Mitogen-activated Protein Kinase siRNA Research Articles

Flavopereirine Inhibits Autophagy via the AKT/p38 MAPK Signaling Pathway in MDA-MB-231 Cells.

Autophagy is a potential target for the treatment of triple negative breast cancer (TNBC). Because of a lack of targeted therapies for TNBC, it is vital to find optimal agents that avoid chemoresistance and metastasis. Flavopereirine has anti-proliferation ability in cancer cells, but whether it regulates autophagy in breast cancer cells remains unclear. A Premo™ Tandem Autophagy Sensor Kit was used to image the stage at which flavopereirine affects autophagy by confocal microscopy. A plasmid that constitutively expresses p-AKT and siRNA targeting p38 mitogen-activated protein kinase (MAPK) was used to confirm the related signaling pathways by Western blot. We found that flavopereirine induced microtubule-associated protein 1 light chain 3 (LC3)-II accumulation in a dose- and time-dependent manner in MDA-MB-231 cells. Confocal florescent images showed that flavopereirine blocked autophagosome fusion with lysosomes. Western blotting showed that flavopereirine directly suppressed p-AKT levels and mammalian target of rapamycin (mTOR) translation. Recovery of AKT phosphorylation decreased the level of p-p38 MAPK and LC3-II, but not mTOR. Moreover, flavopereirine-induced LC3-II accumulation was partially reduced in MDA-MB-231 cells that were transfected with p38 MAPK siRNA. Overall, flavopereirine blocked autophagy via LC3-II accumulation in autophagosomes, which was mediated by the AKT/p38 MAPK signaling pathway.

Monomeric C-reactive protein affects cell injury and apoptosis through activation of p38 mitogen-activated protein kinase in human coronary artery endothelial cells.

C-reactive protein (CRP) is an important predictor of cardiovascular events and plays a role in vascular inflammation and vessel damage. The aim of this study was to investigate the effect of pentameric CRP (pCRP) and monomeric CRP (mCRP) on the production of atherosclerosis-related factors in cultured human coronary artery endothelial cells (HCAECs). HCAECs were treated with pCRP, mCRP, p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580, or transfected with p38 MAPK siRNA. Western blotting was performed to detect the expression of vascular endothelial growth factor (VEGF), cyclooxygenase-2 (COX-2), intercellular adhesion molecule-2 (ICAM-2) and vascular cell adhesion molecule-1 (VCAM-1). Proliferation, damage, and apoptosis of HCAECs were examined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide, lactate dehydrogenase (LDH), and flow cytometry, respectively. mCRP suppressed VEGF and COX-2 expression and enhanced ICAM-2 and VCAM-1 expression in HCAECs, in both dose-dependent and time-dependent manner. Except at 100 µg/ml concentration and 20-hour or 24-hour incubation, pCRP had no apparent effects. mCRP but not pCRP induced HCAEC injury and phosphorylation of p38 MAPK, and the inhibitor SB203580 reversed the effects of mCRP. mCRP promotes injury and apoptosis of HCAECs through a p38 MAPK-dependent mechanism, which provides a new therapy for the injury of HCAECs in atherosclerosis.

Co-Delivery of Docetaxel and p44/42 MAPK siRNA Using PSMA Antibody-Conjugated BSA-PEI Layer-by-Layer Nanoparticles for Prostate Cancer Target Therapy.

How to overcome the low accumulation of chemotherapeutic agent in tumor tissue and exhibit multitherapeutics remains an ongoing challenge for cancer treatment. Here, a simple method is demonstrated that used to prepare prostate-specific membrane antigen antibody (PSMAab )-conjugated fluorescent bovine serum albumin (BSA)-branched polyethylenimine layer-by-layer nanoparticles (BSA-PEILBL NPs) for co-delivery of docetaxel (DTX) and p44/42 mitogen-activated protein kinase (MAPK) small interfering RNA (p44/42 MAPK siRNA) as synergistic and selective inhibition of cancer cell proliferation platform. The results show the levels of α-tubulin and p44/42 MAPK in CWR22R cells are significantly reduced after treatment with PSMAab -conjugated DTX/BSA-PEILBL /siRNA NPs. Consequently, the 50% cellular growth inhibition (IC50 ) values of the NPs loaded with both DTX and p44/42 MAPK siRNA are ≈2.1-fold less than those for the NPs only loaded with DTX. The median survival significantly prolongs from 18 d to upward 45 d compared to mice that receive same dose (12 mg kg-1 ) of free DTX. The results suggest this synergistic delivery system may be a promising clinical treatment in prostate cancer.

Retraction: Hyperbaric oxygen activates discoidin domain receptor 2 via tumour necrosis factor-α and the p38 MAPK pathway to increase vascular smooth muscle cell migration through matrix metalloproteinase 2.

DDR2 (discoidin domain receptor 2) regulates collagen turnover mediated by SMCs (smooth muscle cells) in atherosclerosis. HBO (hyperbaric oxygen) has been used in medical practice; however, the molecular mechanism of the beneficial effects of HBO is poorly understood. Furthermore, the effect of HBO on DDR2 has not been reported previously. In the present study, we investigated the cellular and molecular mechanisms of DDR2 regulation by HBO in VSMCs (vascular SMCs). Cells were exposed to 2.5 ATA (atmosphere absolute) of oxygen in a hyperbaric chamber. DDR2 protein (3.63-fold) and mRNA (2.34-fold) expression were significantly increased after exposure to 2.5 ATA HBO for 1 h. Addition of SB203580 and p38 MAPK (mitogen-activated protein kinase) siRNA (small interfering RNA) 30 min before HBO inhibited the induction of DDR2 protein. HBO also significantly increased DNA-protein binding activity of Myc/Max. Addition of SB203580 and an anti-TNF-alpha (tumour necrosis factor-alpha) monoclonal antibody 30 min before HBO abolished the DNA-protein binding activity induced by HBO. HBO significantly increased the secretion of TNF-alpha from cultured VSMCs. Exogenous addition of TNF-alpha significantly increased DDR2 protein expression, whereas anti-TNF-alpha and anti-(TNF-alpha receptor) antibodies blocked the induction of DDR2 protein expression. HBO significantly increased VSMC migration and proliferation, whereas DDR2 siRNA inhibited the migration induced by HBO. HBO increased activated MMP2 (matrix metalloproteinase 2) protein expression, and DDR2 siRNA abolished the induction of activated MMP2 expression induced by HBO. In conclusion, HBO activates DDR2 expression in cultured rat VSMCs. HBO-induced DDR2 is mediated by TNF-alpha and at least in part through the p38 MAPK and Myc pathways.

Escherichia coli-derived and Staphylococcus aureus-derived extracellular vesicles induce MUC5AC expression via extracellular signal related kinase 1/2 and p38 mitogen-activated protein kinase in human airway epithelial cells.

Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) release extracellular vesicles (EVs). E. coli-derived and S. aureus-derived EVs are associated with neutrophilic respiratory inflammation. In neutrophilic respiratory inflammation of human, expression of mucin is increased in airway epithelial cells and is associated with increased morbidity and mortality of the affected patients. However, no study on the effects of EVs on expression of mucin genes has been reported in airway epithelial cells. Therefore, this study was conducted in order to examine the effects and the brief signaling pathways of E. coli-derived and S. aureus-derived EVs on MUC5AC expression in human airway epithelial cells. In mucin-producing human NCI-H292 airway epithelial cells and primary cultures of normal nasal epithelial cells, the effects and signaling pathways of E. coli-derived and S. aureus-derived EVs on MUC5AC expression were examined using reverse transcription-polymerase chain reaction (RT-PCR), real-time PCR, enzyme immunoassay, and immunoblot analysis with several specific inhibitors and small interfering RNA (siRNA). E. coli-derived and S. aureus-derived EVs induced MUC5AC expression. E. coli-derived and S. aureus-derived EVs significantly activated phosphorylation of extracellular signal related kinase 1/2 (ERK1/2) mitogen-activated protein kinase (MAPK) and p38 MAPK. ERK1/2 MAPK inhibitor, p38 MAPK inhibitor, ERK1/2 MAPK siRNA, and p38 MAPK siRNA significantly blocked E. coli-derived and S. aureus-derived EVs induced MUC5AC messenger RNA (mRNA) expression. The results of this study suggest that E. coli-derived and S. aureus-derived EVs induced MUC5AC expression via ERK1/2 and p38 MAPK signaling pathways in human airway epithelial cells.

Effect of High Glucose on MUC5B expression in Human Airway Epithelial Cells.

Excessive production of mucus results in plugging of the airway tract, which can increase morbidity and mortality in affected patients. In patients with diabetes, inflammatory airway disease appears with more frequent relapse and longer duration of symptoms. However, the effects of high glucose (HG) on the secretion of mucin in inflammatory respiratory diseases are not clear. Therefore, this study was conducted in order to investigate the effect and the brief signaling pathway of HG on MUC5B expression in human airway epithelial cells. The effect and signaling pathway of HG on MUC5B expression were investigated using reverse transcriptase-polymerase chain reaction (RT-PCR), real-time PCR, enzyme immunoassay, and immunoblot analysis with specific inhibitors and small interfering RNA. HG increased MUC5B expression and epidermal growth factor receptor (EGFR) expression, and activated the phosphorylation of EGFR and p38 mitogen-activated protein kinase (MAPK). Pretreatment with EGFR inhibitor significantly attenuated the HG-induced phosphorylation of p38 MAPK, and pretreatments with p38 inhibitor or EGFR inhibitor significantly attenuated HG-induced MUC5B expression. In addition, knockdown of p38 MAPK by p38 MAPK siRNA significantly blocked HG-induced MUC5B expression. These findings suggest that HG induces MUC5B expression via the sequential activations of the EGFR/p38 MAPK signaling pathway in human airway epithelial cells.

Spleen tyrosine kinase induces MUC5AC expression in human airway epithelial cell.

MUC5AC, a major secreted mucin, is increased in chronic inflammatory airway disease. Spleen tyrosine kinase (SYK) is a mediator, which acts as an important regulator of intracellular signal transduction in the inflammatory response. SYK was originally identified in hematopoietic cells, and its expression in some nonhematopoietic cells, including respiratory epithelial cells, was recently demonstrated. However, the effects of SYK on mucin secretion in human airway epithelial cells have not been studied. The objective of this study was to investigate the effect and brief signaling pathways of SYK on MUC5AC expression in human airway epithelial cells. In mucin-producing human NCI-H292 cells and primary cultures of human nasal epithelial cells, the effects and signaling pathways of SYK on MUC5AC expression were investigated by reverse transcriptase-polymerase chain reaction, real-time polymerase chain reaction, enzyme immunoassay, and immunoblot analysis with several specific inhibitors and small interfering RNA (siRNA). SYK induced MUC5AC expression. SYK activated significant phosphorylation of extracellular signal-related kinase (ERK)1/2 and p38 mitogen-activated protein kinase (MAPK) signaling pathways. SYK-induced MUC5AC expression was significantly attenuated by pretreatment with U0126 (ERK1/2 MAPK inhibitor) and SB203580 (p38 MAPK inhibitor). In addition, the knockdown of ERK2 and p38 MAPK by ERK2 and p38 MAPK siRNA significantly blocked SYK-induced MUC5AC expression. These results indicated that SYK increased MUC5AC expression via ERK2 and p38 MAPK signaling pathways in human airway epithelial cells.

Cadmium induces mucin 8 expression via Toll-like receptor 4-mediated extracellular signal related kinase 1/2 and p38 mitogen-activated protein kinase in human airway epithelial cells.

Inhalation of cadmium can lead to development of inflammatory airway diseases such as acute pulmonary edema and chronic obstructive pulmonary disease. In inflammatory airway diseases, expression of mucins is increased, which leads to increased morbidity and mortality of the affected patients. However, no study on the effect of cadmium on expression of mucin genes in airway epithelial cells has been reported. Therefore, this study was conducted in order to investigate the effect and the brief signaling pathway of cadmium on expression of mucin genes in human airway epithelial cells. In mucin-producing human NCI-H292 airway epithelial cells and primary cultures of normal nasal epithelial cells, the effect and signaling pathway of cadmium on expression of mucin genes were investigated using reverse transcription-polymerase chain reaction (RT-PCR), real-time PCR, enzyme immunoassay, and immunoblot analysis with several specific inhibitors and small interfering RNA (siRNA). Cadmium increased mucin 8 (MUC8) expression and Toll-like receptor (TLR) 4 messenger RNA (mRNA) expression. Cadmium significantly activated phosphorylation of extracellular signal related kinase 1/2 (ERK1/2) mitogen-activated protein kinase (MAPK) and p38 MAPK. ERK1/2 MAPK inhibitor, p38 MAPK inhibitor, TLR4 siRNA, ERK1/2 MAPK siRNA, and p38 MAPK siRNA significantly blocked cadmium-induced MUC8 mRNA expression. TLR4 siRNA significantly blocked cadmium-activated phosphorylation of ERK1/2 MAPK and p38 MAPK. The results of this study suggest for the first time that cadmium induces MUC8 expression via TLR4-mediated ERK1/2 and p38 MAPK signaling pathway in human airway epithelial cells.

TNF-α stimulates System A amino acid transport in primary human trophoblast cells mediated by p38 MAPK signaling.

Maternal obesity and gestational diabetes mellitus (GDM) increase the risk of delivering infants that are large for gestational age with greater adiposity, who are prone to the development of metabolic disease in childhood and beyond. These maternal conditions are also associated with increased levels of the proinflammatory cytokine TNF-α in maternal tissues and the placenta. Recent evidence suggests that changes in placental amino acid transport contribute to altered fetal growth. TNF-α was previously shown to stimulate System A amino acid transport in primary human trophoblasts (PHTs), however the molecular mechanisms remain unknown. In this study, we tested the hypothesis that TNF-α regulates amino acid uptake in cultured PHTs by a mitogen-activated protein kinase (MAPK)-dependent mechanism. Treatment of PHTs with TNF-α significantly increased System A amino acid transport, as well as Erk and p38 MAPK signaling. Pharmacological antagonism of p38, but not Erk MAPK activity, inhibited TNF-α stimulated System A activity. Silencing of p38 MAPK using siRNA transfections prevented TNF-α stimulated System A transport in PHTs. TNF-α significantly increased the protein expression of System A transporters SNAT1 and SNAT2, but did not affect their mRNA expression. The effects of TNF-α on SNAT1 and SNAT2 protein expression were reversed by p38 MAPK siRNA silencing. In conclusion, TNF-α regulates System A activity through increased SNAT1 and SNAT2 transporter protein expression in PHTs. These findings suggest that p38 MAPK may represent a critical mechanistic link between elevated proinflammatory cytokines and increased placental amino acid transport in obese and GDM pregnancies associated with fetal overgrowth.

Palmitoleic acid prevents palmitic acid-induced macrophage activation and consequent p38 MAPK-mediated skeletal muscle insulin resistance

Obesity and saturated fatty acid (SFA) treatment are both associated with skeletal muscle insulin resistance (IR) and increased macrophage infiltration. However, the relative effects of SFA and unsaturated fatty acid (UFA)-activated macrophages on muscle are unknown. Here, macrophages were treated with palmitic acid, palmitoleic acid or both and the effects of the conditioned medium (CM) on C2C12 myotubes investigated. CM from palmitic acid-treated J774s (palm-mac-CM) impaired insulin signalling and insulin-stimulated glycogen synthesis, reduced Inhibitor κBα and increased phosphorylation of p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase in myotubes. p38 MAPK inhibition or siRNA partially ameliorated these defects, as did addition of tumour necrosis factor-α blocking antibody to the CM. Macrophages incubated with both FAs generated CM that did not induce IR, while palmitoleic acid-mac-CM alone was insulin sensitising. Thus UFAs may improve muscle insulin sensitivity and counteract SFA-mediated IR through an effect on macrophage activation.

Effect of thymic stromal lymphopoietin on MUC5B expression in human airway epithelial cells

Thymic stromal lymphopoietin (TSLP) is an interleukin 7-like cytokine and a potent factor for B- and T-cell growth and differentiation. Recent studies have demonstrated an association of TSLP with allergic and inflammatory airway diseases. However, no study on the effect of TSLP on expression of mucin genes in airway epithelial cells has been reported. Therefore, the effects and brief signaling pathways of TSLP on expression of mucin genes in human airway epithelial cells were investigated in this study. In mucin-producing human NCI-H292 airway epithelial cells and primary cultures of normal nasal epithelial cells, the effect and signaling pathway of TSLP on expression of mucin genes were investigated using reverse transcriptase-polymerase chain reaction (RT-PCR), real-time PCR, enzyme immunoassay, and immunoblot analysis with several specific inhibitors and small interfering RNA (siRNA). In human NCI-H292 airway epithelial cells, TSLP increased MUC5B expression. TSLP significantly activated phosphorylation of ERK1/2 and p38 mitogen-activated protein kinase (MAPK). U0126 (ERK1/2 MAPK inhibitor) and SB203580 (p38 MAPK inhibitor) significantly attenuated TSLP-induced MUC5B mRNA expression. Knockdown of ERK1, ERK2, and p38 MAPK by ERK1, ERK2, and p38 MAPK siRNA significantly blocked TSLP-induced MUC5B mRNA expression. In the primary cultures of normal nasal epithelial cells, TSLP significantly increased MUC5B mRNA expression, which was significantly attenuated after pretreatment with U0126 and SB203580. These results suggest that TSLP induces MUC5B expression via the ERK1/2 and p38 MAPK signaling pathway in human airway epithelial cells.

Endothelin-1 induces interleukin-18 expression in human osteoblasts

ObjectiveBoth endothelin-1 (ET-1) and interleukin (IL)-18 induce osteoblast proliferation under normal and pathophysiological conditions. In the present study, we explored the interaction between the two proteins by examining the effect of ET-1 on IL-18 expression in cultured human osteoblasts. MethodsHuman osteoblasts were treated with ET-1 in different concentrations (1, 10, 20, 40, or 50nM) for different length of time (1, 6, 12, 18, or 24h) in the presence or absence of ET A receptor (ETAR) blocker BQ123, ET B receptor (ETBR) blocker BQ788, p38 mitogen-activated protein kinase (MAPK) siRNA, or different kinase inhibitors. ResultsET-1 increased the IL-18 mRNA level in a statistically significant dose- and time-dependent manner within 18h, which was reflected in dose-dependent induction of the human IL-18 gene promoter activity and IL-18 protein/secreted protein expression. BQ123 (1μM) and p38 MAPK siRNA and inhibitor PD169316 (25μM) completely abolished the promoting effect of ET-1 on IL-18 expression. [3H]thymidine incorporation assays showed that ET-1 lost a major part (57%) of its promoting effect on osteoblast proliferation when the endogenous IL-18 expression in osteoblasts was knocked down by 75%. ConclusionsET-1 induces IL-18 expression in human osteoblasts at the gene promoter/transcription level via ETAR by a p38 MAPK-dependent mechanism, and that IL-18 mediates a major part of ET-1 induced osteoblast proliferation. This study provides the first evidence of interaction between ET-1 and IL-18 in osteoblast and adds new insights into bone physiology and pathophysiology.

Effect of Onchocerca Volvulus Chitinase on MUC5B Expression in Human Airway Epithelial Cells

Chitin is an essential structural component of the wall of fungal cells and is present in the exoskeleton of arthropods. It has been generally assumed that mammals lack the ability to produce chitinase proteins, the enzymes responsible for chitin degradation. However, recent studies have indicated that mammals produce chitinases and chitinase-like proteins, and chitinase plays a potential role in human asthma and allergic inflammation. In this study, the effect and brief signaling pathway of chitinase on MUC5B expression were investigated in human airway epithelial cells. In the mucin-producing human NCI-H292 airway epithelial cells and the primary cultures of normal nasal epithelial cells, the effect and signaling pathway of chitinase on MUC5B expression were investigated using the reverse transcriptase-polymerase chain reaction (RT-PCR), real-time PCR, enzyme immunoassay, and immunoblot analysis with several specific inhibitors and small interfering RNA (siRNA). In the mucin-producing human NCI-H292 airway epithelial cells, chitinase increased MUC5B expression. Chitinase significantly activated the phosphorylation of p38 mitogen-activated protein kinase (MAPK) but not the phosphorylation of extracellular signa-l-related kinase (ERK) 1/2. The SB203580 (p38 MAPK inhibitor) significantly attenuated chitinase-induced MUC5B mRNA expression, but U0126 (ERK1/2 inhibitor) did not. Knockdown of p38 MAPK by p38 MAPK siRNA significantly blocked chitinase-induced MUC5B expression. In the primary cultures of normal nasal epithelial cells, chitinase significantly increased MUC5B gene expression and this was significantly attenuated after pretreatment with SB203580. These results suggest that chitinase induces MUC5B expression by activation of the p38 MAPK signaling pathway in human airway epithelial cells.

Phorbol 12-Myristate 13-Acetate Induces MUC16 Expression via PKCδ and p38 in Human Airway Epithelial Cells

ObjectivesPhorbol 12-myristate 13-acetate (PMA) is widely used as a protein kinase C (PKC) activator, PKC is involved in the secretion of mucins. MUC16, one of the membrane-bound mucins, is produced in human airway epithelial cells. However, the effect and signaling pathway of PMA on MUC16 expression in human airway epithelial cells has not been reported. Therefore, the effect and brief signaling pathway of PMA on MUC16 expression were investigated in human airway epithelial cells in this study.MethodsIn the mucin-producing human NCI-H292 airway epithelial cells and the primary cultures of normal nasal epithelial cells, the effect and signaling pathway of PMA on MUC16 expression were investigated using reverse transcriptase-polymerase chain reaction (RT-PCR), real-time PCR, enzyme immunoassay, and immunoblot analysis with several specific inhibitors and small interfering RNA (siRNA) for p38 mitogen-activated protein kinase (MAPK).ResultsPMA increased MUC16 expression, and activated phosphorylation of p38 MAPK. However, it did not activate phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2). SB203580 (p38 MAPK inhibitor) inhibited PMA-induced MUC16 expression, while U0126 (ERK1/2 inhibitor) did not. In addition, the knockdown of p38 MAPK by p38 MAPK siRNA significantly blocked PMA-induced MUC16 mRNA expression. Rottlerin (PKCδ inhibitor) inhibited PMA-induced MUC16 expression, and also inhibited the phosphorylation of activated p38 MAPK by PMA.ConclusionThese results show for the first time that PMA-induced MUC16 expression is regulated by activation of the PKCδ and p38 MAPK signaling pathway in human airway epithelial cells.

AMPK induces MUC5B expression via p38 MAPK in NCI-H292 airway epithelial cells

Adenosine monophosphate-activated protein kinase (AMPK) is a well-known serine/threonine kinase that has been implicated in modulation of glucose and fatty acid metabolism. Recent reports have also implicated AMPK in modulation of mucin secretion. In this study, the effects and signaling pathways of AMPK on MUC5B expression were investigated in human NCI-H292 airway epithelial cells. Metformin, as an activator of AMPK, induced MUC5B expression in a dose-dependent manner. Compound C, as an inhibitor of AMPK, inhibited metformin-induced MUC5B expression in a dose-dependent manner. Metformin significantly activated phosphorylation of AMPK; compound C inhibited metformin-activated phosphorylation of AMPK. Without treatment with metformin, there was no difference in MUC5B mRNA expression between Ad-dnAMPK transfected and wild-type adenovirus transfected NCI-H292 cells. However, after treatment with metformin, MUC5B mRNA expression was increased in wild-type adenovirus transfected NCI-H292 cells; MUC5B mRNA expression was significantly decreased in Ad-dnAMPK transfected NCI-H292 cells. Metformin activated phosphorylation of p38 mitogen-activated protein kinase (MAPK); compound C inhibited metformin-activated phosphorylation of p38 MAPK. SB203580, as an inhibitor of p38 MAPK, significantly inhibited metformin-induced MUC5B mRNA expression, while U0126, as an inhibitor of ERK1/2 MAPK, had no effect. In addition, knockdown of p38 MAPK by p38 MAPK siRNA significantly blocked metformin-induced MUC5B mRNA expression. In conclusion, results of this study show that AMPK induces MUC5B expression through the p38 MAPK signaling pathway in airway epithelial cells.

The Effects of Mitogen-Activated Protein Kinase Inhibitors or Small Interfering RNAs on Gallic Acid-Induced HeLa Cell Death in Relation to Reactive Oxygen Species and Glutathione

Gallic acid (GA) is widely distributed in various plants and foods and has various biological properties including anticancer effects. In this study, we investigated the effects of mitogen-activated protein kinase (MAPK) [MAP 20 kinase or ERK kinase (MEK), c-Jun N-terminal kinase (JNK), or p38)] inhibitors or small interfering RNAs (siRNAs) on GA-induced HeLa cell death in relation to reactive oxygen species (ROS) and glutathione (GSH) levels. GA dose dependently inhibited the growth of HeLa cells via apoptosis and/or necrosis at 24 h, which was accompanied by the loss of mitochondrial membrane potential (MMP; ΔΨ(m)). Treatment with 70 μM GA increased the ROS level including O(2)(•-) and significantly induced GSH depletion in HeLa cells. GA decreased the activity of extracellular signal-regulated kinase (ERK) at 24 h, whereas it increased that of JNK at the same time. While the MEK inhibitor or ERK siRNA did not affect cell growth and death in 70 μM GA-treated HeLa cells at 24 h, JNK and p38 inhibitors enhanced cell growth inhibition and death in these cells. Additionally, p38 siRNA administration augmented growth inhibition, death, and MMP (ΔΨ(m)) loss in 70 μM GA-treated HeLa cells. In relation to ROS and GSH levels, JNK and p38 inhibitors increased ROS levels, and GSH-depleted cell numbers in GA-treated HeLa cells. Moreover, p38 siRNA increased O(2)(•-) levels and GSH depletion in GA-treated HeLa cells. Each MAPK inhibitor and siRNA differentially affected ROS and GSH levels in HeLa control cells. Conclusively, JNK and p38 inhibitors and p38 siRNA enhanced growth inhibition and cell death in GA-treated HeLa cells, which were to some extent related to GSH depletion and ROS levels, especially O(2)(•-).

NSAID Inhibition of Prostate Cancer Cell Migration Is Mediated by Nag-1 Induction via the p38 MAPK-p75NTR Pathway

The nonsteroidal anti-inflammatory drugs (NSAID) R-flurbiprofen and ibuprofen have been shown to induce expression of p75(NTR) (neurotrophin receptor) in prostate cancer cell lines. p75(NTR), a tumor necrosis factor receptor superfamily member, is a proapoptotic protein that functions as a tumor suppressor in the human prostate. Expression of p75(NTR) is lost as prostate cancer progresses and is minimal in several metastatic prostate cancer cell lines. NSAIDs induce p75(NTR) through activation of the p38 mitogen-activated protein kinase (MAPK) pathway, with a concomitant decrease in cell survival. Here, we show that treatment with R-flurbiprofen and ibuprofen induces expression of the NSAID-activated gene-1 (Nag-1) protein, a divergent member of the TGF beta (TGF-β) family, in PC-3 cells. Using the selective pharmacologic inhibitor of p38 MAPK, SB202190, and p38 MAPK-specific siRNA (small interfering RNA), we show that Nag-1 induction following NSAID treatment is mediated by the p38 MAPK pathway. p75(NTR)-specific siRNA pretreatment shows that Nag-1 induction by NSAIDs is downstream of p75(NTR) induction. Decreased survival of NSAID-treated cells is rescued by p75(NTR)-specific siRNA but not by Nag-1 siRNA. Transwell chamber and in vitro wound healing assays demonstrate decreased cell migration upon NSAID treatment. Pretreatment of PC-3 cells with p75(NTR) and Nag-1-specific siRNA shows that NSAID inhibition of cell migration is mediated by Nag-1 and p75(NTR). These results demonstrate a role for Nag-1 in NSAID inhibition of cell migration, but not survival.

Down‐regulation of MMP‐2 through the p38 MAPK‐NF‐κB‐dependent pathway by aloe‐emodin leads to inhibition of nasopharyngeal carcinoma cell invasion

Aloe-emodin (AE), extracted from the rhizome of Rheum palmatum, has an anti-proliferative effect on different human cancer cell lines. Nonetheless, the underlying mechanism by which AE inhibits nasopharyngeal carcinoma (NPC) cell invasion is still unclear. The results of this study show that treatment of NPC cells with growth suppressive concentrations of AE caused cell cycle arrest at the S-G(2)/M phase. Coimmunoprecipitation and small interfering RNA (siRNA) studies demonstrated that AE-induced cell cycle arrest in NPC cells was associated with increasing levels of cyclin B1 bound to cyclin-dependent kinase 1. The inhibition of NPC cell invasion by AE was evidenced through the suppression of matrix metalloproteinases-2 (MMP-2) expression. MMP-2 promoter activity and cell invasion were inhibited by p38 mitogen-activated protein kinase (MAPK) siRNA, inhibitor 4-(4-Fluorophenyl)-2-[4-(methylsulfinyl)phenyl]-5-(4-pyridyl)-1H-imidazole (SB203580), and AE, but not by JNK siRNA and inhibitor 1,9-pyrazoloanthrone. Treatment with AE, SB203580, NF-kappaB inhibitors N-p-tosyl-(L)-phenylalanine chloromethyl ketone (TPCK) and pyrrolidine dithiocarbamate (PDTC) or transfection with p38 MAPK siRNA significantly inhibited NF-kappaB transcriptional activity. In addition, TPCK and PDTC treatment inhibited the expression and promoter activity of MMP-2 and thereby significantly inhibited cell invasion activity. The involvement of p38 MAPK activity in NF-kappaB-mediated MMP-2 function was further confirmed through the attenuation of p38 MAPK by SB203580 and NF-kappaB ectopic expression. Collectively, our results indicate that AE inhibits invasion of NPC cells by suppressing the expression of MMP-2 via the p38 MAPK-NF-kappaB signaling pathway.

Regulation of resistin by cyclic mechanical stretch in cultured rat vascular smooth muscle cells

Resistin has a potential role in atherosclerosis; however, the molecular mechanism underlying the increase in resistin expression in atherosclerosis remains unclear. As mechanical stretch plays an important role in atherosclerosis, in the present study we sought to investigate the cellular and molecular mechanisms underlying the regulation of resistin by cyclic mechanical stretch in VSMCs (vascular smooth muscle cells). VSMCs from thoracic aorta of adult Wistar rats were cultured and subjected to cyclic stretch. Cyclic mechanical stretch significantly increased resistin protein and mRNA expression as compared with control cells without stretch. The specific p38 MAPK (mitogen-activated protein kinase) inhibitor SB203580, the antioxidant N-acetylcysteine and p38 MAPK siRNA (small interfering RNA) attenuated the induction of resistin protein by cyclic stretch. Cyclic stretch significantly increased the phosphorylation of p38 MAPK, whereas pre-treatment with SB203580 and N-acetylcysteine significantly inhibited this effect. Cyclic stretch significantly increased ROS (reactive oxygen species) production, and pre-treatment with N-acetylcysteine significantly inhibited stretch-induced ROS production. Cyclic stretch also increased STAT3 (signal transducer and activator of transcription 3)-binding activity and resistin promoter activity, and resistin promoter activity was abolished when STAT3 in the promoter area was mutated. Pre-treatment with SB203580 and N-acetylcysteine significantly attenuated resistin promoter activity induced by cyclic stretch. Cyclic stretch increased the secretion of AngII (angiotensin II) and resistin from cultured VSMCs. Exogenous AngII increased resistin expression, and AngII receptor inhibition attenuated this effect. In conclusion, cyclic mechanical stretch increases resistin expression in cultured rat VSMCs. Stretch-induced resistin expression is mediated through ROS, and the p38 MAPK and STAT3 pathways. Therefore resistin induced by cyclic stretch may contribute to the pathogenesis of atherosclerosis under haemodynamic overload.Key

Hyperbaric oxygen activates discoidin domain receptor 2 via tumour necrosis factor-α and the p38 MAPK pathway to increase vascular smooth muscle cell migration through matrix metalloproteinase 2

DDR2 (discoidin domain receptor 2) regulates collagen turnover mediated by SMCs (smooth muscle cells) in atherosclerosis. HBO (hyperbaric oxygen) has been used in medical practice; however, the molecular mechanism of the beneficial effects of HBO is poorly understood. Furthermore, the effect of HBO on DDR2 has not been reported previously. In the present study, we investigated the cellular and molecular mechanisms of DDR2 regulation by HBO in VSMCs (vascular SMCs). Cells were exposed to 2.5 ATA (atmosphere absolute) of oxygen in a hyperbaric chamber. DDR2 protein (3.63-fold) and mRNA (2.34-fold) expression were significantly increased after exposure to 2.5 ATA HBO for 1 h. Addition of SB203580 and p38 MAPK (mitogen-activated protein kinase) siRNA (small interfering RNA) 30 min before HBO inhibited the induction of DDR2 protein. HBO also significantly increased DNA-protein binding activity of Myc/Max. Addition of SB203580 and an anti-TNF-alpha (tumour necrosis factor-alpha) monoclonal antibody 30 min before HBO abolished the DNA-protein binding activity induced by HBO. HBO significantly increased the secretion of TNF-alpha from cultured VSMCs. Exogenous addition of TNF-alpha significantly increased DDR2 protein expression, whereas anti-TNF-alpha and anti-(TNF-alpha receptor) antibodies blocked the induction of DDR2 protein expression. HBO significantly increased VSMC migration and proliferation, whereas DDR2 siRNA inhibited the migration induced by HBO. HBO increased activated MMP2 (matrix metalloproteinase 2) protein expression, and DDR2 siRNA abolished the induction of activated MMP2 expression induced by HBO. In conclusion, HBO activates DDR2 expression in cultured rat VSMCs. HBO-induced DDR2 is mediated by TNF-alpha and at least in part through the p38 MAPK and Myc pathways.