High Concentrations Of ATP Research Articles

Abstract 42: A new type of drug resistance in cancer: extracellular ATP-induced resistance through ATP internalization and ATP-drug competition

Abstract Drug resistance is a major problem in cancer therapies and responsible for most relapse, one of the major causes of death in cancer. Several cancer drug resistance mechanisms are known, including genetic mutations in protooncogenes or tumor suppressor genes, cancer stem cells, and multi-drug resistance (MDR). Among these mechanisms, MDR is ATP-dependent. Intratumoral (extracellular) ATP levels of various cancer types are 10⁁3 to 10⁁4 times higher than those in their corresponding normal tissues but the biological significance of the high intratumoral ATP concentrations is unknown. We recently reported that extracellular ATP is internalized by cancer cells via macropinocytosis and enhances their growth and survival (1). Based on these results, we hypothesized that the internalized ATP also promotes drug resistance. Here we report that extracellular ATP, at concentrations in or below the range of reported intratumoral ATP levels, substantially increased intracellular ATP levels and promoted cancer cell drug resistance to tyrosine kinase inhibitor (TKI) sunitinib in human NSCLC A549 cells. The ATP increase and the drug resistance were mediated, at least in part, by macropinocytosis, clathrin- and caveolae-mediated endocytoses. The elevated intracellular ATP induced upregulated phosphorylation of PDGFR and proteins/enzymes in the PDGFR-mediated signaling pathways, such as Akt, mTOR, Raf and MEK, resulting in augmented cell growth and reduced apoptosis induced by sunitinib. The upregulated phosphorylation was not mediated by purinergic receptor signaling. Furthermore, in vitro, ex vivo, and in vivo, extracellular ATP partially restored phosphorylation levels of PDGRF and its downstream proteins/enzymes inhibited by sunitinib, a PDGFR inhibitor (TKI). These results strongly suggest that the extracellular ATP-increased intracellular ATP reversed the inhibition of TKIs by competing with the inhibitors for the ATP-binding site of PDGFR, enhancing the Akt-mTOR and Raf-MEK signaling pathways and promoting cancer cell survival. All these findings significantly expand our understanding of the roles of extracellular ATP in the Warburg effect (2) and drug resistance in cancer and indentify a new anti-drug resistance target. 1. Qian Y, Wang X, et al., Cancer Lett. 351(2014): 242-5. 2. Chen X, Qian Y, Wu S. Free. Radic. Biol. Med. 79(2015): 253-263. Citation Format: Xuan Wang, Yunsheng Li, Yanrong Qian, Yanyang Cao, Xiaozhuo Chen. A new type of drug resistance in cancer: extracellular ATP-induced resistance through ATP internalization and ATP-drug competition. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 42.

Modeling Interactions among Individual P2 Receptors to Explain Complex Response Patterns over a Wide Range of ATP Concentrations.

Extracellular ATP acts on the P2X family of ligand-gated ion channels and several members of the P2Y family of G protein-coupled receptors to mediate intercellular communication among many cell types including bone-forming osteoblasts. It is known that multiple P2 receptors are expressed on osteoblasts (P2X2,5,6,7 and P2Y1,2,4,6). In the current study, we investigated complex interactions within the P2 receptor network using mathematical modeling. To characterize individual P2 receptors, we extracted data from published studies of overexpressed human and rodent (rat and mouse) receptors and fit their dependencies on ATP concentration using the Hill equation. Next, we examined responses induced by an ensemble of endogenously expressed P2 receptors. Murine osteoblastic cells (MC3T3-E1 cells) were loaded with fluo-4 and stimulated with varying concentrations of extracellular ATP. Elevations in the concentration of cytosolic free calcium ([Ca2+]i) were monitored by confocal microscopy. Dependence of the calcium response on ATP concentration exhibited a complex pattern that was not explained by the simple addition of individual receptor responses. Fitting the experimental data with a combination of Hill equations from individual receptors revealed that P2Y1 and P2X7 mediated the rise in [Ca2+]i at very low and high ATP concentrations, respectively. Interestingly, to describe responses at intermediate ATP concentrations, we had to assume that a receptor with a K1∕2 in that range (e.g. P2Y4 or P2X5) exerts an inhibitory effect. This study provides new insights into the interactions among individual P2 receptors in producing an ensemble response to extracellular ATP.

P2X7R modulation of visually evoked synaptic responses in the retina.

P2X7Rs are distributed throughout all layers of the retina, and thus, their localisation on various cell types puts into question their specific site(s) of action. Using a dark-adapted, ex vivo mouse retinal whole mount preparation, the present study aimed to characterise the effect of P2X7R activation on light-evoked, excitatory RGC ON-field excitatory post-synaptic potentials (fEPSPs) and on outer retinal electroretinogram (ERG) responses under comparable conditions. The pharmacologically isolated NMDA receptor-mediated RGC ON-fEPSP was reduced in the presence of BzATP, an effect which was significantly attenuated by A438079 and other selective P2X7R antagonists A804598 or AF27139. In physiological Krebs medium, BzATP induced a significant potentiation of the ERG a-wave, with a concomitant reduction in the b-wave and the power of the oscillatory potentials. Conversely, in the pharmacologically modified Mg2+-free perfusate, BzATP reduced both the a-wave and b-wave. The effects of BzATP on the ERG components were suppressed by A438079. A role for P2X7R function in visual processing in both the inner and outer retina under physiological conditions remains controversial. The ON-fEPSP was significantly reduced in the presence of A804598 but not by A438079 or AF27139. Furthermore, A438079 did not have any effect on the ERG components in physiological Krebs but potentiated and reduced the a-wave and b-wave, respectively, when applied to the pharmacologically modified medium. Therefore, activation of P2X7Rs affects the function in the retinal ON pathway. The presence of a high concentration of extracellular ATP would most likely contribute to the modulation of visual transmission in the retina in the pathophysiological microenvironment.

P2X7 nucleotide receptor signaling potentiates the Wnt/β-catenin pathway in cells of the osteoblast lineage.

The P2X7 and Wnt/β-catenin signaling pathways regulate osteoblast differentiation and are critical for the anabolic responses of bone to mechanical loading. However, whether these pathways interact to control osteoblast activity is unknown. The purpose of this study was to investigate the effects of P2X7 activation on Wnt/β-catenin signaling in osteoblasts. Using MC3T3-E1 cells, we found that combined treatment with Wnt3a and the P2X7 agonist 2'(3')-O-(4-benzoylbenzoyl)adenosine 5'-triphosphate (BzATP) elicited more sustained β-catenin nuclear localization than that induced by Wnt3a alone. Wnt3a-induced increases in β-catenin transcriptional activity were also potentiated by treatment with BzATP. Consistent with involvement of P2X7, a high ATP concentration (1mM) potentiated Wnt3a-induced β-catenin transcriptional activity, whereas a low concentration (10μM) of ATP, adenosine 5'-diphosphate (ADP), or uridine 5'-triphosphate (UTP) failed to elicit a response. The potentiation of β-catenin transcriptional activity elicited by BzATP was also inhibited by two distinct P2X7 antagonists: A 438079 and A 740003. Furthermore, responses to Wnt3a in calvarial cells isolated from P2rx7 knockout mice were significantly less than in cells from wild-type controls. In MC3T3-E1 cells, BzATP increased inhibitory phosphorylation of glycogen synthase kinase 3β (GSK3β), a process that was blocked by A 438079 and diminished by inhibition of protein kinase C. Thus, P2X7 signaling may potentiate the canonical Wnt pathway through GSK3β inhibition. Taken together, we show that P2X7 activation prolongs and potentiates Wnt/β-catenin signaling. Consequently, cross-talk between P2X7 and Wnt/β-catenin pathways may modulate osteoblast activity in response to mechanical loading.

Role of lutoid membrane transport and protein synthesis in the regeneration mechanism of latex in different Hevea clones

Natural rubber (cis-1,4 polyisoprene) is synthesised in the milky cytoplasm, the latex, of specialized cells called laticifers in the bark tissues of the rubber tree (Hevea brasiliensis). Regeneration mechanism of latex after each tapping (controlled wounding of the bark) was studied in relation to lutoid membrane enzymes and protein synthesis in twelve rubber clones with varying yield potentials during the peak rubber yielding season. High activity of membrane enzymes and better availability of biochemical energy [ATP] were observed in clones viz; RRII 105, RRIM 600, PB 260, RRII 422 and RRII 430. The highest protein biosynthetic capacity was noticed in clone PB 260 and RRIM 600. However, high ATP content, increased invertase activity and protein biosynthesis were observed in the medium yielding clone GT1 compared to clones with low rubber yield potential. Very low sugar content and increased invertase activity in the latex of clone PB 260 indicated intense latex metabolism with high protein turnover that implies fast recouping of the cellular metabolites lost during latex harvesting. Clone PB 217 was characterized by very high sucrose and low ATP concentration and ATPase activity in latex indicating slow metabolism and hence be suitable for inducing latex metabolism using ethylene stimulant. Low rubber yielding clones such as RRII 33 and RRII 38 were consistently recorded a high sucrose content but very low activity of membrane enzymes, reduced ATP concentration and low protein biosynthesis in latex. Among the recently released modern clones (RRII 400 series), latex regeneration capacity was higher in RRII 422 and RRII 430. The significance of lutoid membrane transport and protein synthesis is discussed in relation to general latex metabolism of these rubber clones. The outcome of this study would be helpful to design suitable latex harvesting systems and yield stimulation methods for optimizing latex production in each clone based on metabolic profiling.

TRPM7 is a molecular substrate of ATP-evoked P2X7-like currents in tumor cells.

Within the ion channel-coupled purine receptor (P2X) family, P2X7 has gained particular interest because of its role in immune responses and in the growth control of several malignancies. Typical hallmarks of P2X7 are nonselective and noninactivating cation currents that are elicited by high concentrations (0.1-10 mM) of extracellular ATP. Here, we observe spurious ATP-induced currents in HEK293 cells that neither express P2X7 nor display ATP-induced Ca(2+) influx or Yo-Pro-1 uptake. Although the biophysical properties of these ionic currents resemble those of P2X7 in terms of their reversal potential close to 0 mV, nonrectifying current-voltage relationship, current run-up during repeated ATP application, and augmentation in bath solutions containing low divalent cation (DIC) concentrations, they are poorly inhibited by established P2X7 antagonists. Because high ATP concentrations reduce the availability of DICs, these findings prompted us to ask whether other channel entities may become activated by our experimental regimen. Indeed, a bath solution with no added DICs yields similar currents and also a rapidly inactivating Na(+)-selective conductance. We provide evidence that TRPM7 and ASIC1a (acid-sensing ion channel type Ia)-like channels account for these noninactivating and phasic current components, respectively. Furthermore, we find ATP-induced currents in rat C6 glioma cells, which lack functional P2X receptors but express TRPM7. Thus, the observation of an atypical P2X7-like conductance may be caused by the activation of TRPM7 by ATP, which scavenges free DICs and thereby releases TRPM7 from permeation block. Because TRPM7 has a critical role in controlling the intracellular Mg(2+) homeostasis and regulating tumor growth, these data imply that the proposed role of P2X7 in C6 glioma cell proliferation deserves reevaluation.

Using the fluorescent properties of STO-609 as a tool to assist structure-function analyses of recombinant CaMKK2

Calcium/calmodulin-dependent kinase kinase 2 (CaMKK2) has been implicated in the regulation of metabolic activity in cancer and immune cells, and affects whole-body metabolism by regulating ghrelin-signalling in the hypothalamus. This has led to efforts to develop specific CaMKK2 inhibitors, and STO-609 is the standardly used CaMKK2 inhibitor to date. We have developed a novel fluorescence-based assay by exploiting the intrinsic fluorescence properties of STO-609. Here, we report an in vitro binding constant of KD ∼17 nM between STO-609 and purified CaMKK2 or CaMKK2:Calmodulin complex. Whereas high concentrations of ATP were able to displace STO-609 from the kinase, GTP was unable to achieve this confirming the specificity of this association. Recent structural studies on the kinase domain of CaMKK2 had implicated a number of amino acids involved in the binding of STO-609. Our fluorescent assay enabled us to confirm that Phe267 is critically important for this association since mutation of this residue to a glycine abolished the binding of STO-609. An ATP replacement assay, as well as the mutation of the ‘gatekeeper’ amino acid Phe267Gly, confirmed the specificity of the assay and once more confirmed the strong binding of STO-609 to the kinase. In further characterising the purified kinase and kinase-calmodulin complex we identified a number of phosphorylation sites some of which corroborated previously reported CaMKK2 phosphorylation and some of which, particularly in the activation segment, were novel phosphorylation events.In conclusion, the intrinsic fluorescent properties of STO-609 provide a great opportunity to utilise this drug to label the ATP-binding pocket and probe the impact of mutations and other regulatory modifications and interactions on the pocket. It is however clear that the number of phosphorylation sites on CaMKK2 will pose a challenge in studying the impact of phosphorylation on the pocket unless the field can develop approaches to control the spectrum of modifications that occur during recombinant protein expression in Escherichia coli.

Impact of exogenous GABA treatments on endogenous GABA metabolism in anthurium cut flowers in response to postharvest chilling temperature

Anthurium flowers are susceptible to chilling injury, and the optimum storage temperature is 12.5–20 °C. The γ-aminobutyric acid (GABA) shunt pathway may alleviate chilling stress in horticultural commodities by providing energy (ATP), reducing molecules (NADH), and minimizing accumulation of reactive oxygen species (ROS). In this experiment, the impact of a preharvest spray treatment with 1 mM GABA and postharvest treatment of 5 mM GABA stem-end dipping on GABA shunt pathway activity of anthurium cut flowers (cv. Sirion) in response to cold storage (4 °C for 21 days) was investigated. GABA treatments resulted in lower glutamate decarboxylase (GAD) and higher GABA transaminase (GABA-T) activities in flowers during cold storage, which was associated with lower GABA content and coincided with higher ATP content. GABA treatments also enhanced accumulation of endogenous glycine betaine (GB) in flowers during cold storage, as well as higher spathe relative water content (RWC). These findings suggest that GABA treatments may alleviate chilling injury of anthurium cut flowers by enhancing GABA shunt pathway activity leading to provide sufficient ATP and promoting endogenous GB accumulation.

P2X receptor inhibition protects THP‐1 monocytes against cytolysis by α‐hemolysin from Escherichia Coli and leukotoxin from Aggregatibacter actinomycetemcomitans

The Repeats in Toxins (RTX) α‐hemolysin (HlyA) from E. coli and leukotoxin A (LtxA) from A. actinomycetemcomitans are important virulence factors for ascending urinary tract infections and aggressive forms of periodontitis respectively. Upon insertion into the plasma membrane ATP is released through the toxin pore and is as signaling molecule essential for the HlyA‐ and LtxA‐induced erythrocyte damage. The immediate effects after ATP release are a massive erythrocyte shrinkage and phosphatidylserine exposure in the outer leaflet of the plasma membrane. These events are eminent for the erythrocytes to be recognized and engulfed by the monocyte cell line THP‐1. Here, we investigate how these toxins and ATP, affect the survival and function of THP‐1 monocytes.By measurements of [Ca2+]i, we demonstrate that the THP‐1 express functional P2X (P2X1, P2X4 and P2X7) and P2Y2 receptor. We show that desensitization of either P2X1 by either low (0.001 μM) or more pronounced of P2X4/P2X7 by high concentrations of ATP (0.1 to 1 mM) increases the survival rate of THP‐1 cells attacked by either HlyA or LtxA in a calcein release assay. This protection is also found by specific blockage of the ATP sensitive receptors P2X1 (100 μM NF449) or P2X7 (100 μM oxATP or A804589). By erythrocyte phagocytosis assay, we determined that low dose HlyA (EC25) preconditioning is increasing the function of erythrocyte clearance at the place of injury.These findings suggest a new target for protecting monocytes during pore‐former toxemia – both pharmacologically through P2X blockers or receptor desenzitisation. Both mechanisms may be relevant for improving monocyte survival and function in patients suffering from infection with RTX producing bacteria.Support or Funding InformationFunded by the Danish Council for Independent Research, MEMRANES and Lundbeck Foundation

Purinergic signalling in a latent stem cell niche of the rat spinal cord.

The ependyma of the spinal cord harbours stem cells which are activated by traumatic spinal cord injury. Progenitor-like cells in the central canal (CC) are organized in spatial domains. The cells lining the lateral aspects combine characteristics of ependymocytes and radial glia (RG) whereas in the dorsal and ventral poles, CC-contacting cells have the morphological phenotype of RG and display complex electrophysiological phenotypes. The signals that may affect these progenitors are little understood. Because ATP is massively released after spinal cord injury, we hypothesized that purinergic signalling plays a part in this spinal stem cell niche. We combined immunohistochemistry, in vitro patch-clamp whole-cell recordings and Ca(2+) imaging to explore the effects of purinergic agonists on ependymal progenitor-like cells in the neonatal (P1-P6) rat spinal cord. Prolonged focal application of a high concentration of ATP (1mM) induced a slow inward current. Equimolar concentrations of BzATP generated larger currents that reversed close to 0mV, had a linear current-voltage relationship and were blocked by Brilliant Blue G, suggesting the presence of functional P2X7 receptors. Immunohistochemistry showed that P2X7 receptors were expressed around the CC and the processes of RG. BzATP also generated Ca(2+) waves in RG that were triggered by Ca(2+) influx and propagated via Ca(2+) release from internal stores through activation of ryanodine receptors. We speculate that the intracellular Ca(2+) signalling triggered by P2X7 receptor activation may be an epigenetic mechanism to modulate the behaviour of progenitors in response to ATP released after injury.

Enhanced OXPHOS, glutaminolysis and β-oxidation constitute the metastatic phenotype of melanoma cells.

Tumours display different cell populations with distinct metabolic phenotypes. Thus, subpopulations can adjust to different environments, particularly with regard to oxygen and nutrient availability. Our results indicate that progression to metastasis requires mitochondrial function. Our research, centered on cell lines that display increasing degrees of malignancy, focused on metabolic events, especially those involving mitochondria, which could reveal which stages are mechanistically associated with metastasis. Melanocytes were subjected to several cycles of adhesion impairment, producing stable cell lines exhibiting phenotypes representing a progression from non-tumorigenic to metastatic cells. Metastatic cells (4C11+) released the highest amounts of lactate, part of which was derived from glutamine catabolism. The 4C11+ cells also displayed an increased oxidative metabolism, accompanied by enhanced rates of oxygen consumption coupled to ATP synthesis. Enhanced mitochondrial function could not be explained by an increase in mitochondrial content or mitochondrial biogenesis. Furthermore, 4C11+ cells had a higher ATP content, and increased succinate oxidation (complex II activity) and fatty acid oxidation. In addition, 4C11+ cells exhibited a 2-fold increase in mitochondrial membrane potential (ΔΨmit). Consistently, functional assays showed that the migration of cells depended on glutaminase activity. Metabolomic analysis revealed that 4C11+ cells could be grouped as a subpopulation with a profile that was quite distinct from the other cells investigated in the present study. The results presented here have centred on how the multiple metabolic inputs of tumour cells may converge to compose the so-called metastatic phenotype.

Sex effect on insulin secretion and mitochondrial function in pancreatic beta cells of elderly Wistar rats

ABSTRACTAims/hypothesis: Glucose tolerance progressively declines with age, and there is a high prevalence of type 2 diabetes in the elderly people. Previous studies have reported the sex differences in risk for type 2 diabetes, especially in the elderly people, whereas reasons for these sex differences remain poorly understood. This study aims to evaluate the effect of sex on glucose-stimulated insulin secretion and mitochondrial function in pancreatic beta cells of Wistar rats. Methods: 3-month-old and 18-month-old Wistar rats of both sexes were used. Insulin secretion of islets was analyzed by glucose-stimulated insulin secretion and islet perifusion assays; ATP content and oxygen consumption rate of islets were determined to evaluate the mitochondrial function. Results: Insulin secretion of islets under high glucose conditions declined significantly with age in both sexes. Glucose-stimulated insulin secretion of elderly female groups was markedly higher than that of male groups under high glucose conditions. Importantly, islets from elderly female groups showed higher mitochondrial function compared with male counterparts, evidenced by higher ATP content and oxygen consumption rate under high glucose conditions. It was also noted that mitochondrial biogenesis of islets from elderly female rats was significant higher compared with male rats. There were notable increases in expression of genes involved in mitochondrial biogenesis in islets from elderly female rats compared with male rats. Conclusion: This study demonstrates a sex dimorphism in the age-associated impairment of pancreatic beta cell function in elderly rats, while the potential mechanism may be related to the sexual differences in mitochondrial biogenesis and function.

Mechanism of Cooperative Force Generations between Skeletal Myosins

To understand the molecular mechanism of cooperative force generation between skeletal myosin molecules, we measured forces generated by myosin-rod cofilaments, in which approximately 17 myosin molecules interact with single actins at the mixing ratio used in this study. Combined with results from the computational model, three factors are important for synchronization of power strokes between myosin motors. First, strain-dependent kinetics are necessary to couple mechanochemical cycles between myosins. Second, multiple power stroke states further enhance a chance of power stroke synchronization. Finally, the physiological ATP concentration is another important factor to enhance a chance of power stroke synchronization, since the strain-dependent transitions accompanied by the first or second power stroke are the rate limiting steps at higher ATP concentrations. Consequently, our computational model predicts that most of steps were generated by synchronous executions of power strokes between several myosin motors at 1 mM ATP, while they are generated primarily by single myosins. Thus, ensemble average curves of steps obtained from our model were distinctively different between 1 mM and 10 μM ATP. These results were consistent with experimental results, supporting our conclusions.

Region-specific rapid regulation of aromatase activity in zebra finch brain.

Recent studies demonstrate that rapid modulation of the estrogen synthetic enzyme aromatase, regulates hypothalamic (HYP) estrogen production, and subsequent neurophysiology and reproductive behavior. In songbirds, in addition to expression in the HYP, aromatase is expressed at high levels in several brain regions notably in the hippocampus (HP) and caudomedial nidopallium (NCM), where estrogens affect learning and memory and auditory processing, respectively. Previous studies, largely in quail HYP, show that aromatase activity is acutely down-regulated by Ca2+ -dependent phosphorylation. Here, using zebra finches (Taeniopygia guttata), we ask if similar mechanisms are at work in the songbird HYP and if there are sex as well as regional differences in aromatase modulation. Using invitro assays to measure activity in homogenates or in partially purified supernatants containing microsomes and synaptosomes of the HP, HYP, and NCM, we examined effects of Ca2+ , Mg2+ , ATP, NADPH, and an inhibitor of kinase activity. We report a rapid down-regulation of aromatase activity in the presence of phosphorylating conditions across all three brain regions and both sexes. However, regional differences were seen in response to some phosphorylating factors, some of which were improved by partial purification of the homogenates. Furthermore, while low concentrations of ATP inhibited aromatase activity, unexpectedly, inhibition was no longer seen with high ATP concentrations. These results provide evidence for a regional and temporal specificity in the rapid modulation of aromatase activity that may bear on local neuroendocrine function. Aromatase activity in male and female zebra finch hippocampus, hypothalamus, and caudomedial nidopallium is rapidly regulated by Ca2+ -dependent phosphorylation. Low ATP and Mg2+ decrease activity, whereas nicotinamide adenine dinucleotide phosphate (NADPH), high ATP, and inhibition of protein kinase C increase activity. Evidence suggests this may occur at the synapse. These results provide a mechanism for rapid regulation of behavior via brain estrogen synthesis.

A curvilinear Model Approach: Actin Cortex Clustering Due to ATP-induced Myosin Pulls

Abstract: The actomyosin cortex is involved in a range of many cellular processes like cell division, motility or shaping. To obtain this variety of functionalities the membrane-bound actin mesh has to be reconstituted by the motor protein myosin. But little is known about the underlying mechanism, which control the different tasks. An underlying in vitro study of a synthetic actomyosin cortex has shown that the cortex organizes into spatial clusters for certain ATP concentrations. Here we develop a curvilinear model that captures the viscoelastic material behavior and the kinetics of the myosin cross bridge. Further, we suggest a formulation for the active contractile stress produced by the motor protein myosin. We demonstrate that the spatial pattern generated by the curvilinear model is consistent with the experimental observations, including mesh clustering due to contractile forces and an absence of contraction for low and high ATP concentrations. Additionally we show that the cluster positioning can be tuned by the ATP-gradient.

Abstract A180: Discovery of furanone derivatives as novel Cdc7 inhibitors with anti-tumor activity

Abstract Introduction> Cell cycle kinases have been recognized as potential anticancer targets, because loss of regulated cell cycle progression would lead to uncontrolled cell growth and development of cancer. Cdc7 kinase is involved in regulation of the cell cycle and plays important roles in DNA replication. Notably, inhibition of Cdc7 in cancer causes lethal S phase or M phase progression, whereas normal cells can survive, most likely through induction of cell cycle arrest at the DNA replication checkpoints. Therefore Cdc7 kinase has emerged as a promising therapeutic target for the treatment of cancers. Here we present a novel series of furanone derivatives with potent inhibitory effects on Cdc7 kinase. <Methods> To identify hit compounds that are insensitive or resistant to high ATP concentration, a high-throughput screening (HTS) was performed in the presence of 100 microM ATP which corresponds to 36-times higher than the Km value of ATP for Cdc7 kinase activity. To characterize inhibitors, ATP dependency and pre-incubation effects were examined. To determine the efficacy of Cdc7 inhibitors, phosphorylation of MCM2 protein in cells were analyzed by western blotting. Selected compounds were subjected to a kinase selectivity panel assay. <Results> Only a single hit compound was identified from the HTS, reflecting extremely low hit rate compared to that of other kinase targets in our past HTS. Subsequent hit to lead studies led to the identification of a novel furanone compound as a potential lead compound for further optimization study. Interestingly, these furanone derivatives showed slow-binding inhibition against Cdc7, and exhibited strong inhibitory activity for Cdc7 even in the presence of 1 mM ATP that was added after the pre-incubation of the kinase and compound. Notably this pre-incubation effect was not observed with other kinases tested. Detailed results using cancer cells will also be presented in this poster. Citation Format: Takayuki Irie, Tokiko Asami, Ayako Sawa, Chika Taniyama, Yoko Funakoshi, Yuko Uno, Hisao Masai, Masaaki Sawa. Discovery of furanone derivatives as novel Cdc7 inhibitors with anti-tumor activity. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A180.

The P2X7 receptor regulates cell survival, migration and invasion of pancreatic ductal adenocarcinoma cells.

BackgroundPancreatic ductal adenocarcinoma (PDAC) is presently one of the cancers with the worst survival rates and least effective treatments. Moreover, total deaths due to PDAC are predicted to increase in the next 15 years. Therefore, novel insights into basic mechanism of PDAC development and therapies are needed. PDAC is characterized by a complex microenvironment, in which cancer and stromal cells release different molecules, such as ATP. ATP can be transported and/or exocytosed from active cancer cells and released from dying cells in the necrotic core of the cancer. We hypothesized that one of the ATP receptors, the P2X7 receptor (P2X7R) could be an important player in PDAC behaviour.MethodsWe determined the expression (real time PCR and Western blot) and localization (immunofluorescence) of P2X7R in human PDAC cell lines (AsPC-1, BxPC-3, Capan-1, MiaPaCa-2, Panc-1) and a “normal” human pancreatic duct epithelial cell line (HPDE). The function of P2X7R in proliferation (BrdU assay), migration (wound assay) and invasion (Boyden chamber with matrigel) was characterized. Furthermore, we studied P2X7R-dependent pore formation (YoPro-1 assay) and cell death (caspase and annexin V / propidium iodide assays).ResultsWe found higher expression of P2X7R protein in PDAC compared to HPDE cells. P2X7R had notable disparate effects on PDAC survival. Firstly, high concentrations of ATP or the specific P2X7R agonist, BzATP, had cytotoxic effects in all cell lines, and cell death was mediated by necrosis. Moreover, the P2X7R–pore antagonist, A438079, prevented ATP-induced pore formation and cell death. Second, in basal conditions and with low concentrations of ATP/BzATP, the P2X7R allosteric inhibitor AZ10606120 reduced proliferation in all PDAC cell lines. P2X7R also affected other key characteristics of cancer cell behavior. AZ10606120 reduced cell migration and invasion in PDAC cell lines compared to that of untreated/vehicle-treated control cells, and stimulation with sub-millimolar concentrations of ATP or BzATP substantially increased cell invasion.ConclusionsPDAC cell lines overexpress P2X7R and the receptor plays crucial roles in cell survival, migration and invasion. Therefore, we propose that drugs targeting P2X7R could be exploited in therapy of pancreatic cancer.Electronic supplementary materialThe online version of this article (doi:10.1186/s12943-015-0472-4) contains supplementary material, which is available to authorized users.

Adenine nucleotide translocase 1 overexpression protects cardiomyocytes against hypoxia via increased ERK1/2 and AKT activation

The influence of mitochondrial function on intracellular signalling is currently under intense investigation. In this regard, we analysed the effect of adenine nucleotide translocase 1 (ANT1), which facilitates the exchange of ADP and ATP across the mitochondrial membrane, on cell-protective survival signalling under hypoxia. ANT1 overexpression enhanced the survival rate in hypoxic cardiomyocytes. The effect was related to stabilization of the mitochondrial membrane potential, suppression of caspase 3 activity, and a reduction in DNA fragmentation. Activation of the cell-protective signalling proteins extracellular signal-regulated kinases 1 and 2 (ERK1/2) and protein kinase B (AKT) was substantially higher in hypoxic ANT1-transgenic (ANT1-TG) cardiomyocytes than in wild-type cardiomyocytes. Kinase activation was associated with significantly higher expression of hypoxia-inducible factor 1α, which induces glycolytic pathway to stabilize ATP production. Accordingly, ANT1-TG cardiomyocytes exhibited earlier and stronger activation of lactate dehydrogenase and a higher ATP content. Treatment with PD980559 and triciribine, inhibitors of ERK1/2 and AKT activation, respectively, abolished cell protection in hypoxic ANT1-TG cardiomyocytes. Inhibition of ANT by carboxyatractyloside prevented the increase in ERK1/2 and AKT phosphorylation and eliminated the cell protective program in hypoxic ANT1-TG cardiomyocytes.In conclusion, the cytoprotective effect observed in hypoxic ANT1-overexpressing cardiomyocytes involves an interdependence between ANT1, activation of ERK1/ERK2 and AKT, and induction of the survival processes regulated by these kinases.

The P2X7 receptor is a potential therapeutic target for the treatment of Uveitis

PurposeAdenosine‐5′‐triphosphate (ATP) functions as an important extracellular messenger and plays a critical role as a danger signalling molecule during inflammation. In pathological conditions damaged cells leak high concentrations of ATP into the extracellular milieu, activating P2 Purinergic receptors which are highly expressed on immune cells. Pathological stimulation of the P2X7 receptor may be involved in the development of autoimmune disease, so we explored the impact of P2X7R antagonist treatment on the development of Experimental Autoimmune Uveitis (EAU) in mice.MethodsEAU was induced in P2X7‐/‐ and wild‐type mice using IRBP peptide 1‐20(GPTHLFQPSLVLDMAKVLLD) with adjuvant Bordetella pertussis toxin. All procedures were performed under a Home Office License in accordance with the regulations of UK ASPA (1986). EAU was then induced in B10.rIII mice with RBP‐3 161‐180 (SGIPYIISYLHPGNTILHVD) and adjuvant Bordetella pertussis toxin. P2X7R antagonist A439079275 or vehicle were injected i.p. twice daily from day 12. p.i when clinical features of EAU manifested. Disease activity was observed through TEFI imaging and animals were treated until peak disease and termination at day 16 p.i.ResultsP2X7R deficiency protected against the development of EAU, with disease scores significantly lower in P2X7‐/‐ animals compared to control animals. P2X7R antagonist treatment with A439079 prevented development of severe EAU with disease scores in A439079 treated animals significantly lower than vehicle treated animals.ConclusionsP2X7R deficiency protects against the development of EAU in mice and P2X7R antagonist can ameliorate established disease. The P2X7R may represent a viable therapeutic target for ocular inflammatory disease.

Integrated isotope-assisted metabolomics and (13)C metabolic flux analysis reveals metabolic flux redistribution for high glucoamylase production by Aspergillus niger.

BackgroundAspergillus niger is widely used for enzyme production and achievement of high enzyme production depends on the comprehensive understanding of cell’s metabolic regulation mechanisms.ResultsIn this paper, we investigate the metabolic differences and regulation mechanisms between a high glucoamylase-producing strain A. niger DS03043 and its wild-type parent strain A. niger CBS513.88 via an integrated isotope-assisted metabolomics and 13C metabolic flux analysis approach. We found that A. niger DS03043 had higher cell growth, glucose uptake, and glucoamylase production rates but lower oxalic acid and citric acid secretion rates. In response to above phenotype changes, A. niger DS03043 was characterized by an increased carbon flux directed to the oxidative pentose phosphate pathway in contrast to reduced flux through TCA cycle, which were confirmed by consistent changes in pool sizes of metabolites. A higher ratio of ATP over AMP in the high producing strain might contribute to the increase in the PP pathway flux as glucosephosphate isomerase was inhibited at higher ATP concentrations. A. niger CBS513.88, however, was in a higher redox state due to the imbalance of NADH regeneration and consumption, resulting in the secretion of oxalic acid and citric acid, as well as the accumulation of intracellular OAA and PEP, which may in turn result in the decrease in the glucose uptake rate.ConclusionsThe application of integrated metabolomics and 13C metabolic flux analysis highlights the regulation mechanisms of energy and redox metabolism on flux redistribution in A. niger. Graphical abstractAn integrated isotope-assisted metabolomics and 13C metabolic flux analysis was was firstly systematically performed in A. niger. In response to enzyme production, the metabolic flux in A. niger DS03043 (high-producing) was redistributed, characterized by an increased carbon flux directed to the oxidative pentose phosphate pathway as well as an increased pool size of pentose. The consistency in 13C metabolic flux analysis and metabolites quantification indicated that an imbalance of NADH formation and consumption led to the accumulation and secretion of organic acids in A. niger CBS513.88 (wild-type)Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-015-0329-y) contains supplementary material, which is available to authorized users.