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

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

BACKGROUNDSildenafil is one of the selective phosphodiesterase 5 inhibitors that has been proven by many investigators to suppress growth factor stimulated (e.g. platelet-derived growth factor (PDGF) or epidermal growth factor (EGF)) proliferation and hypertrophy of pulmonary artery smooth muscle cells (PASMCs) via cGMP/cGKIa pathway. Serotonin promotes cell cycle progression leading to cell mitogenesis and plays a key role in the pathogenesis of pulmonary artery hypertension. The role of sildenafil in proliferation of PASMCs induced by serotonin has not been investigated so far. In this study we explored the underlying mechanism of the effect of sildenafil on serotonin induced proliferation of porcine PASMCs.

METHODSPASMCs were cells from primary cultures by the explant method from the pulmonary artery of swine and cells at passage 3 - 5 were used in this study. MTT colorimetric assay and flow cytometry analysis were used to evaluate the cell proliferation and alterations in cell cycle progression respectively. Western blotting analysis was applied to determine the expression of phosphorylated extracellular signal-regulated kinase (ERK), proliferating cell nuclear antigen (PCNA) and mitogen activated protein kinase (MAPK) phosphatase-1 (MKP-1).

RESULTSSerotonin (10 µmol/L) induced the upregulation of phosphorylation of ERK1/ERK2 and PCNA, an increase in the percentage of cells in S phase and subsequent cell proliferation. Pretreatment with 1 µmol/L sildenafil potentiated the phosphorylation of ERK1/ERK2, an increase in the percentage of cells in S phase and cell proliferation, compared with serotonin stimulation alone (P < 0.05). Furthermore, 30-minute pretreatment with 10 µmol/L U0126, specific antagonist for ERK kinase (MEK) prevented the increase in phosphorylation of ERK1/ERK2 and abolished cell cycle progression and the proliferation of PASMCs induced by sildenafil.

CONCLUSIONThis study shows that sildenafil potentiated the proliferative effect of serotonin on PASMCs via phosphorylation of ERK1/ERK2.

Animals ; Blotting, Western ; Cell Proliferation ; drug effects ; Cells, Cultured ; Dual Specificity Phosphatase 1 ; metabolism ; Mitogen-Activated Protein Kinase 1 ; metabolism ; Mitogen-Activated Protein Kinase 3 ; metabolism ; Myocytes, Smooth Muscle ; cytology ; Phosphorylation ; drug effects ; Piperazines ; pharmacology ; Proliferating Cell Nuclear Antigen ; metabolism ; Pulmonary Artery ; cytology ; Purines ; pharmacology ; Serotonin ; pharmacology ; Sildenafil Citrate ; Sulfones ; pharmacology ; Swine

The aim of this study was to investigate the effects of angiotensin II (Ang II) on extracellular signal-regulated protein kinase (ERK) signaling pathway in cultured vascular smooth muscle cells (VSMCs) from spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats. VSMCs from SHR and WKY rats were treated with 1x10(-7) mmol/L Ang II for 24 h in the absence or presence of 30 min of pre-treatment of valsartan (1x10(-5) mmol/L) or PD98059 (1x10(-5)mmol/L), selective inhibitor of ERKs- dependent pathways, when they were cultured in 20% calf serum medium. VSMCs of SHR and WKY cultured in serum-free medium were used as control groups. Among the different treatments, VSMCs from the SHR and WKY were devided into four groups: (1) control, (2) Ang II, (3) Ang II + valsartan, (4) Ang II + PD98059. ERK activity in VSMCs was measured by immuno-precipitation. Proteins of total ERK (t-ERK), phosphorylated-ERK (p-ERK) and mitogen-activated protein kinases phosphatase-1 (MKP-1) in VSMCs were detected by Western blot. MKP-1 mRNA in VSMCs was measured by RT-PCR. In VSMCs from WKY or SHR rats, ERK activity, p-ERK, MKP-1 and MKP-1 mRNA in Ang II group were higher than those in control group (P<0.05). In both SHRs and WKYs, there were no significant differences in ERK activity, p-ERK, MKP-1 and MKP-1 mRNA among the control group, Ang II + valsartan group and Ang II + PD98059 group. ERK activity, p-ERK, MKP-1 and MKP-1 mRNA in SHRs were significantly higher than those in WKYs with same treatments (P<0.01). There was no significant difference in t-ERK among different groups and no difference in t-ERK between SHRs and WKYs (P>0.05). Our results show that Ang II activates VSMCs ERK signaling pathways via Ang II type 1 (AT(1)) receptors. Ang II increased ERK activity and p-ERK, but not t-ERK, accompanied by an increase in MKP-1 mRNA expression and protein. Among the different treatments, ERK activity and p-ERK were higher in SHR than in WKY. Valsartan and PD98059 blocked Ang II-stimulated ERK activation. These results suggest that ERK signaling pathway plays an important role in the pathogenesis of hypertension. The effect of Ang II on SHR and WKY VSMCs' ERK signaling pathway may be mediated by AT(1) receptors, enhancing ERK activity and the amount of p-ERK, and then increasing MKP-1 mRNA and its expression.
Angiotensin II ; pharmacology ; Animals ; Cells, Cultured ; Dual Specificity Phosphatase 1 ; metabolism ; Extracellular Signal-Regulated MAP Kinases ; metabolism ; Hypertension ; physiopathology ; Male ; Muscle, Smooth, Vascular ; cytology ; Myocytes, Smooth Muscle ; metabolism ; Rats ; Rats, Inbred SHR ; Rats, Inbred WKY ; Signal Transduction ; drug effects ; Tetrazoles ; pharmacology ; Valine ; analogs & derivatives ; pharmacology ; Valsartan

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

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

AIM AND METHODSTo investigate the role of mitogen-activated protein kinase phosphatase-1 (MKP-1) in the regulation of cells proliferation, the expression of MKP-1 and extracellular signal-regulated kinase-1 (ERK-1) in heart and aorta of spontaneous hypertensive rat (SHR) and WKY were studied. We also investigated the effect of MKP-1 genes,which were transfected into vascular smooth muscle cells (VSMC) using the classical calcium phosphate coprecipitation technique, on the incorporation of 3H-TdR in VSMC stimulated by angiotensin II (Ang II).

RESULTS(1) Compared with that of WKY, MKP-1 expression in heart and aorta were significantly decreased by 53% (P < 0.01) and 45% (P < 0.01) in SHR, respectively. While the expression of ERK-1 in heart and aorta of SHR were higher than that of WKY (P < 0.01). The ratio of ERK-1/MKP-1 in heart and aorta of SHR were significantly higher than that of WKY. (2) 3H-TdR incorporation in VSMC stimulated by Ang II (10(-7) mol/L) was increased by 207% (P < 0.01), compared with control group. In the transfected cells with wild MKP-1 gene, Ang II-induced incorporation of 3H-TdR lowered 63%, compared with untransfected cells (P < 0.05). There were no marked inhibitive role between mutant MKP-1-transfected cells and blank vector-transfected cells in response to Ang II, compared with Ang II group (P > 0.05).

CONCLUSIONThese results showed that the expression of ERK-1 in heart and aorta isolated from SHR, which stimulated proliferation and hypertrophy of cells, is higher than that of MKP-1 which dephosphorylates and inactivated ERK-1. In addition, MKP-1 significantly inhibits Ang II-stimulated proliferation of VSMC.

Angiotensin II ; pharmacology ; Animals ; Aorta ; cytology ; enzymology ; Cell Proliferation ; Cells, Cultured ; Dual Specificity Phosphatase 1 ; metabolism ; Heart ; Hypertension ; metabolism ; Mitogen-Activated Protein Kinase 3 ; metabolism ; Muscle, Smooth, Vascular ; cytology ; Myocardium ; cytology ; enzymology ; Myocytes, Smooth Muscle ; drug effects ; metabolism ; Rats ; Rats, Inbred SHR ; Rats, Inbred WKY

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

OBJECTIVETo investigate the effects of protein tyrosine phosphatase-SHP2 and dual-specificity MAPK phosphatase-MKP5 on the activation of MAPKs and cell invasion induced by P2Y purinergic receptor in human prostate cancer cell lines with different metastatic potentials.

METHODSThe wide type (-wt) SHP2, mutant type (-cs) SHP2 and wide type (-wt) MKP5 cDNA expression vectors were constructed and stably transfected into 1E8 cells (highly metastatic) and/or 2B4 cells (non-metastatic). The tyrosine phosphorylation of SHP2 was examined by immunoprecipitation. The activation of ERK1/2 and p38 induced by P2Y receptor agonist ATP was analyzed by Western blot with phospho-specific antibodies against the dually phosphorylated, active forms of ERK1/2 and p38. The in-vitro invasive ability through Matrigel was measured by boyden-chamber assay.

RESULTSATP induced significant SHP2 phosphorylation, which was stronger and lasted longer in 1E8 than in 2B4. SHP2-wt enhanced the ERK1/2 activation induced by ATP in 2B4 cells, while SHP2-cs delayed and decreased this effect in 1E8 cells. Both SHP2-wt and SHP2-cs had no obvious influence on p38 activation. ATP stimulated cell invasion of both 1E8 and 2B4, while transfection of SHP2-wt into 2B4 cells further increased the invasive-stimulating ability of ATP (18.7% increase compared with ATP treatment alone). Transfection of SHP2-cs into 1E8 cells, however, antagonized the invasive-stimulating ability of ATP (40.9% decrease compared with ATP treated group). Up-regulation of MKP5-wt inhibited phosphorylation of p38 by ATP and reduced cell invasion stimulated by ATP (22.4% and 28.7% decrease compared with ATP treated group of 1E8 and 2B4, respectively).

CONCLUSIONSBoth SHP2 and MKP5 play some roles in P2Y receptor-mediated activation of MEK/ERK, p38 signaling pathways and prostate cancer invasion. SHP2 positively regulates ERK activation and prostate cancer invasion, whereas MKP5 inhibits the invasion by suppressing p38 activation.

Adenosine Triphosphate ; pharmacology ; Cell Line, Tumor ; DNA, Complementary ; genetics ; Dual-Specificity Phosphatases ; Genetic Vectors ; Humans ; Intracellular Signaling Peptides and Proteins ; genetics ; metabolism ; Male ; Mitogen-Activated Protein Kinase 1 ; metabolism ; Mitogen-Activated Protein Kinase 3 ; metabolism ; Mitogen-Activated Protein Kinase Phosphatases ; Neoplasm Invasiveness ; Phosphorylation ; Prostatic Neoplasms ; metabolism ; pathology ; Protein Tyrosine Phosphatase, Non-Receptor Type 11 ; Protein Tyrosine Phosphatases ; genetics ; metabolism ; Receptors, Purinergic P2 ; physiology ; Signal Transduction ; Transfection ; p38 Mitogen-Activated Protein Kinases ; metabolism