmacology ; Dexamethasone ; pharmacology ; Dual Specificity Phosphatase 1 ; metabolism ; Humans ; Immediate-Early Proteins ; metabolism ; Lung Neoplasms ; pathology ; Protein-Serine-Threonine Kinases ; metabolism ; Receptors, Glucocorticoid ; metabolism ; Up-Regulation

OBJECTIVETo evaluate the expression of mitogen activated protein kinase phosphatase-1(MKP-1)in pancreatic cancer.

METHODSTotally 60 cases of normal pancreas, chronic pancreatitis(CP), and pancreatic cancer tissues were collected by operation in our hospital. Pancreatic tissues were analyzed by Northern blot analysis and Western blot analysis. Meanwhile, MKP-1 expression was detected in 6 pancreatic cancer cell lines by Western blot analysis.

RESULTSNorthern blot analysis of total RNA revealed relatively low MKP-1 mRNA expression in 7 of 20(35%)normal pancreatic samples. In the remaining 13 samples, the MKP-1 mRNA was absent to faint detectable. In 7 of the 20 CP samples, MKP-1 was demonstrated moderate to high expression. In contrast, 12 of 20(60%)pancreatic cancer samples MKP-1 mRNA was expressed at high levels, whereas in the remaining 8 cancer tissues this mRNA moiety was present at low to moderate levels. Densitometric analysis with normalization to 7S revealed that the median level of MKP-1 mRNA in CP and cancerous tissues was increased by 6.2 folds(P=0.035)and 8.1 folds(P=0.016)in comparison with the median level in the normal pancreatic samples, respectively. Overexpression of MKP-1 was also found in 6 pancreatic cancer cell lines, in which the expression of MKP-1 was slightly lower in one pancreatic cancer cell line but high in the remaining 5 cell lines.

CONCLUSIONSMKP-1 is over-expressed in pancreatic cancer, CP tissues, and pancreatic cell lines. It is speculated that MKP-1 may play an important role in tumorigenesis of pancreatic cancer.

Blotting, Northern ; Blotting, Western ; Dual Specificity Phosphatase 1 ; metabolism ; Humans ; Immunohistochemistry ; Pancreas ; Pancreatic Neoplasms ; metabolism ; RNA, Messenger

The mitogen-activated protein kinase (MAPK) phosphatase-1 (MKP-1) belongs to the MAPK cascades which are central to cell proliferation and apoptosis. The carcinogenic role of MKP-1 has been reported in many types of cancer but it has rarely been investigated in breast cancer. The present study was designed to evaluate the MKP-1 mRNA expression and its possible regulation by methylation of MKP-1 promoter in the model of several breast cancer cell lines and tissues as well as controls. Our data demonstrate MKP-1 mRNA expression significantly decreased in five breast cancer cell lines compared to breast controls (P < 0.01). Using the methylation-specific PCR (MSP) analysis, the unmethylated reaction (U) is dominant in both normal cell lines and benign breast tumors (100% vs. 86.2%), whereas the methylated reaction (M) is dominant in both breast cancer cell lines and invasive breast tumors (100% vs. 57.2%). In terms of methylation ratio (M/M+U), methylation level in MKP-1 promoter is significantly higher in the invasive breast tumor tissues (n = 152) than in benign breast tumor tissues (n = 29) (P < 0.0001). Assessing the methylation ratio of the promoter of MKP-1 gene to diagnose the breast malignancy (invasive vs. benign), the area under the receiver-operating characteristic (ROC) curve was 0.809 (95% CI: 0.711-0.906, P < 0.001). The best performance for this prediction has a sensitivity of 76.32% and a specificity of 82.76% at the cutoff value of 0.38. Taken together, we firstly demonstrated that the promoter methylation of MKP-1 gene is a potential breast cancer biomarker for breast malignancy.
*Breast Neoplasms/diagnosis/genetics/metabolism ; Cell Line, Tumor ; DNA Methylation/*genetics ; Dual Specificity Phosphatase 1/genetics/*metabolism ; Female ; Gene Expression Regulation, Neoplastic ; Humans ; *Promoter Regions, Genetic ; ROC Curve ; Sensitivity and Specificity ; Tumor Markers, Biological

12(S)-Hydroxyheptadeca-5Z,8E,10E-trienoic acid (12-HHT) is an enzymatic product of prostaglandin H2 (PGH2) derived from cyclooxygenase (COX)-mediated arachidonic acid metabolism. Despite the high level of 12-HHT present in tissues and bodily fluids, its precise function remains largely unknown. In this study, we found that 12-HHT treatment in HaCaT cells remarkably down-regulated the ultraviolet B (UVB) irradiation-induced synthesis of interleukin-6 (IL-6), a pro-inflammatory cytokine associated with cutaneous inflammation. In an approach to identify the down-stream signaling mechanism by which 12-HHT down-regulates UVB-induced IL-6 synthesis in keratinocytes, we observed that 12-HHT inhibits the UVB-stimulated activation of p38 mitogen-activated protein kinase (MAPK) and nuclear factor kappa B (NF-kappaB). In addition, we found that 12-HHT markedly up-regulates MAPK phosphatase-1 (MKP-1), a critical negative regulator of p38 MAPK. When MKP-1 was suppressed by siRNA knock-down, the 12-HHT-mediated inhibitory effects on the UVB-stimulated activation of p38 MAPK and NF-kappaB, as well as the production of IL-6, were attenuated in HaCaT cells. Taken together, our results suggest that 12-HHT exerts anti-inflammatory effect via up-regulation of MKP-1, which negatively regulates p38 MAPK and NF-kappaB, thus attenuating IL-6 production in UVB-irradiated HaCaT cells. Considering the critical role of IL-6 in cutaneous inflammation, our findings provide the basis for the application of 12-HHT as a potential anti-inflammatory therapeutic agent in UV-induced skin diseases.
Anti-Inflammatory Agents, Non-Steroidal/pharmacology ; Cell Line ; Dual Specificity Phosphatase 1/biosynthesis/genetics ; Enzyme Activation ; Fatty Acids, Unsaturated/*pharmacology ; Humans ; Interleukin-6/*biosynthesis ; Keratinocytes/*metabolism/radiation effects ; NF-kappa B/metabolism ; RNA Interference ; RNA, Small Interfering ; Receptors, Leukotriene B4/genetics ; Signal Transduction/drug effects ; Skin Diseases/drug therapy ; *Ultraviolet Rays ; Up-Regulation ; p38 Mitogen-Activated Protein Kinases/metabolism