The role of calcium, calcium-activated K+ channels, and tyrosine/kinase in psoralen-evoked responses in human melanoma cells

8-Methoxy psoralen (8-MOP) exerts a short-term (24 h) mitogenic action, and a long-term (48-72 h) anti-proliferative and melanogenic action on two human melanoma cell lines, SK-Mel 28 and C32TG. An increase of intracellular calcium concentration was observed by spectrofluorometry immediately after the addition of 0.1 mM 8-MOP to both cell lines, previously incubated with calcium probe fluo-3 AM (5 µM). The intracellular Ca2+ chelator BAPTA/AM (1 µM) blocked both early (mitogenic) and late (anti-proliferative and melanogenic) 8-MOP effects on both cell lines, thus revealing the importance of the calcium signal in both short- and long-term 8-MOP-evoked responses. Long-term biological assays with 5 and 10 mM tetraethylammonium chloride (TEA, an inhibitor of Ca2+-dependent K+ channels) did not affect the responses to psoralen; however, in 24-h assays 10 mM TEA blocked the proliferative peak, indicating a modulation of Ca2+-dependent K+ channels by 8-MOP. No alteration of cAMP basal levels or forskolin-stimulated cAMP levels was promoted by 8-MOP in SK-Mel 28 cells, as determined by radioimmunoassay. However, in C32TG cells forskolin-stimulated cAMP levels were further increased in the presence of 8-MOP. In addition, assays with 1 µM protein kinase C and calcium/calmodulin-dependent kinase inhibitors, Ro 31-8220 and KN-93, respectively, excluded the participation of these kinases in the responses evoked by 8-MOP. Western blot with antibodies anti-phosphotyrosine indicated a 92% increase of the phosphorylated state of a 43-kDa band, suggesting that the phosphorylation of this protein is a component of the cascade that leads to the increase of tyrosinase activity.

Reactive oxygen species and angiotensin II signaling in vascular cells: implications in cardiovascular disease

Diseases such as hypertension, atherosclerosis, hyperlipidemia, and diabetes are associated with vascular functional and structural changes including endothelial dysfunction, altered contractility and vascular remodeling. Cellular events underlying these processes involve changes in vascular smooth muscle cell (VSMC) growth, apoptosis/anoikis, cell migration, inflammation, and fibrosis. Many factors influence cellular changes, of which angiotensin II (Ang II) appears to be amongst the most important. The physiological and pathophysiological actions of Ang II are mediated primarily via the Ang II type 1 receptor. Growing evidence indicates that Ang II induces its pleiotropic vascular effects through NADPH-driven generation of reactive oxygen species (ROS). ROS function as important intracellular and intercellular second messengers to modulate many downstream signaling molecules, such as protein tyrosine phosphatases, protein tyrosine kinases, transcription factors, mitogen-activated protein kinases, and ion channels. Induction of these signaling cascades leads to VSMC growth and migration, regulation of endothelial function, expression of pro-inflammatory mediators, and modification of extracellular matrix. In addition, ROS increase intracellular free Ca2+ concentration ([Ca2+]i), a major determinant of vascular reactivity. ROS influence signaling molecules by altering the intracellular redox state and by oxidative modification of proteins. In physiological conditions, these events play an important role in maintaining vascular function and integrity. Under pathological conditions ROS contribute to vascular dysfunction and remodeling through oxidative damage. The present review focuses on the biology of ROS in Ang II signaling in vascular cells and discusses how oxidative stress contributes to vascular damage in cardiovascular disease.

Patient and physician evaluation of the severity of acute asthma exacerbations

We studied the ability of patients not experienced in the use of peak expiratory flow meters to assess the severity of their asthma exacerbations and compared it to the assessment of experienced clinicians. We also evaluated which data of physical examination and medical history are used by physicians to subjectively evaluate the severity of asthma attacks. Fifty-seven adult patients (15 men and 42 women, with a mean (± SD) age of 37.3 ± 14.5 years and 24.0 ± 17.9 years of asthma symptoms) with asthma exacerbations were evaluated in a University Hospital Emergency Department. Patients and physicians independently evaluated the severity of the asthma attack using a linear scale. Patient score, physician score and forced expiratory volume at the first second (FEV1) were correlated with history and physical examination variables, and were also considered as dependent variables in multiple linear regression models. FEV1 correlated significantly with the physician score (rho = 0.42, P = 0.001), but not with patient score (rho = 0.03; P = 0.77). Use of neck accessory muscles, expiratory time and wheezing intensity were the explanatory variables in the FEV1 regression model and were also present in the physician score model. We conclude that physicians evaluate asthma exacerbation severity better than patients and that physician's scoring of asthma severity correlated significantly with objective measures of airway obstruction (FEV1). Some variables (the use of neck accessory muscles, expiratory time and wheezing intensity) persisted as explanatory variables in physician score and FEV1 regression models, and should be emphasized in medical schools and emergency settings.

Increased expression of p38 mitogen- activated protein kinase is related to the acute renal lesions induced by gentamicin

Mitogen-activated protein kinases (MAPK) may be involved in the pathogenesis of acute renal failure. This study investigated the expression of p-p38 MAPK and nuclear factor kappa B (NF-kappaB) in the renal cortex of rats treated with gentamicin. Twenty rats were injected with gentamicin, 40 mg/kg, im, twice a day for 9 days, 20 with gentamicin + pyrrolidine dithiocarbamate (PDTC, an NF-kappaB inhibitor), 14 with 0.15 M NaCl, im, twice a day for 9 days, and 14 with 0.15 M NaCl , im, twice a day for 9 days and PDTC, 50 mg kg-1 day-1, ip, twice a day for 15 days. The animals were killed 5 and 30 days after the last of the injections and the kidneys were removed for histological, immunohistochemical and Western blot analysis and for nitrate determination. The results of the immunohistochemical study were evaluated by counting the p-p38 MAPK-positive cells per area of renal cortex measuring 0.05 mm². Creatinine was measured by the Jaffé method in blood samples collected 5 and 30 days after the end of the treatments. Gentamicin-treated rats presented a transitory increase in plasma creatinine levels. In addition, animals killed 5 days after the end of gentamicin treatment presented acute tubular necrosis and increased nitrate levels in the renal cortex. Increased expression of p-p38 MAPK and NF-kappaB was also observed in the kidneys from these animals. The animals killed 30 days after gentamicin treatment showed residual areas of interstitial fibrosis in the renal cortex, although the expression of p-p38 MAPK in their kidneys did not differ from control. Treatment with PDTC reduced the functional and structural changes induced by gentamicin as well as the expression of p-p38 MAPK and NF-kappaB. The increased expression of p-p38 MAPK and NF-kappaB observed in these rats suggests that these signaling molecules may be involved in the pathogenesis of tubulointerstitial nephritis induced by gentamicin.

Involvement of glutamate and reactive oxygen species in methylmercury neurotoxicity

This review addresses the mechanisms of methylmercury (MeHg)-induced neurotoxicity, specifically examining the role of oxidative stress in mediating neuronal damage. A number of critical findings point to a central role for astrocytes in mediating MeHg-induced neurotoxicity as evidenced by the following observations: a) MeHg preferentially accumulates in astrocytes; b) MeHg specifically inhibits glutamate uptake in astrocytes; c) neuronal dysfunction is secondary to disturbances in astrocytes. The generation of reactive oxygen species (ROS) by MeHg has been observed in various experimental paradigms. For example, MeHg enhances ROS formation both in vivo (rodent cerebellum) and in vitro (isolated rat brain synaptosomes), as well as in neuronal and mixed reaggregating cell cultures. Antioxidants, including selenocompounds, can rescue astrocytes from MeHg-induced cytotoxicity by reducing ROS formation. We emphasize that oxidative stress plays a significant role in mediating MeHg-induced neurotoxic damage with active involvement of the mitochondria in this process. Furthermore, we provide a mechanistic overview on oxidative stress induced by MeHg that is triggered by a series of molecular events such as activation of various kinases, stress proteins and other immediate early genes culminating in cell damage.

Effect of exercise on the caloric intake of breast cancer patients undergoing treatment

The purpose of this study was to examine the effects of an exercise intervention on the total caloric intake (TCI) of breast cancer patients undergoing treatment. A secondary purpose was to determine whether or not a relationship existed between changes in TCI, body fat composition (%BF), and fatigue during the study, which lasted 6 months. Twenty females recently diagnosed with breast cancer, scheduled to undergo chemotherapy or radiation, were assigned randomly to an experimental (N = 10) or control group (N = 10). Outcome measures included TCI (3-day food diary), %BF (skinfolds), and fatigue (revised Piper Fatigue Scale). Each exercise session was conducted as follows: initial cardiovascular activity (6-12 min), followed by stretching (5-10 min), resistance training (15-30 min), and a cool-down (approximately 8 min). Significant changes in TCI were observed among groups (F1,18 = 8.582; P = 0.009), at treatments 2 and 3, and at the end of the study [experimental (1973 ± 419), control (1488 ± 418); experimental (1946 ± 437), control (1436 ± 429); experimental (2315 ± 455), control (1474 ± 294), respectively]. A significant negative correlation was found (Spearman rho(18) = -0.759; P < 0.001) between TCI and %BF and between TCI and fatigue levels (Spearman rho(18) = -0.541; P = 0.014) at the end of the study. In conclusion, the results of this study suggest that an exercise intervention administered to breast cancer patients undergoing medical treatment may assist in the mitigation of some treatment side effects, including decreased TCI, increased fatigue, and negative changes in body composition.

The role of stress-activated protein kinase signaling in renal pathophysiology

Two major stress-activated protein kinases are the p38 mitogen-activated protein kinase (MAPK) and the c-Jun amino terminal kinase (JNK). p38 and JNK are widely expressed in different cell types in various tissues and can be activated by a diverse range of stimuli. Signaling through p38 and JNK is critical for embryonic development. In adult kidney, p38 and JNK signaling is evident in a restricted pattern suggesting a normal physiological role. Marked activation of both p38 and JNK pathways occurs in human renal disease, including glomerulonephritis, diabetic nephropathy and acute renal failure. Administration of small molecule inhibitors of p38 and JNK has been shown to provide protection from renal injury in different types of experimental kidney disease through inhibition of renal inflammation, fibrosis, and apoptosis. In particular, a role for JNK signaling has been identified in macrophage activation resulting in up-regulation of pro-inflammatory mediators and the induction of renal injury. The ability to provide renal protection by blocking either p38 or JNK indicates a lack of redundancy for these two signaling pathways despite their activation by common stimuli. Therefore, the stress-activated protein kinases, p38 and JNK, are promising candidates for therapeutic intervention in human renal diseases.

Nuclear calcium signaling: a cell within a cell

Calcium (Ca2+) is a versatile second messenger that regulates a wide range of cellular functions. Although it is not established how a single second messenger coordinates diverse effects within a cell, there is increasing evidence that the spatial patterns of Ca2+ signals may determine their specificity. Ca2+ signaling patterns can vary in different regions of the cell and Ca2+ signals in nuclear and cytoplasmic compartments have been reported to occur independently. No general paradigm has been established yet to explain whether, how, or when Ca2+ signals are initiated within the nucleus or their function. Here we highlight that receptor tyrosine kinases rapidly translocate to the nucleus. Ca2+ signals that are induced by growth factors result from phosphatidylinositol 4,5-bisphosphate hydrolysis and inositol 1,4,5-trisphosphate formation within the nucleus rather than within the cytoplasm. This novel signaling mechanism may be responsible for growth factor effects on cell proliferation.

Growth inhibitory effect and Chk1-dependent signaling involved in G2/M arrest on human gastric cancer cells induced by diallyl disulfide

Diallyl disulfide (DADS) inhibits growth and induces cell cycle G2/M arrest in human gastric cancer MGC803 cells. In this study, 15 mg/L DADS exerted similar effects on growth and cell cycle arrest in human gastric cancer BGC823 cells. Due to the importance of cell cycle redistribution in DADS-mediated anti-carcinogenic effects, we investigated the role of checkpoint kinases (Chk1 and Chk2) during DADS-induced cell cycle arrest. We hypothesized that DADS could mediate G2/M phase arrest through either Chk1 or Chk2 signal transduction pathways. We demonstrated that DADS induced the accumulation of phosphorylated Chk1, but not of Chk2, and that DADS down-regulated Cdc25C and cyclin B1. The expression of mRNA and total protein for Chkl and Chk2 was unchanged. Chk1 is specifically phosphorylated by ATR (ATM-RAD3-related gene). Western blot analysis showed that phospho-ATR was activated by DADS. Taken together, these data suggest that cell cycle G2/M arrest, which was associated with accumulation of the phosphorylated forms of Chk1, but not of Chk2, was involved in the growth inhibition induced by DADS in the human gastric cancer cell line BGC823. Furthermore, the DADS-induced G2/M checkpoint response is mediated by Chk1 signaling through ATR/Chk1/Cdc25C/cyclin B1, and is independent of Chk2.

The Na+/glucose cotransporters: from genes to therapy

Glucose enters eukaryotic cells via two types of membrane-associated carrier proteins, the Na+/glucose cotransporters (SGLT) and the facilitative glucose transporters (GLUT). The SGLT family consists of six members. Among them, the SGLT1 and SGLT2 proteins, encoded by the solute carrier genes SLC5A1 and SLC5A2, respectively, are believed to be the most important ones and have been extensively explored in studies focusing on glucose fluxes under both physiological and pathological conditions. This review considers the regulation of the expression of the SGLT promoted by protein kinases and transcription factors, as well as the alterations determined by diets of different compositions and by pathologies such as diabetes. It also considers congenital defects of sugar metabolism caused by aberrant expression of the SGLT1 in glucose-galactose malabsorption and the SGLT2 in familial renal glycosuria. Finally, it covers some pharmacological compounds that are being currently studied focusing on the interest of controlling glycemia by antagonizing SGLT in renal and intestinal tissues.

Trans fatty acid intake is associated with insulin sensitivity but independently of inflammation

High saturated and trans fatty acid intake, the typical dietary pattern of Western populations, favors a proinflammatory status that contributes to generating insulin resistance (IR). We examined whether the consumption of these fatty acids was associated with IR and inflammatory markers. In this cross-sectional study, 127 non-diabetic individuals were allocated to a group without IR and 56 to another with IR, defined as homeostasis model assessment-IR (HOMA-IR) >2.71. Diet was assessed using 24-h food recalls. Multiple linear regression was employed to test independent associations with HOMA-IR. The IR group presented worse anthropometric, biochemical and inflammatory profiles. Energy intake was correlated with abdominal circumference and inversely with adiponectin concentrations (r = -0.227, P = 0.002), while saturated fat intake correlated with inflammatory markers and trans fat with HOMA-IR (r = 0.160, P = 0.030). Abdominal circumference was associated with HOMA-IR (r = 0.430, P < 0.001). In multiple analysis, HOMA-IR remained associated with trans fat intake (β = 1.416, P = 0.039) and body mass index (β = 0.390, P < 0.001), and was also inversely associated with adiponectin (β = -1.637, P = 0.004). Inclusion of other nutrients (saturated fat and added sugar) or other inflammatory markers (IL-6 and CRP) into the models did not modify these associations. Our study supports that trans fat intake impairs insulin sensitivity. The hypothesis that its effect could depend on transcription factors, resulting in expression of proinflammatory genes, was not corroborated. We speculate that trans fat interferes predominantly with insulin signaling via intracellular kinases, which alter insulin receptor substrates.

Involvement of Src tyrosine kinase and protein kinase C in the expression of macrophage migration inhibitory factor induced by H2O2 in HL-1 mouse cardiac muscle cells

Macrophage migration inhibitory factor (MIF), a pleiotropic cytokine, plays an important role in the pathogenesis of atrial fibrillation; however, the upstream regulation of MIF in atrial myocytes remains unclear. In the present study, we investigated whether and how MIF is regulated in response to the renin-angiotensin system and oxidative stress in atrium myocytes (HL-1 cells). MIF protein and mRNA levels in HL-1 cells were assayed using immunofluorescence, real-time PCR, and Western blot. The result indicated that MIF was expressed in the cytoplasm of HL-1 cells. Hydrogen peroxide (H2O2), but not angiotensin II, stimulated MIF expression in HL-1 cells. H2O2-induced MIF protein and gene levels increased in a dose-dependent manner and were completely abolished in the presence of catalase. H2O2-induced MIF production was completely inhibited by tyrosine kinase inhibitors genistein and PP1, as well as by protein kinase C (PKC) inhibitor GF109203X, suggesting that redox-sensitive MIF production is mediated through tyrosine kinase and PKC-dependent mechanisms in HL-1 cells. These results suggest that MIF is upregulated by HL-1 cells in response to redox stress, probably by the activation of Src and PKC.

ERKs and mitochondria-related pathways are essential for glycyrrhizic acid-mediated neuroprotection against glutamate-induced toxicity in differentiated PC12 cells

The present study focuses on the neuroprotective effect of glycyrrhizic acid (GA, a major compound separated from Glycyrrhiza Radix, which is a crude Chinese traditional drug) against glutamate-induced cytotoxicity in differentiated PC12 (DPC12) cells. The results showed that GA treatment improved cell viability and ameliorated abnormal glutamate-induced alterations in mitochondria in DPC12 cells. GA reversed glutamate-suppressed B-cell lymphoma 2 levels, inhibited glutamate-enhanced expressions of Bax and cleaved caspase 3, and reduced cytochrome C (Cyto C) release. Exposure to glutamate strongly inhibited phosphorylation of AKT (protein kinase B) and extracellular signal-regulated kinases (ERKs); however, GA pretreatment enhanced activation of ERKs but not AKT. The presence of PD98059 (a mitogen-activated protein/extracellular signal-regulated kinase kinase [MEK] inhibitor) but not LY294002 (a phosphoinositide 3-kinase [PI3K] inhibitor) diminished the potency of GA for improving viability of glutamate-exposed DPC12 cells. These results indicated that ERKs and mitochondria-related pathways are essential for the neuroprotective effect of GA against glutamate-induced toxicity in DPC12 cells. The present study provides experimental evidence supporting GA as a potential therapeutic agent for use in the treatment of neurodegenerative diseases.

Vascular O-GlcNAcylation augments reactivity to constrictor stimuli by prolonging phosphorylated levels of the myosin light chain

O-GlcNAcylation is a modification that alters the function of numerous proteins. We hypothesized that augmented O-GlcNAcylation levels enhance myosin light chain kinase (MLCK) and reduce myosin light chain phosphatase (MLCP) activity, leading to increased vascular contractile responsiveness. The vascular responses were measured by isometric force displacement. Thoracic aorta and vascular smooth muscle cells (VSMCs) from rats were incubated with vehicle or with PugNAc, which increases O-GlcNAcylation. In addition, we determined whether proteins that play an important role in the regulation of MLCK and MLCP activity are directly affected by O-GlcNAcylation. PugNAc enhanced phenylephrine (PE) responses in rat aortas (maximal effect, 14.2±2 vs 7.9±1 mN for vehicle, n=7). Treatment with an MLCP inhibitor (calyculin A) augmented vascular responses to PE (13.4±2 mN) and abolished the differences in PE-response between the groups. The effect of PugNAc was not observed when vessels were preincubated with ML-9, an MLCK inhibitor (7.3±2 vs 7.5±2 mN for vehicle, n=5). Furthermore, our data showed that differences in the PE-induced contractile response between the groups were abolished by the activator of AMP-activated protein kinase (AICAR; 6.1±2 vs 7.4±2 mN for vehicle, n=5). PugNAc increased phosphorylation of myosin phosphatase target subunit 1 (MYPT-1) and protein kinase C-potentiated inhibitor protein of 17 kDa (CPI-17), which are involved in RhoA/Rho-kinase-mediated inhibition of myosin phosphatase activity. PugNAc incubation produced a time-dependent increase in vascular phosphorylation of myosin light chain and decreased phosphorylation levels of AMP-activated protein kinase, which decreased the affinity of MLCK for Ca2+/calmodulin. Our data suggest that proteins that play an important role in the regulation of MLCK and MLCP activity are directly affected by O-GlcNAcylation, favoring vascular contraction.

Epidermal growth factor receptor (EGFR) mutations in lung cancer: preclinical and clinical data

Lung cancer leads cancer-related mortality worldwide. Non-small-cell lung cancer (NSCLC), the most prevalent subtype of this recalcitrant cancer, is usually diagnosed at advanced stages, and available systemic therapies are mostly palliative. The probing of the NSCLC kinome has identified numerous nonoverlapping driver genomic events, including epidermal growth factor receptor (EGFR) gene mutations. This review provides a synopsis of preclinical and clinical data on EGFR mutated NSCLC and EGFR tyrosine kinase inhibitors (TKIs). Classic somatic EGFR kinase domain mutations (such as L858R and exon 19 deletions) make tumors addicted to their signaling cascades and generate a therapeutic window for the use of ATP-mimetic EGFR TKIs. The latter inhibit these kinases and their downstream effectors, and induce apoptosis in preclinical models. The aforementioned EGFR mutations are stout predictors of response and augmentation of progression-free survival when gefitinib, erlotinib, and afatinib are used for patients with advanced NSCLC. The benefits associated with these EGFR TKIs are limited by the mechanisms of tumor resistance, such as the gatekeeper EGFR-T790M mutation, and bypass activation of signaling cascades. Ongoing preclinical efforts for treating resistance have started to translate into patient care (including clinical trials of the covalent EGFR-T790M TKIs AZD9291 and CO-1686) and hold promise to further boost the median survival of patients with EGFR mutated NSCLC.