“cAMP Sponge”: A Buffer for Cyclic Adenosine 3′, 5′-Monophosphate

Background

While intracellular buffers are widely used to study calcium signaling, no such tool exists for the other major second messenger, cyclic AMP (cAMP).

Methods/Principal Findings

Here we describe a genetically encoded buffer for cAMP based on the high-affinity cAMP-binding carboxy-terminus of the regulatory subunit RIβ of protein kinase A (PKA). Addition of targeting sequences permitted localization of this fragment to the extra-nuclear compartment, while tagging with mCherry allowed quantification of its expression at the single cell level. This construct (named “cAMP sponge”) was shown to selectively bind cAMP in vitro. Its expression significantly suppressed agonist-induced cAMP signals and the downstream activation of PKA within the cytosol as measured by FRET-based sensors in single living cells. Point mutations in the cAMP-binding domains of the construct rendered the chimera unable to bind cAMP in vitro or in situ. Cyclic AMP sponge was fruitfully applied to examine feedback regulation of gap junction-mediated transfer of cAMP in epithelial cell couplets.

Conclusions

This newest member of the cAMP toolbox has the potential to reveal unique biological functions of cAMP, including insight into the functional significance of compartmentalized signaling events.     

CSF concentrations of cAMP and cGMP are lower in patients with Creutzfeldt-Jakob disease but not Parkinson's disease and amyotrophic lateral sclerosis

Background

The cyclic nucleotides cyclic adenosine-3′,5′-monophosphate (cAMP) and cyclic guanosine-3′,5′-monophosphate (cGMP) are important second messengers and are potential biomarkers for Parkinson''s disease (PD), amyotrophic lateral sclerosis (ALS) and Creutzfeldt-Jakob disease (CJD).

Methodology/Principal Findings

Here, we investigated by liquid chromatography/tandem mass spectrometry (LC-MS/MS) the cerebrospinal fluid (CSF) concentrations of cAMP and cGMP of 82 patients and evaluated their diagnostic potency as biomarkers. For comparison with a well-accepted biomarker, we measured tau concentrations in CSF of CJD and control patients. CJD patients (n = 15) had lower cAMP (−70%) and cGMP (−55%) concentrations in CSF compared with controls (n = 11). There was no difference in PD, PD dementia (PDD) and ALS cases. Receiver operating characteristic (ROC) curve analyses confirmed cAMP and cGMP as valuable diagnostic markers for CJD indicated by the area under the curve (AUC) of 0.86 (cAMP) and 0.85 (cGMP). We calculated a sensitivity of 100% and specificity of 64% for cAMP and a sensitivity of 67% and specificity of 100% for cGMP. The combination of both nucleotides increased the sensitivity to 80% and specificity to 91% for the term cAMPxcGMP (AUC 0.92) and to 93% and 100% for the ratio tau/cAMP (AUC 0.99).

Conclusions/Significance

We conclude that the CSF determination of cAMP and cGMP may easily be included in the diagnosis of CJD and could be helpful in monitoring disease progression as well as in therapy control.     

Extracellular and Intracellular Mechanisms of Mechanotransduction in Three-Dimensionally Embedded Rat Chondrocytes

Purpose

Articular cartilage homeostasis involves modulation of chondrocyte matrix synthesis in response to mechanical stress (MS). We studied extracellular and intracellular mechanotransduction pathways mediating this response.

Methods

We first confirmed rapid up-regulation of the putative chondro-protective cytokine, interleukin (IL)-4, as an immediate response to MS. We then studied the role of IL-4 by investigating responses to exogenous IL-4 or a specific IL-4 inhibitor, combined with MS. Next we investigated the intracellular second messengers. Since chondrocyte phenotype alters according to the extracellular environment, we characterized the response to mechanotransduction in 3-dimensionally embedded chondrocytes.

Results

Expression of aggrecan and type II collagen was significantly up-regulated by exogenous IL-4 whereas MS-induced matrix synthesis was inhibited by an IL-4 blocker. Further, MS-induced matrix synthesis was completely blocked by a p38 MAPK inhibitor, while it was only partially blocked by inhibitors of other putative second messengers.

Conclusion

IL-4 mediates an extracellular pathway of mechanotransduction, perhaps via an autocrine/paracrine loop, while p38 mediates an intracellular pathway prevalent only in a 3-dimensional environment.     

Somatic 'soluble' adenylyl cyclase isoforms are unaffected in Sacy tm1Lex/Sacy tm1Lex 'knockout' mice

Background

Mammalian Soluble adenylyl cyclase (sAC, Adcy10, or Sacy) represents a source of the second messenger cAMP distinct from the widely studied, G protein-regulated transmembrane adenylyl cyclases. Genetic deletion of the second through fourth coding exons in Sacy^tm1Lex/Sacy^tm1Lex knockout mice results in a male sterile phenotype. The absence of any major somatic phenotype is inconsistent with the variety of somatic functions identified for sAC using pharmacological inhibitors and RNA interference.

Principal Findings

We now use immunological and molecular biological methods to demonstrate that somatic tissues express a previously unknown isoform of sAC, which utilizes a unique start site, and which ‘escapes’ the design of the Sacy^tm1Lex knockout allele.

Conclusions/Significance

These studies reveal increased complexity at the sAC locus, and they suggest that the known isoforms of sAC play a unique function in male germ cells.     

A multivariate analysis of serum nutrient levels and lung function

Background

There is mounting evidence that estimates of intakes of a range of dietary nutrients are related to both lung function level and rate of decline, but far less evidence on the relation between lung function and objective measures of serum levels of individual nutrients. The aim of this study was to conduct a comprehensive examination of the independent associations of a wide range of serum markers of nutritional status with lung function, measured as the one-second forced expiratory volume (FEV₁).

Methods

Using data from the Third National Health and Nutrition Examination Survey, a US population-based cross-sectional study, we investigated the relation between 21 serum markers of potentially relevant nutrients and FEV₁, with adjustment for potential confounding factors. Systematic approaches were used to guide the analysis.

Results

In a mutually adjusted model, higher serum levels of antioxidant vitamins (vitamin A, beta-cryptoxanthin, vitamin C, vitamin E), selenium, normalized calcium, chloride, and iron were independently associated with higher levels of FEV₁. Higher concentrations of potassium and sodium were associated with lower FEV₁.

Conclusion

Maintaining higher serum concentrations of dietary antioxidant vitamins and selenium is potentially beneficial to lung health. In addition other novel associations found in this study merit further investigation.     

The Clinical Correlation of Regulatory T Cells and Cyclic Adenosine Monophosphate in Enterovirus 71 Infection

Background

Brainstem encephalitis (BE) and pulmonary edema (PE) are notable complications of enterovirus 71 (EV71) infection.

Objective

This study investigated the immunoregulatory characterizations of EV71 neurological complications by disease severity and milrinone treatment.

Study Design

Patients <18 years with virologically confirmed EV71 infections were enrolled and divided into 2 groups: the hand, foot, and mouth disease (HFMD) or BE group, and the autonomic nervous system (ANS) dysregulation or PE group. Cytokine and cyclic adenosine monophosphate (cAMP) levels, and the regulatory T cell (Tregs) profiles of the patients were determined.

Results

Patients with ANS dysregulation or PE exhibited significantly low frequency of CD4⁺CD25⁺Foxp3⁺ and CD4⁺Foxp3⁺ T cells compared with patients with HFMD or BE. The expression frequency of CD4⁻CD8⁻ was also significantly decreased in patients with ANS dysregulation or PE. Among patients with ANS dysregulation or PE, the expression frequency of CD4⁺Foxp3⁺ increased markedly after milrinone treatment, and was associated with reduction of plasma levels IL-6, IL-8 and IL-10. Plasma concentrations of cAMP were significantly decreased in patients with ANS dysregulation or PE compared with patients with HFMD or BE; however, cAMP levels increased after milrinone treatment.

Conclusions

These findings suggested decreased different regulatory T populations and cAMP expression correlate with increased EV71 disease severity. Improved outcome after milrinone treatment may associate with increased regulatory T populations, cAMP expression and modulation of cytokines levels.     

Hippocampal deletion of BDNF gene attenuates gamma oscillations in area CA1 by up-regulating 5-HT3 receptor

Background

Pyramidal neurons in the hippocampal area CA3 express high levels of BDNF, but how this BDNF contributes to oscillatory properties of hippocampus is unknown.

Methodology/Principal Findings

Here we examined carbachol-induced gamma oscillations in hippocampal slices lacking BDNF gene in the area CA3. The power of oscillations was reduced in the hippocampal area CA1, which coincided with increases in the expression and activity of 5-HT3 receptor. Pharmacological block of this receptor partially restored power of gamma oscillations in slices from KO mice, but had no effect in slices from WT mice.

Conclusion/Significance

These data suggest that BDNF facilitates gamma oscillations in the hippocampus by attenuating signaling through 5-HT3 receptor. Thus, BDNF modulates hippocampal oscillations through serotonergic system.     

Impact of Acetazolamide and CPAP on Cortical Activity in Obstructive Sleep Apnea Patients

Study Objectives

1) To investigate the impact of acetazolamide, a drug commonly prescribed for altitude sickness, on cortical oscillations in patients with obstructive sleep apnea syndrome (OSAS). 2) To examine alterations in the sleep EEG after short-term discontinuation of continuous positive airway pressure (CPAP) therapy.

Design

Data from two double-blind, placebo-controlled randomized cross-over design studies were analyzed.

Setting

Polysomnographic recordings in sleep laboratory at 490 m and at moderate altitudes in the Swiss Alps: 1630 or 1860 m and 2590 m.

Patients

Study 1: 39 OSAS patients. Study 2: 41 OSAS patients.

Interventions

Study 1: OSAS patients withdrawn from treatment with CPAP. Study 2: OSAS patients treated with autoCPAP. Treatment with acetazolamide (500–750 mg) or placebo at moderate altitudes.

Measurements and Results

An evening dose of 500 mg acetazolamide reduced slow-wave activity (SWA; approximately 10%) and increased spindle activity (approximately 10%) during non-REM sleep. In addition, alpha activity during wake after lights out was increased. An evening dose of 250 mg did not affect these cortical oscillations. Discontinuation of CPAP therapy revealed a reduction in SWA (5–10%) and increase in beta activity (approximately 25%).

Conclusions

The higher evening dose of 500 mg acetazolamide showed the “spectral fingerprint” of Benzodiazepines, while 250 mg acetazolamide had no impact on cortical oscillations. However, both doses had beneficial effects on oxygen saturation and sleep quality.     

Remodeling lipid metabolism and improving insulin responsiveness in human primary myotubes

Objective

Disturbances in lipid metabolism are strongly associated with insulin resistance and type 2 diabetes (T2D). We hypothesized that activation of cAMP/PKA and calcium signaling pathways in cultured human myotubes would provide further insight into regulation of lipid storage, lipolysis, lipid oxidation and insulin responsiveness.

Methods

Human myoblasts were isolated from vastus lateralis, purified, cultured and differentiated into myotubes. All cells were incubated with palmitate during differentiation. Treatment cells were pulsed 1 hour each day with forskolin and ionomycin (PFI) during the final 3 days of differentiation to activate the cAMP/PKA and calcium signaling pathways. Control cells were not pulsed (control). Mitochondrial content, ¹⁴C lipid oxidation and storage were measured, as well as lipolysis and insulin-stimulated glycogen storage. Myotubes were stained for lipids and gene expression measured.

Results

PFI increased oxidation of oleate and palmitate to CO₂ (p<0.001), isoproterenol-stimulated lipolysis (p = 0.01), triacylglycerol (TAG) storage (p<0.05) and mitochondrial DNA copy number (p = 0.01) and related enzyme activities. Candidate gene and microarray analysis revealed increased expression of genes involved in lipolysis, TAG synthesis and mitochondrial biogenesis. PFI increased the organization of lipid droplets along the myofibrillar apparatus. These changes in lipid metabolism were associated with an increase in insulin-mediated glycogen storage (p<0.001).

Conclusions

Activation of cAMP/PKA and calcium signaling pathways in myotubes induces a remodeling of lipid droplets and functional changes in lipid metabolism. These results provide a novel pharmacological approach to promote lipid metabolism and improve insulin responsiveness in myotubes, which may be of therapeutic importance for obesity and type 2 diabetes.     

Phosphodiesterase 7 inhibition preserves dopaminergic neurons in cellular and rodent models of Parkinson disease

Background

Phosphodiesterase 7 plays a major role in down-regulation of protein kinase A activity by hydrolyzing cAMP in many cell types. This cyclic nucleotide plays a key role in signal transduction in a wide variety of cellular responses. In the brain, cAMP has been implicated in learning, memory processes and other brain functions.

Methodology/Principal Findings

Here we show a novel function of phosphodiesterase 7 inhibition on nigrostriatal dopaminergic neuronal death. We found that S14, a heterocyclic small molecule inhibitor of phosphodiesterase 7, conferred significant neuronal protection against different insults both in the human dopaminergic cell line SH-SY5Y and in primary rat mesencephalic cultures. S14 treatment also reduced microglial activation, protected dopaminergic neurons and improved motor function in the lipopolysaccharide rat model of Parkinson disease. Finally, S14 neuroprotective effects were reversed by blocking the cAMP signaling pathways that operate through cAMP-dependent protein kinase A.

Conclusions/Significance

Our findings demonstrate that phosphodiesterase 7 inhibition can protect dopaminergic neurons against different insults, and they provide support for the therapeutic potential of phosphodiesterase 7 inhibitors in the treatment of neurodegenerative disorders, particularly Parkinson disease.     

Root cortical aerenchyma inhibits radial nutrient transport in maize (Zea mays)

Background and Aims

Formation of root cortical aerenchyma (RCA) can be induced by nutrient deficiency. In species adapted to aerobic soil conditions, this response is adaptive by reducing root maintenance requirements, thereby permitting greater soil exploration. One trade-off of RCA formation may be reduced radial transport of nutrients due to reduction in living cortical tissue. To test this hypothesis, radial nutrient transport in intact roots of maize (Zea mays) was investigated in two radiolabelling experiments employing genotypes with contrasting RCA.

Methods

In the first experiment, time-course dynamics of phosphate loading into the xylem were measured from excised nodal roots that varied in RCA formation. In the second experiment, uptake of phosphate, calcium and sulphate was measured in seminal roots of intact young plants in which variation in RCA was induced by treatments altering ethylene action or genetic differences.

Key Results

In each of three paired genotype comparisons, the rate of phosphate exudation of high-RCA genotypes was significantly less than that of low-RCA genotypes. In the second experiment, radial nutrient transport of phosphate and calcium was negatively correlated with the extent of RCA for some genotypes.

Conclusions

The results support the hypothesis that RCA can reduce radial transport of some nutrients in some genotypes, which could be an important trade-off of this trait.     

Life or death: prognostic value of a resting EEG with regards to survival in patients in vegetative and minimally conscious States

Objective

To investigate the potentially prognostic value of a resting state electroencephalogram (EEG) with regards to the clinical outcome from vegetative and minimally conscious states (VS and MCS) in terms of survival six months after a brain injury.

Methods

We quantified a dynamic repertoire of EEG oscillations in resting condition with eyes closed in patients in VS and MCS. The exact composition of EEG oscillations was assessed by analysing the probability-classification of short-term EEG spectral patterns.

Results

Results demonstrated that (a) the diversity and the variability of EEG for Non-Survivors were significantly lower than for Survivors; and (b) a higher probability of mostly delta and slow-theta oscillations occurring either alone or in combination were found during the first assessment for patients with a bad outcome (i.e., those who died) within six months of an injury compared to patients who survived. At the same time, patients with a good outcome (i.e., those who survived) after six months post-injury had a higher probability of mostly fast-theta and alpha oscillations occurring either alone or in combination during the first assessment when compared to patients who died within six months of an injury.

Conclusions

Resting state EEGs properly analysed may have a potentially prognostic value with regards to the outcome from VS or MCS in terms of survival six months after a brain injury.

Significance

This work may have implications for clinical care, rehabilitative programmes and medical–legal decisions for patients with impaired consciousness states after being in a coma due to acute brain injuries.     

Prelimbic and infralimbic prefrontal cortex interact during fast network oscillations

Background

The medial prefrontal cortex has been implicated in a variety of cognitive and executive processes such as decision making and working memory. The medial prefrontal cortex of rodents consists of several areas including the prelimbic and infralimbic cortex that are thought to be involved in different aspects of cognitive performance. Despite the distinct roles in cognitive behavior that have been attributed to prelimbic and infralimbic cortex, little is known about neuronal network functioning of these areas, and whether these networks show any interaction during fast network oscillations.

Methodology/Principal Findings

Here we show that fast network oscillations in rat infralimbic cortex slices occur at higher frequencies and with higher power than oscillations in prelimbic cortex. The difference in oscillation frequency disappeared when prelimbic and infralimbic cortex were disconnected.

Conclusions/Significance

Our data indicate that neuronal networks of prelimbic and infralimbic cortex can sustain fast network oscillations independent of each other, but suggest that neuronal networks of prelimbic and infralimbic cortex are interacting during these oscillations.     

Effects of surface viscoelasticity on cellular responses of endothelial cells

Background:

One area of nanoscience deals with nanoscopic interactions between nanostructured materials and biological systems. To elucidate the effects of the substrate surface morphology and viscoelasticity on cell proliferation, fractal analysis was performed on endothelial cells cultured on nanocomposite samples based on silicone rubber (SR) and various concentrations of organomodified nanoclay (OC).

Methods:

The nanoclay/SR ratio was tailored to enhance cell behavior via changes in sample substrate surface roughness and viscoelasticity.

Results:

Surface roughness of the cured SR filled with negatively-charged nanosilicate layers had a greater effect than elasticity on cell growth. The surface roughness of SR nanocomposite samples increased with increasing the OC content, leading to enhanced cell growth and extracellular matrix (ECM) remodeling. This was consistent with the decrease in SR segmental motions and damping factor as the primary viscoelastic parameters by the nanosilicate layers with increasing clay concentrations.

Conclusions:

The inclusion of clay nanolayers affected the growth and behavior of endothelial cells on microtextured SR.Key Words: Silicone rubber, Nanoclay, Elastic Modulus, Roughness, Cell proliferation     

Dependency of Calcium Alternans on Ryanodine Receptor Refractoriness

Background

Rapid pacing rates induce alternations in the cytosolic calcium concentration caused by fluctuations in calcium released from the sarcoplasmic reticulum (SR). However, the relationship between calcium alternans and refractoriness of the SR calcium release channel (RyR2) remains elusive.

Methodology/Principal Findings

To investigate how ryanodine receptor (RyR2) refractoriness modulates calcium handling on a beat-to-beat basis using a numerical rabbit cardiomyocyte model. We used a mathematical rabbit cardiomyocyte model to study the beat-to-beat calcium response as a function of RyR2 activation and inactivation. Bi-dimensional maps were constructed depicting the beat-to-beat response. When alternans was observed, a novel numerical clamping protocol was used to determine whether alternans was caused by oscillations in SR calcium loading or by RyR2 refractoriness. Using this protocol, we identified regions of RyR2 gating parameters where SR calcium loading or RyR2 refractoriness underlie the induction of calcium alternans, and we found that at the onset of alternans both mechanisms contribute. At low inactivation rates of the RyR2, calcium alternans was caused by alternation in SR calcium loading, while at low activation rates it was caused by alternation in the level of available RyR2s.

Conclusions/Significance

We have mapped cardiomyocyte beat-to-beat responses as a function of RyR2 activation and inactivation, identifying domains where SR calcium load or RyR2 refractoriness underlie the induction of calcium alternans. A corollary of this work is that RyR2 refractoriness due to slow recovery from inactivation can be the cause of calcium alternans even when alternation in SR calcium load is present.     

β2-Agonist induced cAMP is decreased in asthmatic airway smooth muscle due to increased PDE4D

Background and Objective

Asthma is associated with airway narrowing in response to bronchoconstricting stimuli and increased airway smooth muscle (ASM) mass. In addition, some studies have suggested impaired β-agonist induced ASM relaxation in asthmatics, but the mechanism is not known.

Objective

To characterize the potential defect in β-agonist induced cAMP in ASM derived from asthmatic in comparison to non-asthmatic subjects and to investigate its mechanism.

Methods

We examined β₂-adrenergic (β₂AR) receptor expression and basal β-agonist and forskolin (direct activator of adenylyl cyclase) stimulated cAMP production in asthmatic cultured ASM (n = 15) and non-asthmatic ASM (n = 22). Based on these results, PDE activity, PDE4D expression and cell proliferation were determined.

Results

In the presence of IBMX, a pan PDE inhibitor, asthmatic ASM had ∼50% lower cAMP production in response to isoproterenol, albuterol, formoterol, and forskolin compared to non-asthmatic ASM. However when PDE4 was specifically inhibited, cAMP production by the agonists and forskolin was normalized in asthmatic ASM. We then measured the amount and activity of PDE4, and found ∼2-fold greater expression and activity in asthmatic ASM compared to non-asthmatic ASM. Furthermore, inhibition of PDE4 reduced asthmatic ASM proliferation but not that of non-asthmatic ASM.

Conclusion

Decreased β-agonist induced cAMP in ASM from asthmatics results from enhanced degradation due to increased PDE4D expression. Clinical manifestations of this dysregulation would be suboptimal β-agonist-mediated bronchodilation and possibly reduced control over increasing ASM mass. These phenotypes appear to be “hard-wired” into ASM from asthmatics, as they do not require an inflammatory environment in culture to be observed.     

Calcium Supplementation Increases Blood Creatinine Concentration in a Randomized Controlled Trial

Background

Calcium supplements are widely used among older adults for osteoporosis prevention and treatment. However, their effect on creatinine levels and kidney function has not been well studied.

Methods

We investigated the effect of calcium supplementation on blood creatinine concentration in a randomized controlled trial of colorectal adenoma chemoprevention conducted between 2004–2013 at 11 clinical centers in the United States. Healthy participants (N = 1,675) aged 45–75 with a history of colorectal adenoma were assigned to daily supplementation with calcium (1200 mg, as carbonate), vitamin D₃ (1000 IU), both, or placebo for three or five years. Changes in blood creatinine and total calcium concentration were measured after one year of treatment and multiple linear regression was used to estimate effects on creatinine concentrations.

Results

After one year of treatment, blood creatinine was 0.013±0.006 mg/dL higher on average among participants randomized to calcium compared to placebo after adjustment for other determinants of creatinine (P = 0.03). However, the effect of calcium treatment appeared to be larger among participants who consumed the most alcohol (2–6 drinks/day) or whose estimated glomerular filtration rate (eGFR) was less than 60 ml/min/1.73 m² at baseline. The effect of calcium treatment on creatinine was only partially mediated by a concomitant increase in blood total calcium concentration and was independent of randomized vitamin D treatment. There did not appear to be further increases in creatinine after the first year of calcium treatment.

Conclusions

Among healthy adults participating in a randomized clinical trial, daily supplementation with 1200 mg of elemental calcium caused a small increase in blood creatinine. If confirmed, this finding may have implications for clinical and public health recommendations for calcium supplementation.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00153816","term_id":"NCT00153816"}}NCT00153816     

Six types of multistability in a neuronal model based on slow calcium current

Background

Multistability of oscillatory and silent regimes is a ubiquitous phenomenon exhibited by excitable systems such as neurons and cardiac cells. Multistability can play functional roles in short-term memory and maintaining posture. It seems to pose an evolutionary advantage for neurons which are part of multifunctional Central Pattern Generators to possess multistability. The mechanisms supporting multistability of bursting regimes are not well understood or classified.

Methodology/Principal Findings

Our study is focused on determining the bio-physical mechanisms underlying different types of co-existence of the oscillatory and silent regimes observed in a neuronal model. We develop a low-dimensional model typifying the dynamics of a single leech heart interneuron. We carry out a bifurcation analysis of the model and show that it possesses six different types of multistability of dynamical regimes. These types are the co-existence of 1) bursting and silence, 2) tonic spiking and silence, 3) tonic spiking and subthreshold oscillations, 4) bursting and subthreshold oscillations, 5) bursting, subthreshold oscillations and silence, and 6) bursting and tonic spiking. These first five types of multistability occur due to the presence of a separating regime that is either a saddle periodic orbit or a saddle equilibrium. We found that the parameter range wherein multistability is observed is limited by the parameter values at which the separating regimes emerge and terminate.

Conclusions

We developed a neuronal model which exhibits a rich variety of different types of multistability. We described a novel mechanism supporting the bistability of bursting and silence. This neuronal model provides a unique opportunity to study the dynamics of networks with neurons possessing different types of multistability.     

Epigallocatechin-3-gallate (EGCG) inhibits the migratory behavior of tumor bronchial epithelial cells

Background

Many studies associated the main polyphenolic constituent of green tea, (-)-Epigallocatechin-3-gallate (EGCG), with inhibition of cancers, invasion and metastasis. To date, most of the studies have focused on the effect of EGCG on cell proliferation or death. Since cell migration is an important mechanism involved in tumor invasion, the aim of the present work was to target another approach of the therapeutic effect of EGCG, by investigating its effect on the cell migratory behavior.

Methods

The effect of EGCG (at concentrations lower than 10 μg/ml) on the migration speed of invasive cells was assessed by using 2D and 3D models of cell culture. We also studied the effects of EGCG on proteinases expression by RT-PCR analysis. By immunocytochemistry, we analyzed alterations of vimentin organization in presence of different concentrations of EGCG.

Results

We observed that EGCG had an inhibitory effect of cell migration in 2D and 3D cell culture models. EGCG also inhibited MMP-2 mRNA and protein expression and altered the intermediate filaments of vimentin.

Conclusion

Taken together, our results demonstrate that EGCG is able to inhibit the migration of bronchial tumor cells and could therefore be an attractive candidate to treat tumor invasion and cell migration.