The morphological response of Emiliania huxleyi to seawater carbonate chemistry changes: an inter-strain comparison

Four strains of the coccolithophore Emiliania huxleyi (RCC1212, RCC1216, RCC1238, RCC1256) were grown in dilute batch culture at four CO2 levels ranging from ~200 µatm to ~1200 µatm. Coccolith morphology was analyzed based on scanning electron micrographs. Three of the four strains did not exhibit a change in morphology over the CO2 range tested. One strain (RCC1256) displayed an increase in the percentage of malformed coccoliths with increasing CO2 concentration. We conclude that the sensitivity of the coccolith-shaping machinery to carbonate chemistry changes is strain-specific. Although it has been shown before that carbonate chemistry related changes in growth- and calcification rate are strain-specific, there seems to be no consistent correlation between coccolith morphology and growth or calcification rate. We did not observe an increase in the percentage of incomplete coccoliths in RCC1256, indicating that the coccolith-shaping machinery per se is affected by acidification and not the signalling pathway that produces the stop-signal for coccolith growth.

Characterising granuloma regression and liver recovery in a murine model of schistosomiasis japonica

For hepatic schistosomiasis the egg-induced granulomatous response and the development of extensive fibrosis are the main pathologies. We used a Schistosoma japonicum-infected mouse model to characterise the multi-cellular pathways associated with the recovery from hepatic fibrosis following clearance of the infection with the anti-schistosomal drug, praziquantel. In the recovering liver splenomegaly, granuloma density and liver fibrosis were all reduced. Inflammatory cell infiltration into the liver was evident, and the numbers of neutrophils, eosinophils and macrophages were significantly decreased. Transcriptomic analysis revealed the up-regulation of fatty acid metabolism genes and the identification of Peroxisome proliferator activated receptor alpha as the upstream regulator of liver recovery. The aryl hydrocarbon receptor signalling pathway which regulates xenobiotic metabolism was also differentially up-regulated. These findings provide a better understanding of the mechanisms associated with the regression of hepatic schistosomiasis.

Rôle du facteur de transcription PITX1 dans les pathogenèses de l'ostéoporose et des maladies parodontales

L’ostéoporose est une maladie caractérisée par une faible masse osseuse et une détérioration du tissu osseux. Cette condition entraîne une plus grande fragilité osseuse et des risques de fractures. Plusieurs études ont associé l’ostéoporose à la faible densité osseuse des mandibules, à la perte d’attache parodontale, à l’augmentation de la hauteur de la crête alvéolaire et à la chute des dents. Cette étude vise à comprendre les mécanismes sous-jacents cette perte osseuse. En effet, au cours du développement des souris, PITX1 joue un rôle clé dans l'identité des membres postérieurs et dans le bon développement des mandibules et des dents. Son inactivation complète chez la souris mène à un phénotype squelettique sévère. Tandis que, son inactivation partielle provoque des symptômes apparentés à l'arthrose avec une augmentation de la masse osseuse au niveau de l’os cortical et de l’os trabéculaire. Inversement, une étude antérieure chez des jumelles monozygotiques discordantes pour l’ostéoporose, montrent une augmentation d’environ 8.6 fois du niveau d’expression du gène Pitx1 chez la jumelle ostéoporotique. Collectivement, ces données nous ont poussés à investiguer sur le rôle du facteur de transcription PITX1 dans le métabolisme osseux normal et pathologique. Dans ce contexte, des souris transgéniques Col1α1-Pitx1 sur-exprimant Pitx1 spécifiquement dans le tissu osseux sous le promoteur du collagène de type-I (fragment 2.1kpb) ont été générées et phénotypiquement caractérisées. Ces résultats ont révelé que les souris transgéniques Col1α1-Pitx1 présentaient un phénotype similaire à celui des patients ostéoporotiques accompagné d'une perte de dents et des problèmes dentaires et parodontaux. De plus, cette étude a révélé que la surexpression de Pitx1 induit une altération de l’homéostasie osseuse via l’inactivation de la voie de signalisation Wnt/β-caténine canonique. Cette hypothèse a été appuyée par le fait que le traitement des souris transgéniques Col1α1-Pitx1 avec du chlorure de lithium, un activateur de la voie Wnt canonique, prévient le phénotype ostéoporotique chez ces souris. Finalement, cette étude établit un rôle crucial de PITX1 dans la régulation de la masse osseuse et une implication possible dans l’ostéoporose et les maladies parodontales via l’inactivation de la voie de signalisation Wnt/β-caténine canonique.

Rôle des cellules myéloïdes dans des modèles murins de la neuroinflammation

L’inflammation du système nerveux central (SNC), appelée neuroinflammation, est un aspect inséparable des maladies neurodégénératives chroniques comme la sclérose en plaques (SEP) et la maladie d’Alzheimer (MA). La caractérisation de la signature moléculaire spécifique à chaque population cellulaire dans des pathologies distinctes va aboutir à la compréhension et donc au contrôle de la neuroinflammation. Le présent ouvrage a pour but de mieux comprendre les mécanismes d’action de deux types cellulaires myéloïdes, la microglie et les neutrophiles, au cours des affections neuroinflammatoires du SNC. Ainsi, le premier objectif a été de comprendre le rôle des cytokines IL-36 dans la neuroinflammation établie au cours de l’encéphalomyélite auto-immune expérimentale (EAE). Dans une seconde partie, l’objectif a été d’explorer l’action du GPR84, un récepteur couplé à la protéine G spécifique à la microglie dans le SNC, lors de l’altération des fonctions cérébrales dans un modèle de souris transgénique de la MA. Nos résultats démontrent que la voie de signalisation IL-36/IL36R est augmentée dans trois modèles différents de l’EAE, mais ne contribue pas au développement ni à la progression de la pathologie. En utilisant l’approche de cytométrie en flux nous identifions les neutrophiles comme la source majeure de l’IL-36γ. De plus, nous démontrons que la microglie exprime l’IL-36R et sa stimulation par l’IL-36γ conduit à la production de cytokines pro-inflammatoires. Dans un second temps, nous caractérisons l’augmentation de l’expression du GPR84 par la microglie dans le modèle murin de la MA APP/PS1. Ainsi, le croisement de ces souris avec des souris déficientes en GPR84 diminue l’activation et le recrutement de la microglie autour des plaques d’amyloïde-β et accélère le déclin cognitif. Nos études impliquent le GPR84 comme un acteur important dans le maintien de l’homéostasie neuronale puisque son absence favorise la dégénérescence des dendrites dans le cerveau. Les résultats obtenus dans cette thèse apportent de nouveaux éléments qui peuvent contribuer au développement des thérapies qui ciblent les cellules myéloïdes dans diverses pathologies du SNC. Ces données ouvrent de nouvelles pistes pour élucider le rôle de l’IL-36γ dans des maladies neurodégénératives. Enfin, pour une première fois, nous présentons un modèle murin permettant d’identifier le(s) ligand(s) endogène(s) du GPR84, une cible thérapeutique potentielle pour la prévention et/ou le traitement de la MA.

The Influence of Estrogens on the Biological and Therapeutic Actions of Growth Hormone in the Liver

[EN]GH is main regulator of body growth and composition, somatic development, intermediate metabolism and gender-dependent dimorphism in mammals. The liver is a direct target of estrogens because it expresses estrogen receptors which are connected with development, lipid metabolism and insulin sensitivity, hepatic carcinogenesis, protection from drug-induced toxicity and fertility. In addition, estrogens can modulate GH actions in liver by acting centrally, regulating pituitary GH secretion, and, peripherally, by modulating GHR-JAK2-STAT5 signalling pathway

The Role of Morphogens in B Cell Development

Morphogens are signalling molecules that play a significant role in modulation of cell fate and development. Hedgehog proteins (Hh) are morphogens that have been shown to be involved in the development of immune cells. In this study, it is demonstrated that treatment of B cells with rShh, can increase B cell activation and also promote survival of B cells at 18hours post-stimulus. Also, at this time point, there was found to be an increase in secretion of antibody isotypes and IL-6. By 40hours post-stimulus, it was observed that the level of B cell activation was apparently arrested in treated B cells, whereas the level of activation continued to rise in untreated B cells. Interestingly, it was observed that there was an increase in the percentage of; CD23-CD25+ B cells when B cells were treated with rShh and this was accompanied by an increase in apoptosis. Consistent with this finding in relation to apoptosis, there was an increased expression of the pro-apoptotic protein Bnip3 in B cells treated with rShh by 40hours post-stimulus. It was observed that there were three subsets of B cells arising in our culture at 40hours, which were all found to possess different characteristics. It was demonstrated that treatment with rShh can increase B cell differentiation towards FO-I at 18hours post-stimulus. By 40hours post-stimulus, Hh signalling can divert differentiation away from the FO-I B cell towards the T2-MZP, which was accompanied by an increase in IL-10 secretion. Gene expression analysis revealed that Hh signalling could modulate a number of molecules involved in delivering the BCR signal into the cells such as Btk, Nfatc1 and Traf2. Additionally, deletion of Dhh, showed that there was a skewed peripheral B cell development in the Dhh-/- mice. Overall, our data demonstrate that Hh signalling can regulate the development of B cells in response to an activation stimulus by strengthening the BCR signalling pathway.

Interleukin-4 receptor alpha gene variants and allergic disease

The interleukin-4 (IL-4) signalling cascade has been identified as a pathway potentially important in the development of asthma. Genetic variants within this signalling pathway might contribute to the risk of developing asthma in a given individual. A number of polymorphisms have been described within the IL-4 receptor alpha (IL-4Ralpha) gene. In addition polymorphism occurs in the promoter for the IL-4 gene itself. This commentary accompanies a paper by C Ober et al describing the contribution of IL-4Ralpha polymorphism to susceptibility to asthma and atopy in the Hutterite population and other outbred populations collected during the collaborative studies on the genetics of asthma (CSGA) programme

Avaliação do potencial cancerigénico de microcistinas (cianotoxinas)

Tese de doutoramento em Farmácia (Toxicologia), apresentada à Faculdade de Farmácia da Universidade de Lisboa, 2009.

Host cellular regulatory networks in dengue virus-human interactions

Dengue fever is one of the most important mosquito-borne diseases worldwide and is caused by infection with dengue virus (DENV). The disease is endemic in tropical and sub-tropical regions and has increased remarkably in the last few decades. At present, there is no antiviral or approved vaccine against the virus. Treatment of dengue patients is usually supportive, through oral or intravenous rehydration, or by blood transfusion for more severe dengue cases. Infection of DENV in humans and mosquitoes involves a complex interplay between the virus and host factors. This results in regulation of numerous intracellular processes, such as signal transduction and gene transcription which leads to progression of disease. To understand the mechanisms underlying the disease, the study of virus and host factors is therefore essential and could lead to the identification of human proteins modulating an essential step in the virus life cycle. Knowledge of these human proteins could lead to the discovery of potential new drug targets and disease control strategies in the future. Recent advances of high throughput screening technologies have provided researchers with molecular tools to carry out investigations on a large scale. Several studies have focused on determination of the host factors during DENV infection in human and mosquito cells. For instance, a genome-wide RNA interference (RNAi) screen has identified host factors that potentially play an important role in both DENV and West Nile virus replication (Krishnan et al. 2008). In the present study, a high-throughput yeast two-hybrid screen has been utilised in order to identify human factors interacting with DENV non-structural proteins. From the screen, 94 potential human interactors were identified. These include proteins involved in immune signalling regulation, potassium voltage-gated channels, transcriptional regulators, protein transporters and endoplasmic reticulum-associated proteins. Validation of fifteen of these human interactions revealed twelve of them strongly interacted with DENV proteins. Two proteins of particular interest were selected for further investigations of functional biological systems at the molecular level. These proteins, including a nuclear-associated protein BANP and a voltage-gated potassium channel Kv1.3, both have been identified through interaction with the DENV NS2A. BANP is known to be involved in NF-kB immune signalling pathway, whereas, Kv1.3 is known to play an important role in regulating passive flow of potassium ions upon changes in the cell transmembrane potential. This study also initiated a construction of an Aedes aegypti cDNA library for use with DENV proteins in Y2H screen. However, several issues were encountered during the study which made the library unsuitable for protein interaction analysis. In parallel, innate immune signalling was also optimised for downstream analysis. Overall, the work presented in this thesis, in particular the Y2H screen provides a number of human factors potentially targeted by DENV during infection. Nonetheless, more work is required to be done in order to validate these proteins and determine their functional properties, as well as testing them with infectious DENV to establish a biological significance. In the long term, data from this study will be useful for investigating potential human factors for development of antiviral strategies against dengue.

Rôle du facteur de transcription PITX1 dans les pathogenèses de l'ostéoporose et des maladies parodontales

L’ostéoporose est une maladie caractérisée par une faible masse osseuse et une détérioration du tissu osseux. Cette condition entraîne une plus grande fragilité osseuse et des risques de fractures. Plusieurs études ont associé l’ostéoporose à la faible densité osseuse des mandibules, à la perte d’attache parodontale, à l’augmentation de la hauteur de la crête alvéolaire et à la chute des dents. Cette étude vise à comprendre les mécanismes sous-jacents cette perte osseuse. En effet, au cours du développement des souris, PITX1 joue un rôle clé dans l'identité des membres postérieurs et dans le bon développement des mandibules et des dents. Son inactivation complète chez la souris mène à un phénotype squelettique sévère. Tandis que, son inactivation partielle provoque des symptômes apparentés à l'arthrose avec une augmentation de la masse osseuse au niveau de l’os cortical et de l’os trabéculaire. Inversement, une étude antérieure chez des jumelles monozygotiques discordantes pour l’ostéoporose, montrent une augmentation d’environ 8.6 fois du niveau d’expression du gène Pitx1 chez la jumelle ostéoporotique. Collectivement, ces données nous ont poussés à investiguer sur le rôle du facteur de transcription PITX1 dans le métabolisme osseux normal et pathologique. Dans ce contexte, des souris transgéniques Col1α1-Pitx1 sur-exprimant Pitx1 spécifiquement dans le tissu osseux sous le promoteur du collagène de type-I (fragment 2.1kpb) ont été générées et phénotypiquement caractérisées. Ces résultats ont révelé que les souris transgéniques Col1α1-Pitx1 présentaient un phénotype similaire à celui des patients ostéoporotiques accompagné d'une perte de dents et des problèmes dentaires et parodontaux. De plus, cette étude a révélé que la surexpression de Pitx1 induit une altération de l’homéostasie osseuse via l’inactivation de la voie de signalisation Wnt/β-caténine canonique. Cette hypothèse a été appuyée par le fait que le traitement des souris transgéniques Col1α1-Pitx1 avec du chlorure de lithium, un activateur de la voie Wnt canonique, prévient le phénotype ostéoporotique chez ces souris. Finalement, cette étude établit un rôle crucial de PITX1 dans la régulation de la masse osseuse et une implication possible dans l’ostéoporose et les maladies parodontales via l’inactivation de la voie de signalisation Wnt/β-caténine canonique.

Elevated hepatocyte growth factor levels in osteoarthritis osteoblasts contribute to their altered response to bone morphogenetic protein-2 and reduced mineralization capacity

International audience

Phosphodiesterase 6 generates intracellular cGMP microdomains in the native endothelium

Endothelial cells (EC) are essential regulator of vascular homeostasis through the generation and release of various bioactive agents, including nitric oxide (NO). NO modulates several vascular functions such as vascular tone and permeability, through the stimulation of soluble guanylate cyclase (sGC) leading to the production of cGMP. Conversely, phosphodiesterases (PDEs) are enzymes metabolizing cyclic nucleotides (cGMP and cAMP) and are therefore major regulatory players for cGMP and cAMP signalling pathways. Although ECs are the main source of NO, little is known on the endothelial NO-cGMP signalling pathway and cellular outcomes. It was then hypothesized that a specific population of cGMP-phosphodiesterases allows ECs to stabilize cGMP levels despite the elevated production of NO. Expression of cGMP-phosphodiesterases was initially studied in resistance mesenteric arteries from mice. PDE5 and PDE6 were both found at mRNA and protein levels in native arteries but PDE6 is not found in cultured ECs. Interestingly, subcellular distributions of both enzymes were distinct. PDE5 appeared to be homogeneously distributed whilst PDE6 catalytic subunits (PDE6 and PDE6) showed a preferential staining in the perinuclear region. These results suggest that PDE6 might be involved in the regulation of cGMP microdomains. Based on these findings, a mathematical model was developed. Simulations of dynamic cGMP levels in ECs support the notion of cGMP microdomains dependent on PDE6 expression and localization. In the absence of PDE6, application of NO either as a single bolus or repetitive pulses led to a homogeneous increase in cGMP levels in ECs despite PDE5 homogeneous distribution. However, PDE6 subcellular targeting to the perinuclear membrane generated a cGMP-depleted perinuclear space. The findings from this study provide the first evidence of the expression and specific intracellular distribution of PDE6 in native endothelial cells that strongly support their involvement in the generation of cGMP microdomains

Phosphodiesterase 6 generates intracellular cGMP microdomains in the native endothelium

Endothelial cells (EC) are essential regulator of vascular homeostasis through the generation and release of various bioactive agents, including nitric oxide (NO). NO modulates several vascular functions such as vascular tone and permeability, through the stimulation of soluble guanylate cyclase (sGC) leading to the production of cGMP. Conversely, phosphodiesterases (PDEs) are enzymes metabolizing cyclic nucleotides (cGMP and cAMP) and are therefore major regulatory players for cGMP and cAMP signalling pathways. Although ECs are the main source of NO, little is known on the endothelial NO-cGMP signalling pathway and cellular outcomes. It was then hypothesized that a specific population of cGMP-phosphodiesterases allows ECs to stabilize cGMP levels despite the elevated production of NO. Expression of cGMP-phosphodiesterases was initially studied in resistance mesenteric arteries from mice. PDE5 and PDE6 were both found at mRNA and protein levels in native arteries but PDE6 is not found in cultured ECs. Interestingly, subcellular distributions of both enzymes were distinct. PDE5 appeared to be homogeneously distributed whilst PDE6 catalytic subunits (PDE6 and PDE6) showed a preferential staining in the perinuclear region. These results suggest that PDE6 might be involved in the regulation of cGMP microdomains. Based on these findings, a mathematical model was developed. Simulations of dynamic cGMP levels in ECs support the notion of cGMP microdomains dependent on PDE6 expression and localization. In the absence of PDE6, application of NO either as a single bolus or repetitive pulses led to a homogeneous increase in cGMP levels in ECs despite PDE5 homogeneous distribution. However, PDE6 subcellular targeting to the perinuclear membrane generated a cGMP-depleted perinuclear space. The findings from this study provide the first evidence of the expression and specific intracellular distribution of PDE6 in native endothelial cells that strongly support their involvement in the generation of cGMP microdomains

Kinetic modelling of in vitro data of PI3K, mTOR1, PTEN enzymes and on-target inhibitors Rapamycin, BEZ235, and LY294002

The phosphatidylinositide 3-kinases (PI3K) and mammalian target of rapamycin-1 (mTOR1) are two key targets for anti-cancer therapy. Predicting the response of the PI3K/AKT/mTOR1 signalling pathway to targeted therapy is made difficult because of network complexities. Systems biology models can help explore those complexities but the value of such models is dependent on accurate parameterisation. Motivated by a need to increase accuracy in kinetic parameter estimation, and therefore the predictive power of the model, we present a framework to integrate kinetic data from enzyme assays into a unified enzyme kinetic model. We present exemplar kinetic models of PI3K and mTOR1, calibrated on in vitro enzyme data and founded on Michaelis-Menten (MM) approximation. We describe the effects of an allosteric mTOR1 inhibitor (Rapamycin) and ATP-competitive inhibitors (BEZ2235 and LY294002) that show dual inhibition of mTOR1 and PI3K. We also model the kinetics of phosphatase and tensin homolog (PTEN), which modulates sensitivity of the PI3K/AKT/mTOR1 pathway to these drugs. Model validation with independent data sets allows investigation of enzyme function and drug dose dependencies in a wide range of experimental conditions. Modelling of the mTOR1 kinetics showed that Rapamycin has an IC50 independent of ATP concentration and that it is a selective inhibitor of mTOR1 substrates S6K1 and 4EBP1: it retains 40% of mTOR1 activity relative to 4EBP1 phosphorylation and inhibits completely S6K1 activity. For the dual ATP-competitive inhibitors of mTOR1 and PI3K, LY294002 and BEZ235, we derived the dependence of the IC50 on ATP concentration that allows prediction of the IC50 at different ATP concentrations in enzyme and cellular assays. Comparison of the drug effectiveness in enzyme and cellular assays showed that some features of these drugs arise from signalling modulation beyond the on-target action and MM approximation and require a systems-level consideration of the whole PI3K/PTEN/AKT/mTOR1 network in order to understand mechanisms of drug sensitivity and resistance in different cancer cell lines. We suggest that using these models in systems biology investigation of the PI3K/AKT/mTOR1 signalling in cancer cells can bridge the gap between direct drug target action and the therapeutic response to these drugs and their combinations.

Regulation of prostate cancer cell function by activators of AMP-activated protein kinase

AMP-activated protein kinase (AMPK) is a key regulator of cell energy homeostasis. More recently, it has become apparent that AMPK regulates cell proliferation, migration and inflammation. Previous evidence has suggested that AMPK may influence proliferation and invasion by regulating the pro-proliferative mitogen-activated protein kinases (MAPKs). However, the mechanisms underlying this crosstalk between AMPK and MAPK signalling are not fully understood. As AMPK activation has been reported to have anti-proliferative effects, there has been increasing interest in AMPK activation as a therapeutic target for tumourigenesis. The aim of this study was to investigate whether AMPK activation influenced prostate cancer (PC) cell line proliferation, migration and signalling. Therefore, different PC cell lines were incubated with two structurally-unrelated molecules that activate AMPK by different mechanisms, AICAR and A769662. Both chemicals activated AMPK in a concentration- and time-dependent manner in PC3, DU145 and LNCaP cell lines. AMPK activity as assessed by AMPK activating phosphorylation as well as phosphorylation of the AMPK substrate ACC increased along with tumour severity in PC biopsies. Furthermore, both activators of AMPK decreased cell proliferation and migration in the androgen-independent PC cell lines PC3 and DU145. Inhibition of proliferation by A769662 was attenuated in AMPK α1-/- AMPK α2-/- knockout (KO) mouse embryonic fibroblasts (MEFs) compared to wild type (WT) MEFs, and the inhibitory effect on migration of AICAR lost significance in PC3 cells infected with adenoviruses expressing a dominant negative AMPK α mutant, indicating these effects are partially mediated by AMPK. Furthermore, long-term activation of AMPK was associated with inhibition of both the phosphatidylinositol 3’-kinase/protein kinase B (PI3K/Akt) signalling pathway in addition to the extracellular signal-regulated kinase 1/2 (ERK1/2) signalling pathway. Indeed, the actions of AMPK activators on PC cell line viability were mimicked by selective inhibitors of Akt and ERK1/2 pathways. In contrast to the effects of prolonged incubation with AMPK activators, short-term incubation with AMPK activators had no effect on epidermal growth factor (EGF)-stimulated ERK1/2 phosphorylation in PC cell lines. In addition, AMPK activation did not influence phosphorylation of the other MAPK family members p38 and JNK. Interestingly, both AICAR and A769662 decreased EGF-stimulated ERK5 phosphorylation in PC3, DU145 and LNCaP cells as assessed with an anti-phospho-ERK5 antibody. Further characterisation of this effect indicated that prior stimulation with the AMPK activators had no effect on ERK5 phosphorylation stimulated by transient transfection with a constitutively active ERK5 kinase (MEK5DD), which represents the only known canonical kinase for ERK5. Intriguingly, the pattern of EGF-stimulated ERK5 phosphorylation was distinct from that mediated by MEK5DD activation of ERK5. This finding indicates that AMPK activation inhibits EGF-stimulated ERK5 phosphorylation at a point at or above the level of MEK5, although why EGF and constitutively active MEK5 stimulate markedly different immunoreactive species recognised by the anti-phospho-ERK5 antibody requires further study. A769662 had a tendency to reduce EGF-stimulated ERK5 phosphorylation in WT MEFs, yet was without effect in MEFs lacking AMPK. These data indicate that AMPK may underlie the effect of A769662 to reduce EGF-stimulated ERK5 phosphorylation. Prolonged stimulation of PC cell lines with AICAR or A769662 inhibited EGF-stimulated Akt Ser473 phosphorylation, whereas only incubation with A769662 rapidly inhibited Akt phosphorylation. This difference in the actions of the different AMPK activators may suggest an AMPK-independent effect of A769662. Furthermore, AICAR increased phosphorylation of Akt in WT MEFs, an effect that was absent in MEFs lacking AMPK, indicating that this effect of AICAR may be AMPK-dependent. Taken together, the data presented in this study suggest that AMPK activators markedly inhibit proliferation and migration of PC cell lines, reduce EGF-stimulated ERK1/2 and Akt phosphorylation after prolonged incubation and rapidly inhibit ERK5 phosphorylation. Both AMPK activators exhibit a number of effects that are likely to be independent of AMPK in PC cell lines, although inhibition of ERK1/2, ERK5 and Akt may underlie the effects of AMPK activators on proliferation, viability and migration. Further studies are required to understand the crosstalk between those signalling pathways and their underlying significance in PC progression.