Modulation of Plasma Metabolite Biomarkers of the MAPK Pathway with MEK Inhibitor RO4987655: Pharmacodynamic and Predictive Potential in Metastatic Melanoma.

MAPK pathway activation is frequently observed in human malignancies, including melanoma, and is associated with sensitivity to MEK inhibition and changes in cellular metabolism. Using quantitative mass spectrometry-based metabolomics, we identified in preclinical models 21 plasma metabolites including amino acids, propionylcarnitine, phosphatidylcholines, and sphingomyelins that were significantly altered in two B-RAF-mutant melanoma xenografts and that were reversed following a single dose of the potent and selective MEK inhibitor RO4987655. Treatment of non-tumor-bearing animals and mice bearing the PTEN-null U87MG human glioblastoma xenograft elicited plasma changes only in amino acids and propionylcarnitine. In patients with advanced melanoma treated with RO4987655, on-treatment changes of amino acids were observed in patients with disease progression and not in responders. In contrast, changes in phosphatidylcholines and sphingomyelins were observed in responders. Furthermore, pretreatment levels of seven lipids identified in the preclinical screen were statistically significantly able to predict objective responses to RO4987655. The RO4987655 treatment-related changes were greater than baseline physiological variability in nontreated individuals. This study provides evidence of a translational exo-metabolomic plasma readout predictive of clinical efficacy together with pharmacodynamic utility following treatment with a signal transduction inhibitor. Mol Cancer Ther; 16(10); 2315-23. ©2017 AACR.

Ageing related periostin expression increase from cardiac fibroblasts promotes cardiomyocytes senescent.

Periostin, as an extracellular matrix (ECM) protein, plays a critical role in myocardial fibrosis and also might be involved in the heart inflammatory process since it is a downstream molecule of IL4 and IL13. Considering the possible important role of periostin in heart aging, this study explored periostin expression pattern in both rat and human, the effect of periostin expression on cardiomyocyte senescent and expression of three cytokines (IL13, IL4 and IL6) in different age groups of human. This study found heart aging is associated with increased expression of periostin from cardiac fibroblasts and serum inflammatory cytokines (IL13 and IL6). Excessive periostin expression contributed to cardiomyocyte senescent, which could be alleviated through blocking the Ang-II-TGF ß1-MAPK/ERK pathway. Thus, periostin might play an important role in a vicious circle (aging-fibrosis-inflammation-aging) of heart through promoting myocardial fibrosis and cardiomyocyte senescent simultaneously. It is a potential aging marker that could be directly measured in serum.

Cardiac signaling molecules and plasma biomarkers after cardiac transplantation: impact of tacrolimus versus cyclosporine.

BACKGROUND: We investigated cardiac proinflammatory, mitogenic, and apoptotic signaling events, and plasma biomarkers of inflammation and oxidative stress in de novo adult cardiac transplant (CTX) patients receiving tacrolimus (TAC) or cyclosporine A (CsA). METHODS: One hundred CTX recipients were randomized 1:1 to TAC/CsA in a prospective, randomized open-label multicenter study. Biomarkers of inflammation, immunity, oxidative stress, and cardiac signaling underlying growth and inflammation (extracellular signal-related kinase 1/2, p38 mitogen-activated protein kinase, mitogen-activated protein kinase kinases [MEK] 1/2 and 3/6, c-Src), and apoptosis and survival (c-Jun NH2-terminal kinases [JNK], Bax/Bcl2, Akt) were assessed at 2, 4, 12, 26, and 52 weeks post-CTX. Plasma from healthy controls (n = 30) and tissue from explanted non-failing hearts (n = 6) were used as controls. RESULTS: Biomarkers of inflammation/immunity (interleukin -6 and -18, soluble intercellular adhesion molecule, E-selectin, monocyte chemoattractant protein-1, osteopontin, fibrinogen, N-terminal prohormone brain natriuretic peptide, high-sensitive C-reactive protein) and oxidative stress (thiobarbituric acid reactive substances, nitrotyrosine) were increased, and antioxidant capacity was (glutathione/glutathione disulfide) decreased in patients vs healthy controls (p < 0.05). Phosphorylation of mitogen-activated protein kinases and Akt was increased, and Bax/Bcl was decreased in transplanted vs non-transplanted hearts. Except for plasma fibrinogen, which was lower in TAC vs. CsA, (p = 0.01), there were no significant differences in parameters studied between TAC vs CsA immunoprophylaxis. CONCLUSIONS: De novo CTX recipients exhibit significant sub-clinical inflammation and oxidative stress that persists 12 months after transplantation. Associated with this is activation of myocardial growth and inflammatory signaling and decreased apoptosis. Our findings suggest that CTX is an inflammatory condition associated with oxidative stress and myocardial growth regardless of CsA or TAC immunoprophylaxis and independently of rejection status.

Neurons activated during fear memory consolidation and reconsolidation are mapped to a common and new topography in the lateral amygdala.

A key question in neuroscience is how memory is selectively allocated to neural networks in the brain. This question remains a significant research challenge, in both rodent models and humans alike, because of the inherent difficulty in tracking and deciphering large, highly dimensional neuronal ensembles that support memory (i.e., the engram). In a previous study we showed that consolidation of a new fear memory is allocated to a common topography of amygdala neurons. When a consolidated memory is retrieved, it may enter a labile state, requiring reconsolidation for it to persist. What is not known is whether the original spatial allocation of a consolidated memory changes during reconsolidation. Knowledge about the spatial allocation of a memory, during consolidation and reconsolidation, provides fundamental insight into its core physical structure (i.e., the engram). Using design-based stereology, we operationally define reconsolidation by showing a nearly identical quantity of neurons in the dorsolateral amygdala (LAd) that expressed a plasticity-related protein, phosphorylated mitogen-activated protein kinase, following both memory acquisition and retrieval. Next, we confirm that Pavlovian fear conditioning recruits a stable, topographically organized population of activated neurons in the LAd. When the stored fear memory was briefly reactivated in the presence of the relevant conditioned stimulus, a similar topography of activated neurons was uncovered. In addition, we found evidence for activated neurons allocated to new regions of the LAd. These findings provide the first insight into the spatial allocation of a fear engram in the LAd, during its consolidation and reconsolidation phase.

Obesity, inflammation and resting energy expenditure: possible mechanism of progranulin in this pathway.

AIM: The aim of the study was to measure circulating PGRN levels and to investigate its potential correlation with resting metabolic rate and obesity related complications. Moreover, to investigate on the PGRN and some important gene expressions in energy expenditure in vitro in samples of PBMCs derived from all participants of our study in a cellular model. METHODS: Of the 163 participants who were recruited for the current cross-sectional study, 37 (22.69%) were normal weight (18.5≤BMI<25), 53 (32.51%) were overweight (25≤BMI<30), 48 (29.44%) were categorized as class I obese (BMI 30 -34.9) and 25 (15.33%) were classified as class II and III obese (BMI≥35). All participants were assessed for the measurement of RMR by means of indirect calorimetry following an overnight fasting. Body composition was analyzed with the Bioelectrical Impedance technique by the BODY COMPOSITION ANALYZER BC-418M -Tanita. The PBMCs were separated from whole blood by Ficoll-hypaque technique. Total cellular RNA was extracted and the cDNA was synthesized. This process was followed by real-time PCR using specific primer pairs for PGRN, AKT, MAPK and mRNA, and beta actin mRNA was used as the internal control. Circulating PGRN was measured with the use of ELISA method. RESULTS: The circulating levels and gene expressions of PGRN rose in parallel with the increase of body weight. However, there was significant difference in the strength of association between circulating PGRN as well as PGRN gene expression and obesity-related variables. Moreover, PGRN gene expression had significant correlation with BMI, visceral fat, MAPK and AKT gene expression. The increased mass of visceral fat in correlation with the increased PGRN levels was more pronounced in high or normal resting metabolic rate group compared with the group with low resting metabolic rate. After adjusting for BMI and gender, we found that circulating PGRN can predict the RMR/kg independent of other variables such as TG, HDL, and hs-CRP (P=0.03). CONCLUSION: PGRN associated with obesity and glucose homeostasis and may predict the resting metabolic rate levels independent of confounder factors. Experimental study may clarify the PGRN role in obesity etiology through metabolism regulation.

Cyclic nucleotides and mitogen-activated protein kinases: regulation of simvastatin in platelet activation.

BACKGROUND: 3-Hydroxy-3-methyl-glutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) have been widely used to reduce cardiovascular risk. These statins (i.e., simvastatin) may exert other effects besides from their cholesterol-lowering actions, including inhibition of platelet activation. Platelet activation is relevant to a variety of coronary heart diseases. Although the inhibitory effect of simvastatin in platelet activation has been studied; the detailed signal transductions by which simvastatin inhibit platelet activation has not yet been completely resolved. METHODS: The aim of this study was to systematically examine the detailed mechanisms of simvastatin in preventing platelet activation. Platelet aggregation, flow cytometric analysis, immunoblotting, and electron spin resonance studies were used to assess the antiplatelet activity of simvastatin. RESULTS: Simvastatin (20-50 microM) exhibited more-potent activity of inhibiting platelet aggregation stimulated by collagen than other agonists (i.e., thrombin). Simvastatin inhibited collagen-stimulated platelet activation accompanied by [Ca2+]i mobilization, thromboxane A2 (TxA2) formation, and phospholipase C (PLC)gamma2, protein kinase C (PKC), and mitogen-activated protein kinases (i.e., p38 MAPK, JNKs) phosphorylation in washed platelets. Simvastatin obviously increased both cyclic AMP and cyclic GMP levels. Simvastatin markedly increased NO release, vasodilator-stimulated phosphoprotein (VASP) phosphorylation, and endothelial nitric oxide synthase (eNOS) expression. SQ22536, an inhibitor of adenylate cyclase, markedly reversed the simvastatin-mediated inhibitory effects on platelet aggregation, PLCgamma2 and p38 MAPK phosphorylation, and simvastatin-mediated stimulatory effects on VASP and eNOS phosphorylation. CONCLUSION: The most important findings of this study demonstrate for the first time that inhibitory effect of simvastatin in platelet activation may involve activation of the cyclic AMP-eNOS/NO-cyclic GMP pathway, resulting in inhibition of the PLCgamma2-PKC-p38 MAPK-TxA2 cascade, and finally inhibition of platelet aggregation.

Effect of different antihypertensive treatments on Ras, MAPK and Akt activation in hypertension and diabetes.

Ras GTPases function as transducers of extracellular signals regulating many cell functions, and they appear to be involved in the development of hypertension. In the present study, we have investigated whether antihypertensive treatment with ARBs (angiotensin II receptor blockers), ACEi (angiotensin-converting enzyme inhibitors) and diuretics induce changes in Ras activation and in some of its effectors [ERK (extracellular-signal-regulated kinase) and Akt] in lymphocytes from patients with hypertension without or with diabetes. ACEi treatment transiently reduced Ras activation in the first month of treatment, but diuretics induced a sustained increase in Ras activation throughout the 3 months of the study. In patients with hypertension and diabetes, ARB, ACEi and diuretic treatment increased Ras activation only during the first week. ACEi treatment increased phospho-ERK expression during the first week and also in the last 2 months of the study; however, diuretic treatment reduced phospho-ERK expression during the last 2 months of the study. In patients with hypertension and diabetes, antihypertensive treatments did not induce changes in phospho-ERK expression in lymphocytes. ACEi treatment reduced phospho-Akt expression in patients with hypertension and diabetes only in the first month of treatment. In conclusion, these findings show that antihypertensive treatments with ACEi, and diuretics to a lesser extent, modify Ras activation and some of its signalling pathways, although in different directions, whereas ARBs do not appear to have any influence on Ras signalling pathways.

Constitutive activation of the 41- and 43-kDa mitogen-activated protein (MAP) kinases in the progression of prostate cancer to an androgen-independent state.

AIM: The 41- and 43-kDa mitogen-activated protein kinases (MAPK; ERK2 and ERK1, respectively) play pivotal roles in the mitogenic signal transduction pathway. We previously demonstrated that constitutive activation of the MAPK cascade was related to the carcinogenesis of human tumors. In this study, we examined whether constitutive activation of MAPK was related to the progression to androgen independence of prostate cancer. METHODS: MAPK activation was examined by the appearance of phosphorylated forms and an in vitro kinase assay in four human (androgen-dependent and independent) prostate cancer cell lines and rat prostate cancer cell line Dunning (androgen-sensitive G line, and androgen-independent AT-3, AT-6 sublines). In addition, when androgen-dependent mouse Shionogi Carcinoma 115 (SC115) cells were serially cultured without androgen to obtain androgen-independent cells, the time and degree of MAPK activation were examined. RESULTS: One of three human androgen-independent cell lines (DU145) showed constitutive activation of MAPK, while an androgen-dependent cell line (LNCaP) did not show MAPK activation. While MAPK were not activated in an androgen-sensitive Dunning G cell line, MAPK were activated in androgen-independent sublines (AT-3 and AT-6) derived from a G cell line. In addition, when SC115 cells were serially cultured without androgen, the cells 16-24 weeks after androgen removal showed MAPK activation. Furthermore, in subcloned cells, MAPK activation was observed even in the cells maintained for 9 weeks in medium without testosterone. CONCLUSIONS: The present fi ndings suggest that constitutive activation of MAPK may be associated with the acquisition of hormone independence in prostate cancer and that clonal selection after androgen removal and hormone-independent growth through the MAPK signal transduction pathway could begin at a relatively early period in the individual cells.

Suppression of PMN apoptosis by hypoxia is dependent on Mcl-1 and MAPK activity.

BACKGROUND: Hypoxia has been shown to delay the onset of neutrophil (polymorphonuclear leukocytes [PMNs]) apoptosis. With the use of antisense oligonucleotides, we have previously demonstrated that Mcl-1 is necessary for this effect. We wanted to further characterize the expression of Mcl-1 and examine signaling pathways required for the delay in apoptosis that is mediated by hypoxia. METHODS: For kinase signaling inhibition, PMNs were incubated for 12 hours with the following inhibitors: PD98059 Mitogen Activated Protein Kinase Kinase (MEK), SB202190 (p38 mitogen-activated protein kinase [MAPK]), and LY294002 (phosphatidyl inositol-3-kinase [PI3K]). PMNs that were treated with inhibitors were assessed for apoptosis by morphologic features or were lysed for Western blot analysis. RESULTS: Western blot analyses, immunofluorescent staining, and quantification showed an upregulation of Mcl-1 expression after 12 hours of incubation in response to hypoxia. When inhibitors of either MEK or p38 MAPK were incubated with PMNs during hypoxia, apoptosis increased to similar levels as normoxia. We further wanted to determine whether signaling through p38 MAPK or MEK led to increased Mcl-1 expression. Western blot analysis confirmed that the inhibition of p38 MAPK led to a significant decrease in Mcl-1 expression. CONCLUSIONS: We have documented a novel mechanism by which hypoxia can modify PMN apoptosis in the wound site by the activation of p38 MAPK signaling, thereby inducing the anti-apoptotic protein Mcl-1.