A new prognosis prediction model combining TNM stage with MAP2K4 and JNK in postoperative pancreatic cancer patients.

Mitogen-activated protein kinase kinase 4 (MAP2K4) is a tumor suppressor in many cancers. However, its roles and action mechanisms in pancreatic ductal adenocarcinoma (PDAC) remain unclear. Here, we analyzed MAP2K4 and its downstream kinases (c-Jun N-terminal kinase (JNK) and p38) using immunohistochemical staining and their prognostic significances using univariate and multivariate Cox proportional hazards regression analysis in our PDAC cohort. Then, we validated MAP2K4/JNK/p38 mRNA levels and prognostic significances using The Cancer Genome Atlas (TCGA) database. Finally, we evaluated the effects of MAP2K4 on the proliferation and invasion of PDAC cells. MAP2K4, JNK, and p38 proteins were expressed in 97.3 % (72/74), 95.6 % (65/68), and 88.6 % (62/70) of the samples, respectively, and their levels in tumor tissues were significantly higher than those in normal ducts. MAP2K4 protein expression was lower in male patients (p = 0.028). In our PDAC cohort, advanced TNM stage, low MAP2K4, and high JNK protein levels were significant prognostic factors for poor overall survival (OS) based on a univariate survival analysis (p = 0.006, p < 0.001, and p = 0.004, respectively). N stage and MAP2K4 and JNK protein levels were independent prognostic factors for OS based on multivariate analysis. We then built a prognosis prediction nomogram combining the standard TNM staging system with MAP2K4 and JNK expression that had a Harrell's C-index of 0.645. The new prognosis prediction model effectively stratified the resected patients with PDAC, from both our cohort and TCGA database, into low- and high-risk groups. Finally, MAP2K4 overexpression inhibited pancreatic cancer cell proliferation and migration in vitro. This study shows that reduced protein and mRNA levels of MAP2K4 found in PDAC patients, coupled to in vitro effects observed, support the tumor suppressor role of MAP2K4 in PDAC. Importantly, combining MAP2K4 and JNK expression with the TNM staging system results in a better prediction of postoperative survival of patients with PDAC.

Ensemble structural analyses depict the regulatory mechanism of non-phosphorylated human MAP2K4.

Mitogen-activated protein kinase kinase 4 (MAP2K4) plays a critical role in regulating the stress-activated protein kinase signaling cascade. A small angle X-ray scattering experiment, a powerful technique for analyzing a solution structure cleared from the structural artifacts due to crystal packing, provided the ensemble structures of human non-phosphorylated MAP2K4 in three states involving the apo form, the binary complex with an ATP analogue, and the ternary complex with the ATP analogue and substrate peptide. These ensemble structures provided more detailed mechanisms for regulating MAP2K4 in addition to those delineated only by the crystal structures in three states.

Propofol post-conditioning protects the blood brain barrier by decreasing matrix metalloproteinase-9 and aquaporin-4 expression and improves the neurobehavioral outcome in a rat model of focal cerebral ischemia-reperfusion injury.

Propofol, an intravenous anesthetic, inhibits neuronal apoptosis induced by ischemic stroke, protects the brain from ischemia/reperfusion injury and improves neuronal function. However, whether propofol is able to protect the blood brain barrier (BBB) and the underlying mechanisms have remained to be elucidated. In the present study, a rat model of cerebral ischemia/reperfusion was established, using a thread embolism to achieve middle cerebral artery occlusion. Rats were treated with propofol (propofol post-conditioning) or physiological saline (control) administered by intravenous injection 30 min following reperfusion. Twenty-four hours following reperfusion, neurobehavioral manifestations were assessed. The levels of cephaloedema, damage to the BBB and expression levels of matrix metalloproteinase-9 (MMP-9), aquaporin-4 (AQP-4) and phosphorylated c-Jun N-terminal kinase (pJNK) were determined in order to evaluate the effects of propofol on the BBB. In comparison to the cerebral ischemia/reperfusion group, the levels of brain water content and Evans blue content, as well as the expression levels of MMP-9, AQP-4 and pJNK were significantly reduced in the propofol post-conditioning group. These results indicated that propofol post-conditioning improved the neurobehavioral manifestations and attenuated the BBB damage and cephaloedema induced following cerebral ischemia/reperfusion. This effect may be due to the inhibition of MMP-9 and AQP-4 expression, and the concurrent decrease in JNK phosphorylation.

Differential expression patterns of metastasis suppressor proteins in basal cell carcinoma.

BACKGROUND: Basal cell carcinomas (BCCs) are common malignant skin tumors. Despite having a significant invasion capacity, they metastasize only rarely. Our aim in this study was to detect the expression patterns of the NM23-H1, NDRG1, E-cadherin, RHOGDI2, CD82/KAI1, MKK4, and AKAP12 metastasis suppressor proteins in BCCs. METHODS: A total of 96 BCC and 10 normal skin samples were included for the immunohistochemical study. Eleven frozen BCC samples were also studied by quantitative real time polymerase chain reaction (qRT-PCR) to detect the gene expression profile. RESULTS: NM23-H1 was strongly and diffusely expressed in all types of BCC. Significant cytoplasmic expression of NDRG1 and E-cadherin was also detected. However, AKAP12 and CD82/KAI1 expression was significantly decreased. The expressions of the other proteins were somewhere between the two extremes. Similarly, qRT-PCR analysis showed down-regulation of AKAP12 and up-regulation of NM23-H1 and NDRG1 in BCC. Morphologically aggressive BCCs showed significantly higher cytoplasmic NDRG1 expression scores and lower CD82/KAI1 scores than non-aggressive BCCs. CONCLUSION: The relatively preserved levels of NM23-H1, NDRG1, and E-cadherin proteins may have a positive effect on the non-metastasizing features of these tumors.

Biochemical mechanisms of bornyl caffeate induced cytotoxicity in rat pheochromocytoma PC12 cells.

The chemopreventive and antineoplastic activities of caffeic acid derivatives are highly dependent on the chemical structures and cancer cell types. The objective of the present study was to investigate the cytotoxicity of bornyl caffeate and the underlying molecular mechanisms in rat pheochromocytoma PC12 cells. Our initial studies demonstrated that bornyl caffeate exhibited potent cytotoxicity in PC12 cells in a concentration- and time-dependent manner. By examining the cell morphology on a fluorescence microscope and detecting the cell surface phosphoserine with Annexin V-FITC, we proposed that bornyl caffeate could induce apoptosis in PC12 cells. We tested this hypothesis by investigating the effects of bornyl caffeate on several apoptosis-related biomarkers. These experiments showed that bornyl caffeate induced the up-regulation of Bax and down-regulation of Bcl-xl, the disruption of mitochondrial membrane potential, the activation of caspase 3 and the cleavage of PARP. Mechanistic studies further revealed that bornyl caffeate caused the depletion of glutathione (GSH), generation of superoxide ion and progressive activation of p38 mitogen-activate protein kinase (MAPK) and c-Jun N-terminal kinase (JNK) in a concentration-dependent manner. In particular, GSH depletion appeared to be the most important mechanism underlying the cytotoxicity of bornyl caffeate. The preservation of the intracellular GSH contents with N-acetyl-L-cysteine (NAC), GSH and vitamin C abolished the effect of bornyl caffeate on the activation of p38 MAPK and JNK, preserved the integrity of mitochondrial membrane and ultimately rescued the cells from drug-induced cell death. These results suggest that bornyl caffeate induces apoptosis in PC12 cells via stimulating the depletion of GSH, the generation of reactive oxygen species (ROS) and the dissipation of mitochondrial transmembrane potential.

Influence of sleep deprivation on expression of MKK4 and c-fos in the mandibular condylar cartilage of rats.

The aim of this study was to investigate the changes in expression of mitogen-activated protein kinase kinase 4 (MKK4) and c-fos in the mandibular condylar cartilage of rats that had been subjected to sleep deprivation. One hundred and twenty female Wistar rats were randomly divided into 6 groups with 20 in each: sleep deprivation for 2 days, 4 days, 6 days, and 8 days, large-platform controls, and cage controls. After sleep deprivation by the modified multiple platform method the sleep-deprived rats were killed. The large-platform and cage control rats were killed at the same time as the rats deprived of sleep for 8 days. Haematoxylin and eosin were used to record the morphological changes in cartilage, and immunohistochemistry and real-time quantitative polymerase chain reaction (PCR) were used to detect the expression of MKK4 and c-fos. Pathological alterations were apparent after 6 and 8 days of sleep deprivation. Compared with control groups, the expression of MKK4 in the sleep-deprived groups was lower, while that of c-fos was higher. As the duration of sleep deprivation increased, the expression of MKK4 decreased. These results indicate that the variation in expression of MKK4 and c-fos may be correlated with pathological changes induced by sleep deprivation in mandibular condylar cartilage in rats.

Sitagliptin exerts an antinflammatory action.

CONTEXT: Sitagliptin is an inhibitor of the enzyme dipeptidyl peptidase-IV (DPP-IV), which degrades the incretins, glucagon-like peptide-1 and glucose-dependent insulinotropic polypeptide, and thus, sitagliptin increases their bioavailability. The stimulation of insulin and the suppression of glucagon secretion that follow exert a glucose lowering effect and hence its use as an antidiabetic drug. Because DPP-IV is expressed as CD26 on cell membranes and because CD26 mediates proinflammatory signals, we hypothesized that sitagliptin may exert an antiinflammatory effect. PATIENTS AND METHODS: Twenty-two patients with type 2 diabetes were randomized to receive either 100 mg daily of sitagliptin or placebo for 12 wk. Fasting blood samples were obtained at baseline and at 2, 4, and 6 hours after a single dose of sitagliptin and at 2, 4, 8, and 12 wk of treatment. RESULTS: Glycosylated hemoglobin fell significantly from 7.6 ± 0.4 to 6.9 ± 3% in patients treated with sitagliptin. Fasting glucagon-like peptide-1 concentrations increased significantly, whereas the mRNA expression in mononuclear cell of CD26, the proinflammatory cytokine, TNFα, the receptor for endotoxin, Toll-like receptor (TLR)-4, TLR-2, and proinflammatory kinases, c-Jun N-terminal kinase-1 and inhibitory-κB kinase (IKKß), and that of the chemokine receptor CCR-2 fell significantly after 12 wk of sitagliptin. TLR-2, IKKß, CCR-2, and CD26 expression and nuclear factor-κB binding also fell after a single dose of sitagliptin. There was a fall in protein expression of c-Jun N-terminal kinase-1, IKKß, and TLR-4 and in plasma concentrations of C-reactive protein, IL-6, and free fatty acids after 12 wk of sitagliptin. CONCLUSIONS: These effects are consistent with a potent and rapid antiinflammatory effect of sitagliptin and may potentially contribute to the inhibition of atherosclerosis. The suppression of CD26 expression suggests that sitagliptin may inhibit the synthesis of DPP-IV in addition to inhibiting its action.

Biological and prognostic relevance of mitogen-activated protein kinases in pancreatic adenocarcinoma.

OBJECTIVES: The aims of this study were to study the biological and clinical significance of 3 main proteins of the mitogen-activated protein kinase (MAPK) signaling pathway, ERK1/2, P38, and MKK4, in a series of patients having pancreatic adenocarcinomas treated by surgery. METHODS: We examined the immunohistochemical expression of 3 MAPK proteins, ERK1/2, P38, and MKK4 in 99 surgically resected pancreatic ductal adenocarcinomas. Tumor protein expression was studied with regard to pathological characteristics and to postsurgical recurrence-free and overall survivals. RESULTS: MKK4 expression was related to tumor cell proliferation, evaluated by the Ki67 index (P < 0.01). ERK1/2 expression was related to a shorter recurrence-free survival on both univariate and multivariate analysis (P < 0.01; odds ratio, 8.39; 95% confidence interval, 2.68-26.26) independently of lymph node metastases and tumor size, and to a shorter overall survival (P = 0.01) on univariate analysis. In patients without postsurgical treatment, both ERK1/2 and P38 tumor expression correlated with a shorter recurrence-free survival (P < 0.01 and P = 0.02, respectively). CONCLUSIONS: The results of our study suggest that in pancreatic ductal adenocarcinomas, the MKK4 protein was directly related to high cell proliferation, and that tumor ERK1/2 and P38 expression correlated to shorter postsurgical recurrence-free and overall survivals.

Potential characteristics of stem cells from human exfoliated deciduous teeth compared with bone marrow-derived mesenchymal stem cells for mineralized tissue-forming cell biology.

INTRODUCTION: Tissue engineering and regenerative medicine using stem cell biology has been a promising field for treatment of local and systemic intractable diseases. Recently, stem cells from human exfoliated deciduous teeth (SHED) have been identified as a novel population of stem cells. This study focused on the characterization of SHED as compared with bone marrow-derived mesenchymal stem cells (BMMSCs). METHODS: We investigated potential characteristics of SHED by using DNA microarray, real-time reverse transcriptase polymerase chain reaction, and immunofluorescence analysis. RESULTS: Multiple gene expression profiles indicated that the expression of 2753 genes in SHED had changed by ≥2.0-fold as compared with that in BMMSCs. One of the most significant pathways that accelerated in SHED was that of bone morphogenetic protein (BMP) receptor signaling, which contains several cascades such as PKA, JNK, and ASK1. When the BMP signaling pathway was stimulated by BMP-2, the expression of BMP-2, BMP-4, Runx2, and DSPP was up-regulated significantly in SHED than that in BMMSCs. Furthermore, the BMP-4 protein was expressed much higher in SHED but not in BMMSCs, as confirmed by immunofluorescence. CONCLUSIONS: By using the gene expression profiles, this study indicates that SHED is involved in the BMP signaling pathway and suggests that BMP-4 might play a crucial role in this. These results might be useful for effective cell-based tissue regeneration, including that of bone, pulp, and dentin, by applying the characteristics of SHED.

Mitogen-activated protein kinase phosphatase 2 regulates the inflammatory response in sepsis.

Sepsis results from a dysregulation of the regulatory mechanisms of the pro- and anti-inflammatory response to invading pathogens. The mitogen-activated protein (MAP) kinase cascades are key signal transduction pathways involved in the cellular production of cytokines. The dual-specific phosphatase 1 (DUSP 1), mitogen-activated protein kinase phosphatase-1 (MKP-1), has been shown to be an important negative regulator of the inflammatory response by regulating the p38 and Jun N-terminal protein kinase (JNK) MAP kinase pathways to influence pro- and anti-inflammatory cytokine production. MKP-2, also a dual-specific phosphatase (DUSP 4), is a phosphatase highly homologous with MKP-1 and is known to regulate MAP kinase signaling; however, its role in regulating the inflammatory response is not known. We hypothesized a regulatory role for MKP-2 in the setting of sepsis. Mice lacking the MKP-2 gene had a survival advantage over wild-type mice when challenged with intraperitoneal lipopolysaccharide (LPS) or a polymicrobial infection via cecal ligation and puncture. The MKP-2(-/-) mice also exhibited decreased serum levels of both pro-inflammatory cytokines (tumor necrosis factor alpha [TNF-alpha], interleukin-1beta [IL-1beta], IL-6) and anti-inflammatory cytokines (IL-10) following endotoxin challenge. Isolated bone marrow-derived macrophages (BMDMs) from MKP-2(-/-) mice showed increased phosphorylation of the extracellular signal-regulated kinase (ERK), decreased phosphorylation of JNK and p38, and increased induction of MKP-1 following LPS stimulation. The capacity for cytokine production increased in MKP-2(-/-) BMDMs following MKP-1 knockdown. These data support a mechanism by which MKP-2 targets ERK deactivation, thereby decreasing MKP-1 and thus removing the negative inhibition of MKP-1 on cytokine production.

Overexpression of mitogen-activated protein kinase kinase 4 and nuclear factor-kappaB in laryngeal squamous cell carcinoma: a potential indicator for poor prognosis.

This study aimed to investigate the expression and clinical significance of mitogen-activated protein kinase kinase 4 MKK4 and nuclear factor-kappaB (NF-kappaB) in patients with laryngeal squamous cell carcinoma (LSCC). We used immunohistochemistry (IHC) to examine the expression of MKK4 and NF-kappaB in 78 LSCCs and their adjacent normal tissues. To clarify the validity of MKK4 and NF-kappaB as determined by the IHC analysis, RT-PCR was performed on 21 tissues randomly selected from the 78 LSCCs. The positive expression rates of MKK4 and NF-kappaB in patients with LSCC were 67.9% (53/78) and 60.3% (47/78) respectively, which were significantly higher than those in the adjacent normal tissue (both P<0.01). The positive expression of MKK4 and NF-kappaB tended to be associated positively with lymph node metastasis (both P<0.01) as well as T stage (both P<0.01). The Spearman analysis indicated that the expression level of MKK4 was positively correlated with that of NF-kappaB significantly (rs=0.368, P<0.01). Overall survival curves estimated by Kaplan-Meier showed that tumor patients with low MKK4 and NF-kappaB expression in their tumor cells survive significantly longer than patients with high MKK4 and NF-kappaB levels (P=0.027, and P<0.01, respectively). In addition, multivariate Cox regression analysis showed that N stage, T stage and NF-kappaB expression are significant independent prognostic factors for overall survival (P<0.01, P=0.014, and P=0.027, respectively). These findings suggested that the expression of MKK4 and NF-kappaB may be considered as a useful prognostic marker of LSCC after surgical resection.

Quantitative cell signalling analysis reveals down-regulation of MAPK pathway activation in colorectal cancer.

Mitogen-activated protein kinases (MAPK) are considered to play significant roles in colonic carcinogenesis and kinase inhibitor therapy has been proposed as a potential tool in the treatment of this disease. Reverse-phase microarray assays using phospho-specific antibodies can directly measure levels of phosphorylated protein isoforms. In the current study, samples from 35 cases of untreated colorectal cancer colectomies were laser capture-microdissected to isolate epithelium and stroma from cancer as well as normal (i.e. uninvolved) mucosa. Lysates generated from these four tissue types were spotted onto reverse-phase protein microarrays and probed with a panel of antibodies to ERK, p-ERK, p38, p-p38, p-JNK, MEK and p-MEK. Whereas total protein levels were unchanged, or slightly elevated (p38, p = 0.0025) in cancers, activated isoforms, including p-ERK, p-p38 and p-JNK, were decreased two- to four-fold in cancers compared with uninvolved mucosa (p < 0.0023 in all cases except for p-JNK in epithelium, where decrement was non-significant). This was backed up by western blotting. Dukes' stage B and C cancers displayed lower p-ERK and p-p38 expression than Dukes' stage A cancers, although this was not statistically significant. It is concluded that MAPK activity may be down-regulated in colorectal cancer and that further exploration of inhibitory therapy in this system should be carefully evaluated if this finding is confirmed in larger series.

Survival signaling and terminal differentiation in cholesteatoma epithelium.

CONCLUSION: There is a strong indication that epithelial keratinocytes in cholesteatoma are protected against apoptosis. The late terminal differentiation program in cholesteatoma epithelium is disturbed. OBJECTIVES: Previously, minimal apoptosis has been demonstrated in cholesteatoma epithelium. The phosphoinositide 3-kinase/Akt/protein kinase B (PI3K/Akt/PKB) and the mitogen activated protein kinases (MAPK) signaling transduction pathways have been reported to protect epithelial cells against apoptosis. Both pathways have also been proven to regulate late terminal differentiation of keratinocytes. In cholesteatoma epithelium, MAPK activation has been shown to be associated with terminal differentiation. The purpose of this study was to investigate whether in human cholesteatoma epithelium protection against programmed cell death by means of PI3K/Akt survival signaling is present and associated with MAPK activation and terminal differentiation. MATERIALS AND METHODS: Fifteen human cholesteatoma and patient-matched retro-auricular skin samples were immunohistochemically stained for pAkt/PKB, phosphorylated extracellular regulated kinase1/2 (pERK1/2), phosphorylated JNK/SAPK, phosphorylated p38, involucrin and filaggrin. Positive cells were counted by computer-assisted digital image analysis. RESULTS: Protein expressions of pAkt/PKB, pERK1/2, pp38, and involucrin in cholesteatoma epithelium were significantly increased when compared with retro-auricular skin (p<0.01). Filaggrin expression was significantly decreased (p=0.03). The positive correlation was confirmed between both pERK1/2 and pp38, and involucrin (p < or = 0.05).

Deletion of the inducible 70-kDa heat shock protein genes in mice impairs cardiac contractile function and calcium handling associated with hypertrophy.

BACKGROUND: Hspa1a and Hspa1b genes encode stress-inducible 70-kDa heat shock proteins (Hsp70) that protect cells from insults such as ischemia. Mice with null mutations of both genes (KO) were generated, and their cardiac phenotype was explored. METHODS AND RESULTS: Heart rate and blood pressures were normal in the KO mice. Hearts from KO mice were more susceptible to both functional and cellular damage by ischemia/reperfusion. Cardiac hypertrophy developed in Hsp70-KO mice. Ca2+ transients in cardiomyocytes of KO mice showed a delayed (120%) calcium decline and decreased sarcoplasmic reticulum calcium content. Cell shortening was decreased by 35%, and rates of contraction and relaxation were slower by 40%. These alterations can be attributed to the absence of Hsp70 because viral expression of Hsp70 in KO cultured cardiomyocytes restored these parameters. One mechanism underlying myocyte dysfunction could be decreased SERCA2a expression. This hypothesis was supported by a prolonged calcium decline and decreased SERCA2a protein. Viral SERCA2a expression restored contractility and Ca2+ transients. We examined the involvement of Jun N-terminal kinase (JNK), p38-mitogen-activated protein kinase (p38-MAPK), Raf-1, and extracellular signal-regulated kinase (ERK) in SERCA2a downregulation and the cardiac phenotype of KO mice. Levels of phosphorylated JNK, p38-MAPK, Raf-1, and ERK were elevated in KO hearts. Activation of the Raf-1-ERK pathway in normal cardiomyocytes resulted in decreased SERCA2a. CONCLUSIONS: Absence of Hsp70 leads to dysfunctional cardiomyocytes and impaired stress response of Hsp70-KO hearts against ischemia/reperfusion. In addition, deletion of Hsp70 genes might induce cardiac dysfunction and development of cardiac hypertrophy through the activation of JNK, p38-MAPK, Raf-1, and ERK.

Adrenomedullin regulates expressions of transforming growth factor-beta1 and beta1-induced matrix metalloproteinase-2 in hepatic stellate cells.

Adrenomedullin (AM), a peptide isolated from human pheochromocytoma, can be produced and secreted by various types of cells including hepatic stellate cells (HSCs), and its possible role in HSCs is not clear now. In the present study, the interactive regulation between transforming growth factor (TGF)-beta1 and AM and the effect of AM on TGF-beta1-induced matrix metalloproteinase (MMP)-2 expression in HSCs were investigated. TGF-beta1 and AM inhibited gene transcript level mutually (real-time reverse transcription-polymerase chain reaction). AM suppressed the protein expression level of TGF-beta1 (Western blot), but TGF-beta1 might have no effect on AM secretion level. MMP-2 protein expression in HSCs was increased in response to TGF-beta1, and upregulation of MMP-2 expression stimulated with TGF-beta1 was suppressed by AM in dose-dependent manner (Western blot). AM decreased the phosphorylation level of extracellular signal-regulated kinase (ERK) in HSCs treated with TGF-beta1, and TGF-beta1-induced MMP-2 expression was suppressed by adding Mitogen-activated protein Kinase/ERK (MEK) inhibitor U(0126) (Western blot)(.) Our results suggest that AM may intervene the activation of HSCs by inhibiting TGF-beta1 production and TGF-beta1-induced MMP-2 expression; AM may suppress the upregulation of MMP-2 expression induced by TGF-beta1 partially through ERK pathway.

Thermal injury-induced peroxynitrite production and pulmonary inducible nitric oxide synthase expression depend on JNK/AP-1 signaling.

OBJECTIVE: To determine whether burn-induced peroxynitrite production and expression of lung inducible nitric oxide synthase (iNOS), intercellular adhesion molecule (ICAM)-1, vascular cell adhesion molecule (VCAM)-1, CXCR2, macrophage inflammatory protein (MIP)-2, and neutrophil chemokine (KC) are mediated by the c-Jun NH2-terminal kinase (JNK). DESIGN: Prospective, experimental study. SETTING: Research laboratory at a university hospital. SUBJECTS: Thermal injury models in the mice. INTERVENTIONS: In experiment 1, specific pathogen-free C57/BL6 mice were subjected to 30% total body surface area third-degree burn over shaved back. At 0 hr, 2 hrs, 4 hrs, and 6 hrs after burn, lung tissues of those mice were harvested for JNK activity assay, AP-1 DNA-binding activity, and pJNK immunohistochemistry. In experiment 2, a specific JNK inhibitor, SP600125, was given (30 mg/kg intraperitoneally) to mice immediately postburn to suppress the JNK activity. At 8 hrs after burn, blood was assayed for the peroxynitrite-mediated dihydrorhodamine (DHR) 123 oxidation. Lung tissues were harvested for myeloperoxidase (MPO) determination, ICAM-1, VCAM-1, CXCR2, KC, MIP-2, interleukin-1beta, and interleukin-6 messenger RNA expression; iNOS immunohistochemical staining; and histologic studies. Pulmonary microvascular dysfunction was quantified by measuring the extravasations of Evans blue dye. MEASUREMENTS AND MAIN RESULTS: The JNK activity and AP-1 DNA-binding activity of lung tissue significantly increased to a peak at 2 hrs and 4 hrs, respectively, after thermal injury. Immunohistochemical study demonstrated that the increase of the pJNK was mostly from the bronchiole epithelial cells. This increase of MPO activity in lung, blood DHR 123 oxidation level, and lung permeability increased six-fold, nine-fold, and four-fold after burn. SP600125 administration obliterated the thermal injury-induced JNK activity, AP-1 DNA-binding activity, and iNOS expression in lung tissue. SP600125 treatment also significantly decreased MPO activity, blood DHR 123 oxidation, and lung permeability by 54%, 8%, and 47%, respectively, and markedly decreased the thermal injury-induced perivascular and interstitial inflammatory cell infiltration and septum edema. Furthermore, SP600125 abolished thermal injury-induced ICAM-1, VCAM-1, CXCR2, MIP-2, and KC but not interleukin-1beta and interleukin-6 messenger RNA levels of lung tissues. CONCLUSIONS: Thermal injury induces lung tissue JNK activation and AP-1 DNA-binding activity mainly from airway epithelial cells. Thermal injury-induced peroxynitrite production and lung iNOS, ICAM-1, and VCAM-1 expression are mediated by the JNK signaling. JNK inhibition decreases thermal injury-induced lung neutrophil infiltration and subsequently pulmonary hyperpermeability.

Insulin-like growth factor-1 mediates stretch-induced upregulation of myostatin expression in neonatal rat cardiomyocytes.

OBJECTIVES: Myostatin, a negative regulator of muscle growth, is increased in hypertrophied and infarcted heart. However, the mechanism of regulation is not known. Mechanical stress is an important regulatory factor for cardiomyocyte growth. The aim of the study was to investigate the effect of cyclic stretch on the expression of myostatin gene in cardiomyocytes. METHODS: Neonatal Wistar rat cardiomyocytes grown on a flexible membrane base were stretched by vacuum to 20% of maximum elongation at 60 cycles/min. An in vivo model of aorta-caval shunt in adult rats was used to investigate the myostatin expression. RESULTS: Cyclic stretch significantly increased myostatin protein and mRNA expression after 6 to 18 h of stretch. Addition of the p38 mitogen-activated protein (MAP) kinase inhibitor SB203580, insulin-like growth factor-1 (IGF-1) monoclonal antibody, and p38 siRNA 30 min before stretch inhibited the induction of myostatin protein. Cyclic stretch increased, while SB203580, IGF-1, and IGF-1 receptor antibody abolished, the phosphorylated p38 protein. Gel shift assays showed significant increase of DNA-protein binding activity of myocyte enhancer factor 2 (MEF2) after stretch, and transfection with p38 siRNA abolished the DNA-protein binding activity induced by cyclic stretch. Cyclic stretch significantly increased the IGF-1 secretion from myocytes. Both conditioned media from stretched myocytes and exogenous administration of IGF-1 recombinant protein to the non-stretched myocytes increased myostatin protein expression similar to that seen after cyclic stretch. An in vivo model of aorta-caval shunt in adult rats also demonstrated the increased myostatin expression in the myocardium. CONCLUSIONS: Cyclic mechanical stretch enhances myostatin expression in cultured rat neonatal cardiomyocytes. The stretch-induced myostatin is mediated by IGF-1 at least in part through a p38 MAP kinase and MEF2 pathway.

Decreased glycolytic metabolism accelerates apoptosis in response to 2-acetyl furanonaphthoquinone in K1735 melanoma irrespective of bcl-2 overexpression.

Solid tumors are often placed under stress conditions, such as glucose starvation which may result in topoisomerase II drug resistance. In this study, we investigated whether glucose deprivation or substitution by fructose regulates tumor cell apoptosis induced by 2-acetyl furanonaphthoquinone (FNQ). We now show that FNQ exerts much greater antitumor activity than either 7-methoxy 2-ethyl FNQ or 2-ethyl FNQ. Whereas 0.8 microM FNQ induces apoptosis after 16 hours in glucose-supplemented conditions irrespective of bcl-2 overexpression in K1735 melanoma, 0.5 microM FNQ is also effective within 12 hours in low glucose or in fructose-supplemented medium. Under the latter conditions, apoptosis-associated PARP cleavage and cytosolic cytochrome C are increased, together with induction and partial translocation to mitochondria of phosphorylated Jun-N-terminal kinase and massive upregulation of mitochondrial Mn superoxide dismutase. We propose that mitochondrial colocalization of these activities is important in this synergistic anti-tumor effect of FNQ and glucose depletion. Since glucose limitation slows proliferation and decreases efficacy of some genotoxic drugs that trigger apoptosis in rapidly dividing cells, we propose evaluating FNQ as a novel therapeutic anti-cancer adjuvant against slowly proliferating tumors.

Reduced metastasis-suppressor gene mRNA-expression in breast cancer brain metastases.

PURPOSE: Brain metastases are an increasingly common complication in breast cancer patients. The Metastasis Suppressor Genes (MSG) Nm23, KISS1, KAI1, BRMS1, and Mkk4 have been associated with the metastatic potential of breast cancer in vitro and in vivo. METHODS: The mRNA expression of Nm23, KISS1, KAI1, BRMS1, and Mkk4 in fresh frozen tissue samples of brain metastases from ductal invasive breast cancer specimens was examined in relation to primary tumors. In a first step, mRNA expression screening was carried out using a semi-quantitative RT-PCR approach, in a second step quantitative real-time RT-PCR was performed on selected specimens. By immunohistochemical staining, gene products were visualized on the protein level. RESULTS: Semi-quantitative RT-PCR revealed reduced mRNA expression of Nm23, KISS1, KAI1, BRMS, and Mkk4 in brain metastases. Results for KISS1, KAI1, BRMS, and Mkk4 were confirmed by real-time RT-PCR. In detail, mRNA expression reduction in breast cancer brain metastases was tenfold. Expression of MSG could be confirmed by immunohistochemical staining on protein level. CONCLUSIONS: Our investigations revealed significantly reduced mRNA expression of metastases suppressor genes KISS1, KAI1, BRMS1, and Mkk4 in breast cancer brain metastasis. Particularly, in the case of KISS1 and Mkk4, an important role for future treatment of patients with breast cancer brain metastatic lesions can be assumed.