Increase In Intracellular ATP Research Articles

Bioprinting-associated pulsatile hydrostatic pressure elicits a mild proinflammatory response in epi- and endothelial cells

During nozzle-based bioprinting, like inkjet and microextrusion, cells are subjected to hydrostatic pressure for up to several minutes. The modality of the bioprinting-related hydrostatic pressure is either constant or pulsatile depending on the technique. We hypothesized that the difference in the modality of hydrostatic pressure affects the biological response of the processed cells differently. To test this, we used a custom-made setup to apply either controlled constant or pulsatile hydrostatic pressure on endothelial and epithelial cells. Neither bioprinting procedure visibly altered the distribution of selected cytoskeletal filaments, cell-substrate adhesions, and cell-cell contacts in either cell type. In addition, pulsatile hydrostatic pressure led to an immediate increase of intracellular ATP in both cell types. However, the bioprinting-associated hydrostatic pressure triggered a pro-inflammatory response in only the endothelial cells, with an increase of interleukin 8 (IL-8) and a decrease of thrombomodulin (THBD) transcripts. These findings demonstrate that the settings adopted during nozzle-based bioprinting cause hydrostatic pressure that can trigger a pro-inflammatory response in different barrier-forming cell types. This response is cell-type and pressure-modality dependent. The immediate interaction of the printed cells with native tissue and the immune system in vivo might potentially trigger a cascade of events. Our findings, therefore, are of major relevance in particular for novel intra-operative, multicellular bioprinting approaches.

Polar mutagenesis of polycistronic bacterial transcriptional units using Cas12a

BackgroundFunctionally related genes in bacteria are often organized and transcribed as polycistronic transcriptional units. Examples are the fim operon, which codes for biogenesis of type 1 fimbriae in Escherichia coli, and the atp operon, which codes for the FoF1 ATP synthase. We tested the hypothesis that markerless polar mutations could be efficiently engineered using CRISPR/Cas12a in these loci.ResultsCas12a-mediated engineering of a terminator sequence inside the fimA gene occurred with efficiencies between 10 and 80% and depended on the terminator’s sequence, whilst other types of mutations, such as a 97 bp deletion, occurred with 100% efficiency. Polar mutations using a terminator sequence were also engineered in the atp locus, which induced its transcriptional shutdown and produced identical phenotypes as a deletion of the whole atp locus (ΔatpIBEFHAGDC). Measuring the expression levels in the fim and atp loci showed that many supposedly non-polar mutants induced a significant polar effect on downstream genes. Finally, we also showed that transcriptional shutdown or deletion of the atp locus induces elevated levels of intracellular ATP during the exponential growth phase.ConclusionsWe conclude that Cas12a-mediated mutagenesis is an efficient simple system to generate polar mutants in E. coli. Different mutations were induced with varying degrees of efficiency, and we confirmed that all these mutations abolished the functions encoded in the fim and atp loci. We also conclude that it is difficult to predict which mutagenesis strategy will induce a polar effect in genes downstream of the mutation site. Furthermore the strategies described here can be used to manipulate the metabolism of E. coli as showcased by the increase in intracellular ATP in the markerless ΔatpIBEFHAGDC mutant.

Febuxostat, a Xanthine Oxidoreductase Inhibitor, Decreases NLRP3-dependent Inflammation in Macrophages by Activating the Purine Salvage Pathway and Restoring Cellular Bioenergetics

The nucleotide-binding oligomerization domain–like receptor family, pyrin domain–containing 3 (NLRP3) inflammasome mediates caspase-1 activation and IL-1β processing and is implicated in autoinflammatory as well as other chronic inflammatory diseases. Recent studies have demonstrated that xanthine oxidoreductase (XOR) inhibition attenuated IL-1β secretion in activated macrophages, but the detailed mechanism of inhibition remains unclear. In this study, we report that febuxostat, an inhibitor of XOR, suppressed NLRP3 inflammasome-mediated IL-1β secretion and cell death by two mechanisms: in a mitochondrial ROS (mitoROS)-dependent and mitoROS-independent manner. MitoROS-independent effects of febuxostat were mediated by an increase of intracellular ATP and improved mitochondrial energetics via the activation of purine salvage pathway. Our findings suggest that cellular bioenergetics are important in regulating NLRP3 activation, and XOR inhibition may be clinically relevant in NLRP3-related inflammatory diseases.

A temporal transcriptomic dynamics study reveals the reason of enhanced ε-poly-L-lysine production in Streptomyces albulus M-Z18 by pH shock

ε-Poly-L-lysine (ε-PL), a natural food preservative, is mainly produced by Streptomyces in aerobic fermentation. Previously, we have significantly enhanced ε-PL production through a pH shock strategy. To understand the underlying reason between pH shock and high ε-PL production, RNA-seq technology was conducted in this study to investigate the temporal transcription at three time-points corresponding to early, mid-term and late stages of fermentation. Transcription of genes related to signal transduction, electron respiratory chain, membrane transport, nitrogen metabolism, energy metabolism and redox homeostasis were significantly up-regulated, accompanied by the increase of intracellular ATP, respiratory activity. The assay of intracellular reactive oxygen species and malonaldehyde showed that pH shock could enhance the antioxidant capacity and reduce oxidative damage of cells effectively. We deduced that signal transduction systems play a major role in responding to pH shock, and result in the enhancement of the metabolic vitality and intracellular redox homeostasis. The obtained information will facilitate future studies for ε-PL production improvement and lay a foundation for molecular mechanism investigation of pH shock.

Inhibitors of energy metabolism interfere with antibiotic-induced death in mycobacteria

Energy metabolism has recently gained interest as a target space for antibiotic drug development in mycobacteria. Of particular importance is bedaquiline (Sirturo), which kills mycobacteria by inhibiting the F1F0 ATP synthase. Other components of the electron transport chain such as the NADH dehydrogenases (NDH-2 and NdhA) and the terminal respiratory oxidase bc1:aa3 are also susceptible to chemical inhibition. Because antituberculosis drugs are prescribed as part of combination therapies, the interaction between novel drugs targeting energy metabolism and classical first and second line antibiotics must be considered to maximize treatment efficiency. Here, we show that subinhibitory concentration of drugs targeting the F1F0 ATP synthase and the cytochrome bc1:aa3, as well as energy uncouplers, interfere with the bactericidal potency of isoniazid and moxifloxacin. Isoniazid- and moxifloxacin-induced mycobacterial death correlated with a transient increase in intracellular ATP that was dissipated by co-incubation with energy metabolism inhibitors. Although oxidative phosphorylation is a promising target space for drug development, a better understanding of the link between energy metabolism and antibiotic-induced mycobacterial death is essential to develop potent drug combinations for the treatment of tuberculosis.

Endocytosis of KATP Channels Drives Glucose-Stimulated Excitation of Pancreatic β Cells

Insulin secretion from pancreatic β cells in response to high glucose (HG) critically depends on the inhibition of KATP channel activity in HG. It is generally believed that HG-induced effects are mediated by the increase in intracellular ATP, but here, we showed that, in INS-1 cells, endocytosis of KATP channel plays a major role. Upon HG stimulation, resting membrane potential depolarized by 30.6mV (from -69.2 to -38.6mV) and KATP conductance decreased by 91% (from 0.243 to 0.022nS/pF), whereas intracellular ATP was increased by only 47%. HG stimulation induced internalization of KATP channels, causing a significant decrease in surface channel density, and this decrease was completely abolished by inhibiting endocytosis using dynasore, a dynamin inhibitor, or a PKC inhibitor. These drugs profoundly inhibited HG-induced depolarization. Our results suggest that the control of KATP channel surface density plays a greater role than ATP-dependent gating in regulating β cell excitability.

Molecular events leading to death of Leishmania donovani under spermidine starvation after hypericin treatment.

We have previously reported that the hypericin treatment caused spermidine starvation and death of Leishmania parasite. Here, we report different molecular events under spermidine starvation and potential role of spermidine in processes other than redox homeostasis of the parasite. We have analyzed changes in expression of several genes by using quantitative gene expression analysis. Further, these changes at molecular level were also confirmed by using biochemical and cellular studies. Altered expression of several genes involved in redox metabolism, hypusine modification of eIF5A, DNA repair pathway and autophagy was observed. There was decrease in Sir2RP expression after hypericin treatment and this decrease has been found to be associated with induced ROS due to hypericin treatment as it has been rescued by either trypanothione or spermidine supplementation. Translation initiation in the parasite was decreased upon spermidine starvation. We also observed increased AMPK expression upon hypericin treatment. The increase in intracellular ATP and NAD+ levels as well as decrease in Sir2RP expression of the parasite are cytoprotective mechanism towards generated ROS due to hypericin treatment possibly by inducing autophagy as indicated by increase in autophagy related gene expression and acridine orange staining. However, the autophagy needs to be established using more rigorous methodologies.

Existence of ATP sensitive potassium currents on human periodontal ligament cells

ObjectivePotassium channels of the ATP-sensitive family (KATP channel) are inhibited by increase in intracellular ATP. Electrophysiological studies have demonstrated that the kinetics and pharmacological properties of KATP channels vary among different tissues, suggesting structurally and functionally distinct types. There are studies showing human periodontal ligament (PDL) cells respond to mechanical stress by increasing ATP release, which participates in bone resorption or bone homeostasis. So, in this study we investigated the existence of KATP channel subunit and their single channel properties in human periodontal ligaments. Materials & methodThe human PDL cells were isolated from healthy erupted third molar. For patch-clamp experiments, human PDL fibroblasts were seeded on 3.5cm plastic dishes. The inside-out patch clamp recordings were performed under voltage clamp mode. Reverse transcriptase polymerase chain reaction (RT-PCR) was conducted to identify the channel subunits. All pair-wise comparisons were performed by Paired t-test. A P value <0.05 was considered significant. ResultsWe observed mRNA transcripts for Kir6.1, Kir6.2 and Sur2B subuits in the human PDL cells. In inside-out patch mode, the single channel conductance was 163pS at symmetrical K+ concentration of 140mM and inward rectification was seen in ATP-free bath solution. The reversal potential of the currents was found to be 0mV at symmetrical concentration (140mM) of K+ in bath solution. The single channel currents were almost blocked by adding 5mM ATP in the bath solution. However, the currents were not blocked by 100μM glibenclamide, a subunit specific KATP channel blocker. ConclusionsThese results indicate that human PDL cells express KATP channels subunit including Sur2B and Kir6.1 and Kir6.2 which are sensitive to ATP but insensitive to glibenclamide.

Abstract 1173: Extracellular ATP is internalized through not only macropinocytosis but also other endocytic process and promotes growth of human lung cancer cells with Ras mutations

Abstract Within a tumor, extracellular ATP (exATP) concentrations are in the range of several hundred micromolars or higher, 103-104 times higher than those in normal tissues1. We recently showed that extracellular ATP, within the intratumoral concentrations, was internalized by Ras-mutated (Rasmut) A549 human lung cancer cells through macropinocytosis, elevated intracellular ATP levels and rescued cancer cells under various stresses2. However, macropinocytosis3 alone could not account for all the elevated intracellular ATP increase. Here, we show that other endocytic processes, in addition to macropinocytosis, also contributed to the internalization of exATP in A549 and H1299 human lung cancer cells. Cell viability assays, intracellular ATP luminescence measurement assays, fluorescent microscopy were used in this study. First, high-molecular-weight (HMW) dextran uptake assay indicated that A549 and H1299, both Rasmut cancer cells, exhibited a phenotype of macropinocytosis. The colocalization of the red fluorescent HMW dextran and green nonhydrolyzable ATP analog inside the cells indicated that extracellular ATP was internalized by macropinocytosis. In contrast, significantly fewer macropinosomes were found in NL-20 noncancerous lung cells. In addition, the colocalization of low-molecular-weight (LMW) dextran, an endocytic tracer, and ATP analog indicated the internalization of exATP through non-macropinocytic-endocytosis in both lung cancer cell lines. Interestingly, when both red HMW dextran and green LMW dextran were applied to cancer cells, not only colocalization, but also some green-only endosomes were observed, strongly suggesting that both endocytic processes were responsible for exATP internalization. Collectively, these data indicated for the first time that other endocytic processes in addition to macropinocytosis contributed to the internalization of exATP in human lung cancer cells with Rasmut. This finding may significantly enhance our understanding of energy hemostasis within cancer cells and the Warburg effect, which in turn facilitates the development of anticancer therapeutics targeting the endocytic processes and Ras mutations as well as novel anticancer strategies. 1. Pellegatti, et al. (2008). PLoS ONE 3, e2599. 2. Qian, et al. (2014). Cancer Letters 351, 242-251. 3. Commisso, et al. (2013). Nature 497, 633-637. Citation Format: Yanrong Qian, Xuan Wang, Yunsheng Li, Yanyang Cao, Shiyong Wu, Xiaozhuo Chen. Extracellular ATP is internalized through not only macropinocytosis but also other endocytic process and promotes growth of human lung cancer cells with Ras mutations. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1173. doi:10.1158/1538-7445.AM2015-1173

Abstract 3364: Extracellular ATP-induced intracellular ATP concentration elevation mediated by macropinocytosis promotes growth, survival, and drug resistance of cancer cells

Abstract Cancer cells switch from highly efficient mitochondrial oxidative phosphorylation (OXPHOS) to low efficiency glycolysis for ATP synthesis even when oxygen is abundant, a phenomenon called the Warburg effect. Because of the higher rates of cell growth and proliferation, cancer cells need more ATP than normal cells of the same tissues. However, the switch is counterintuitive since cancer cells need more ATP but cancer cells prefer to use a low efficiency pathway to produce ATP. How cancer cells secure all their ATP needs is not well understood. On the other hand, extracellular ATP concentration in cancer (intratumoral ATP concentration) are found to be in the range of several hundred μM, ∼10^3-10^4 times higher than those found in normal tissues. Huge differences exist between cancer and normal cells regarding extracellular ATP. Based on these observations, we hypothesized that cancer cells take up ATP from extracellular space to supplement their energy needs. Here we report that in multiple cancer cell lines belonging to multiple cancer types, 0.5-3 mM ATP, within the intratumoral ATP concentration range, elevated intracellular ATP levels by more than 50%. Extracellular ATP also reduced stress and promoted survival of cancer cells that were under OXPHOS and glucose metabolism inhibitions. Furthermore, extracellular ATP increased the viability of cancer cells treated with sunitinib or pazopanib, anticancer drugs working as ATP analogs by competing with ATP for the ATP binding site of targeted tyrosine kinases (TKs). In contrast, extracellular ATP did not affect the activity of paclitaxel, a drug unrelated to ATP. Inhibitor studies revealed that the extracellular ATP-induced intracellular ATP increase shows no dependence on OXPHOS, glycolysis, master ATP regulator AMPK, transcription or translation. These results strongly suggest that not all the increased intracellular ATP is synthesized inside of cancer cells. Fluorescence microscopy study showed that human lung cancer A549 cells, which are KRas oncogene mutation-positive (KRas+), exhibit macropinotcytosis, a type of endocytosis that non-specifically take in extracellular nutritional molecules by “fluid drinking”. Inhibition of macropinocytosis resulted in decrease in dextran take-up and intracellular ATP levels, and alleviated the drug resistance to TKIs in dose-dependent manners. These strongly suggest that macropinocytosis is responsible, at least in part, for transporting extracellular ATP into cancer cells, which led to drug resistance. All these results demonstrate, for the first time, unrecognized roles of extracellular ATP in contributing to the intracellular ATP pool and increase drug resistance in cancer cells and suggest a novel ATP transport mechanism. These findings challenge our traditional view on ATP and shed new lights on ATP homeostasis and ATP-induced drug resistance in cancer cells. Citation Format: Yanrong Qian, Xuan Wang, Yi Liu, Yunsheng Li, Robert A. Colvin, Lingying Tong, Shiyong Wu, Xiaozhuo Chen. Extracellular ATP-induced intracellular ATP concentration elevation mediated by macropinocytosis promotes growth, survival, and drug resistance of cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3364. doi:10.1158/1538-7445.AM2014-3364

Abstract 5257: Cancer cells have not only sweet tooth but also large mouths: Evidence for uptake of ATP into KRas oncogene-expressing cancer cells by macropinocytosis

Abstract Cancer cells are known to exhibit reprogrammed metabolism and prefer to use glycolygsis for their ATP synthesis even when oxygen is abundant, a phenomenon called the Warburg effect. However, cancer cells are also known for more rapid growth and proliferation, therefore requiring more ATP than normal cells of the same tissues. How cancer cells manage to use low efficiency glycolysis but still meet their higher ATP demands is enigmatic. In our previous study, we found that extracellular ATP induced large intracellular ATP increase and drug resistance to tyrosine kinase inhibitors (TKIs) that function as ATP analogs / competitors in cancer cells. The induced ATP increase was from neither OXPHOS nor glycolysis, and was not dependent on AMPK, the master ATP regulator. Furthermore, the increase did not involve new gene expression or new protein synthesis. Recently, it was reported that cells transformed with an oncogenic form of KRas gene (KRas+) exhibit a phenotype of macropinocytosis, a type of endocytosis. KRas-induced macropinocytosis nonspecifically takes up extracellular proteins. All these led us to hypothesize that the increased intracellular ATP induced by extracellular ATP is, at least in part, not produced intracellularly but is transported into cancer cells. To test this hypothesis, high molecular weight fluorescent dextran was use to determine if KRas+ human non-small cell lung cancer A549 cells exhibit macropinocytosis. Nonhydrolyzable fluorescent ATP analog was used to visualize if the ATP analog can be transported into A549 cells along with dextran. Fluorescence microscopy studies revealed that A549 cells exhibit the phenotype of macropinocytosis, which can be reduced by a macropinocytosis inhibitor EIPA. HMW dextran and the nonhydrolysable fluorescent ATP analog were found co-localized inside A549 cells, demonstrating that they were transported into A549 cells via macropinosomes. It was also found that the ATP increase cannot be explained by purinergic receptor signaling alone. In comparison, KRas- human breast cancer MCF7 cells do not take up HMW dextran, indicating that they do not exhibit macropinocytosis. All these provide strong and visual evidence that ATP can be transported into KRas+ A549 cancer cells by macropinocytosis. Other experiments using radioactive ATP or purinergic receptor inhibitors also strongly support this conclusion. This is the first time that ATP (ATP analog) is shown to be taken into cancer cells. This finding will have major impact on how we view cancer cells in terms of their energy exchange with other cancer or noncancer cells in the tumor and on the Warburg effect. It will also have profound implications on how to more effectively inhibiting KRas+ or KRas- cancer cells by targeting ATP transport and ATP metabolism. Citation Format: Yanrong Qian, Xuan Wang, Yi Liu, Yunsheng Li, Robert A. Colvin, Xiaozhuo Chen. Cancer cells have not only sweet tooth but also large mouths: Evidence for uptake of ATP into KRas oncogene-expressing cancer cells by macropinocytosis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5257. doi:10.1158/1538-7445.AM2014-5257

55: IFN-ß regulation of glucose metabolism is PI3K/Akt dependent and important for antiviral activity against coxsackievirus B3

An effective type I interferon (IFN)-mediated immune response requires the rapid expression of antiviral proteins that are necessary to inhibit viral replication and virus spread. We provide evidence that IFN-ß regulates metabolic events important for the induction of a rapid antiviral response. IFN-ß decreases the phosphorylation of AMP-activated protein kinase (AMPK), coincident with an increase in intracellular ATP. Our data suggest that IFN-ß regulates glucose metabolism mediated by signaling effectors similarly to activation by insulin. Our studies reveal a biphasic IFN-ß-inducible uptake of glucose by cells, mediated by PI3K/Akt, and IFN-ß-inducible regulation of GLUT4 translocation to the cell surface. Additionally, we provide evidence that IFN-ß-regulated glycolytic metabolism is important for the acute induction of an antiviral response during infection with cardiotropic coxsackievirus B3 (CVB3). Interference with IFN-ß-inducible glucose metabolism diminishes the antiviral response, whereas treatment with metformin, a drug that increases insulin sensitivity, enhances the antiviral potency of IFN-ß against CVB3 both in vitro and in vivo . Taken together, these findings highlight an important role for IFN-ß in modulating glucose metabolism during a virus infection and suggest that the use of metformin in combination with IFN-ß during acute virus infection may result in enhanced antiviral responses.

Extracellular ATP is internalized by macropinocytosis and induces intracellular ATP increase and drug resistance in cancer cells

ATP plays central roles in cancer metabolism and the Warburg effect. Intratumoral ATP concentrations are up to 104 times higher than those of interstitial ATP in normal tissues. However, extracellular ATP is not known to enter cancer cells. Here we report that human A549 lung cancer cells internalized extracellular ATP by macropinocytosis as demonstrated by colocalization of a nonhydrolyzable fluorescent ATP and a macropinocytosis tracer high-molecular-weight dextran, as well as by a macropinocytosis inhibitor study. Extracellular ATP also induced increase of intracellular ATP levels, without involving transcription and translation at significant levels, and cancer cells’ resistance to ATP-competitor anticancer drugs, likely through the mechanism of ATP internalization. These findings, described for the first time, have profound implications in ATP-sharing among cancer cells in tumors and highlight a novel anticancer target.

Beta interferon regulation of glucose metabolism is PI3K/Akt dependent and important for antiviral activity against coxsackievirus B3.

An effective type I interferon (IFN)-mediated immune response requires the rapid expression of antiviral proteins that are necessary to inhibit viral replication and virus spread. We provide evidence that IFN-β regulates metabolic events important for the induction of a rapid antiviral response: IFN-β decreases the phosphorylation of AMP-activated protein kinase (AMPK), coincident with an increase in intracellular ATP. Our studies reveal a biphasic IFN-β-inducible uptake of glucose by cells, mediated by phosphatidylinositol 3-kinase (PI3K)/Akt, and IFN-β-inducible regulation of GLUT4 translocation to the cell surface. Additionally, we provide evidence that IFN-β-regulated glycolytic metabolism is important for the acute induction of an antiviral response during infection with coxsackievirus B3 (CVB3). Last, we demonstrate that the antidiabetic drug metformin enhances the antiviral potency of IFN-β against CVB3 both in vitro and in vivo. Taken together, these findings highlight an important role for IFN-β in modulating glucose metabolism during a virus infection and suggest that the use of metformin in combination with IFN-β during acute virus infection may result in enhanced antiviral responses. Type I interferons (IFN) are critical effectors of an antiviral response. These studies describe for the first time a role for IFN-β in regulating metabolism--glucose uptake and ATP production--to meet the energy requirements of a robust cellular antiviral response. Our data suggest that IFN-β regulates glucose metabolism mediated by signaling effectors similarly to activation by insulin. Interference with IFN-β-inducible glucose metabolism diminishes the antiviral response, whereas treatment with metformin, a drug that increases insulin sensitivity, enhances the antiviral potency of IFN-β.

Resistance to hypoxia-induced necroptosis is conferred by glycolytic pyruvate scavenging of mitochondrial superoxide in colorectal cancer cells

Cancer cells may survive under oxygen and nutrient deprivation by metabolic reprogramming for high levels of anaerobic glycolysis, which contributes to tumor growth and drug resistance. Abnormally expressed glucose transporters (GLUTs) are colocalized with hypoxia (Hx) inducible factor (HIF)1α in peri-necrotic regions in human colorectal carcinoma. However, the underlying mechanisms of anti-necrotic resistance conferred by glucose metabolism in hypoxic cancer cells remain poorly understood. Our aim was to investigate signaling pathways of Hx-induced necroptosis and explore the role of glucose pyruvate metabolite in mechanisms of death resistance. Human colorectal carcinoma cells were Hx exposed with or without glucose, and cell necroptosis was examined by receptor-interacting protein (RIP)1/3 kinase immunoprecipitation and 32P kinase assays. Our results showed increased RIP1/3 complex formation and phosphorylation in hypoxic, but not normoxic cells in glucose-free media. Blocking RIP1 signaling, by necrostatin-1 or gene silencing, decreased lactodehydrogenase (LDH) leakage and plasma membrane disintegration. Generation of mitochondrial superoxide was noted after hypoxic challenge; its reduction by antioxidants inhibited RIP signaling and cell necrosis. Supplementation of glucose diminished the RIP-dependent LDH leakage and morphological damage in hypoxic cells, whereas non-metabolizable sugar analogs did not. Hypoxic cells given glucose showed nuclear translocation of HIF1α associated with upregulation of GLUT-1 and GLUT-4 expression, as well as increase of intracellular ATP, pyruvate and lactate levels. The glucose-mediated death resistance was ablated by iodoacetate (an inhibitor to glyceraldehyde-3-phosphate dehydrogenase), but not by UK5099 (an inhibitor to mitochondrial pyruvate carrier), suggesting that glycolytic pathway was involved in anti-necrotic mechanism. Lastly, replacing glucose with cell-permeable pyruvate derivative also led to decrease of Hx-induced necroptosis by suppression of mitochondrial superoxide in an energy-independent manner. In conclusion, glycolytic metabolism confers resistance to RIP-dependent necroptosis in hypoxic cancer cells partly through pyruvate scavenging of mitochondrial free radicals.

Rejuvenation of ATP during storage does not reverse effects of the hypothermic storage lesion

Hypothermic storage (HS) of red blood cells (RBCs) leads to a progressive deterioration of cell quality. Mitigating these deleterious changes could allow maintenance or even an improvement of RBC in vitro quality. The aim was to determine the effect of a cold rejuvenation treatment of RBCs and in particular to assess the connection between ATP levels, RBC deformability, and morphology during RBC storage. A pool-and-split design of leukoreduced CPD-saline-adenine-glucose-mannitol-packed RBC units was used to generate three groups: untreated controls, sham-treated units, and units treated with a cold (1-6 °C) rejuvenation solution on Day 28, 35, or 42 of cold storage. Units were followed until Day 49 of storage and assessed for ATP concentration, morphology, deformability, and other in vitro quality variables including hemolysis, pH, and supernatant potassium levels. At every treatment time, rejuvenation was associated with a significant increase in intracellular ATP (p < 0.01). On Day 28, rejuvenation was accompanied by a significant decrease in deformability 1 week after treatment (p < 0.01). Rejuvenation on Day 28, but not Day 35 or 42, was also associated with a significant change in morphology (p < 0.01). Of the in vitro quality variables measured, most changed during cold storage, but differences among treatment groups were not observed. The results of our study demonstrate a cold rejuvenation of RBCs during HS increases intracellular ATP, but that this change does not ameliorate, or exacerbate, the metabolic or biochemical symptoms of the storage lesion.

Abstract 1703: Extracellular ATP mediated intracellular ATP increase: Evidence for transport of extracellular ATP into cancer cells.

Abstract ATP as a most common cofactor of enzymatic reactions and a universal source of bioenergy transfer plays central metabolic and signal transduction roles in normal and cancer cells. Almost all cancer cells switch from the highly efficient mitochondrial oxidative phosphorylation to low efficiency glycolysis for ATP synthesis, a phenomenon called the Warburg effect that remains to be fully understood. Despite the low efficiency glycolysis, cancer cells appear to have no shortage of ATP under any circumstance examined. Different from cell culture media, high concentrations of extracellular ATP is present in the blood and in cancer. Although ample evidence indicates intratumoral (extracellular) ATP concentration is higher than other parts of the body, it is presently unclear about the sources and fates of intratumoral/extracellular ATP and how they affect intracellular ATP. ATP is not known to cross plasma membrane and there is no known plasma membrane associated ATP transporter. We previously reported drastic increases of intracellular ATP concentration and rescue of glucose deprived A549 human lung cancer cells by addition of extracellular ATP.1 We hypothesized that the increased intracellular ATP is a result of extracellular ATP taken up by cancer cells. To test this hypothesis, we studied extracellular ATP-induced intracellular ATP increase in A549 lung and other cancer and non-tumorigenic cells using the ATP assay. Here we report (i) Extracellular ATP increased intracellular ATP of A549 cells in a dose- and time-dependent manners. (ii) The ATP increase was also observed even when cells’ mitochondrial ATP synthesis and/or glycolysis were blocked. (iii) Extracellular ATP at 3 mM also induced a sharp and temporary ATP surge (peak) several hours after the inhibition described in (ii) and the surge was absent in the nontumorigenic cell lines under the same conditions. (iv) The timing of the surge appeared to be dependent upon extracellular ATP concentration. (v) Extracellular ATP led to changes in energy status sensing protein AMPK and its phosphorylation. All these results indicate that extracellular ATP exerts profound effects on intracellular ATP and suggest a possible direct ATP transport mechanism into cancer cells. These results strongly suggest that cancer cells supplement their ATP need with extracellular ATP and extracellular ATP may play some vitally important but previously unrecognized roles in the Warburg effect and tumorigenesis. These new findings challenge the current view that extracellular ATP functions only through plasma membrane receptors and force us to rethink the roles of extracellular ATP played in cancer metabolism, energy homeostasis, as well as the symbiotic relationship between cancer cells and between cancer and normal / stromal cells. They also imply a new strategy for cancer treatment. 1.Liu, Y., et al., Mol. Cancer Ther. 2012, 11:1672-1682. Citation Format: Yanrong Qian, Yi Liu, Xuan Wang, Xiaozhuo Chen. Extracellular ATP mediated intracellular ATP increase: Evidence for transport of extracellular ATP into cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1703. doi:10.1158/1538-7445.AM2013-1703

The P2Y2/Src/p38/COX-2 pathway is involved in the resistance to ursolic acid-induced apoptosis in colorectal and prostate cancer cells

One of the hallmarks of cancer is resistance to apoptosis. Elucidating the mechanisms of how cancer cells evade or delay apoptosis should lead to novel therapeutic strategies. Previously, we showed that HT-29 colorectal cancer cells undergoing apoptosis overexpressed cyclooxygenase-2 (COX-2), in a p38 dependent pathway, to delay ursolic acid-induced apoptosis. Here, we focused on elucidating the upstream signaling pathways regulating this resistance mechanism. The role of ATP as an extracellular signaling molecule took a long time to be accepted. In recent years, ATP and its analogs, via the activation of specific purinergic receptors, have been implicated in many biological processes including cell proliferation, differentiation and apoptosis. In the present report, we have demonstrated a novel role involving purinergic receptors and particularly the P2Y(2) receptor in resistance to ursolic acid-induced apoptosis in both colorectal HT-29 and prostate DU145 cancer cells. We found that ursolic acid induced an increase in intracellular ATP and P2Y(2) transcript levels. Upon activation, P2Y(2) activated Src which in turn phosphorylated p38 leading to COX-2 overexpression which induced resistance to apoptosis in both HT-29 and DU145 cells. Furthermore, Ca(2+)-independent PLA(2) (iPLA(2)) and Ca(2+)-dependent secretory PLA(2) (sPLA(2)) were responsible for arachidonic acid release, the substrate of COX-2. Our findings document that apoptosis triggering was dependent on protein kinase C (PKC) activation in both cell lines after ursolic acid treatment.

An ATP signalling pathway in plant cells: extracellular ATP triggers programmed cell death in Populus euphratica

We elucidated the extracellular ATP (eATP) signalling cascade active in programmed cell death (PCD) using cell cultures of Populus euphratica. Millimolar amounts of eATP induced a dose- and time-dependent reduction in viability, and the agonist-treated cells displayed hallmark features of PCD. eATP caused an elevation of cytosolic Ca(2+) levels, resulting in Ca(2+) uptake by the mitochondria and subsequent H(2) O(2) accumulation. P. euphratica exhibited an increased mitochondrial transmembrane potential, and cytochrome c was released without opening of the permeability transition pore over the period of ATP stimulation. Moreover, the eATP-induced increase of intracellular ATP, essential for the activation of caspase-like proteases and subsequent PCD, was found to be related to increased mitochondrial transmembrane potential. NO is implicated as a downstream component of the cytosolic Ca(2+) concentration but plays a negligible role in eATP-stimulated cell death. We speculate that ATP binds purinoceptors in the plasma membrane, leading to the induction of downstream intermediate signals, as the proposed sequence of events in PCD signalling was terminated by the animal P2 receptor antagonist suramin.

Prediction of Lympho-Hematopoietic Regulatory Responses to Different Agents Using a 7-Population In Vitro Proliferation Assay.

The organizational hierarchy and the different population components of the lympho-hematopoietic system have been developed over five decades by studying the response of the system to a variety of perturbations from different sources, including radiation and chemical and drug administration. Indeed, the stem cell compartment hierarchy as we know it today would not have been possible without such studies. Many of these have utilized the in vitro functional ability of the cells to differentiate into colonies of cells in semi-solid medium using the colony-forming assay. However, without initial proliferation of the cells, differentiation would not occur. Therefore, the colony-forming assay implies proliferation, but does not directly measure it. In this communication we describe the proliferative response of 7-lympho-hematopoietic in vitro populations measured directly and simultaneously in mouse and human bone marrow to a number of different compounds, including chemicals, antibiotics, anti-inflammatory and anti-cancer drugs. The majority of these agents are part of 347 compounds listed in the Registry of Cytotoxicity (RC) for which the IC50 values (detected by neutral red uptake in mouse 3T3 and normal human keritinocyte cells) and LD50 (oral rat or mouse values) are known. Each of these compounds were tested over a dose range from 1pM to 10μM on 2 stem cell (HPP-SP and CFC-GEMM), 3 hematopoietic (BFU-E, GM-CFC and MK-CFC) and 2 lymphopoietic (T-CFC and B-CFC) populations simultaneously. Combinations of growth factors used routinely in the colony-forming assay were used to induce proliferation in the absence (controls) or presence of the test compounds. Each compound dose response was tested on a single human or mouse bone marrow population by suspending the cells in serum components, methyl cellulose and the respective growth factors and dispensing 8 replicates of 100μl for each dose into a 96-well plate. During the 5 (mouse) or 7 day (human) incubation period after which proliferation, with little or no differentiation, is measured, there is an increase in intracellular ATP (iATP) concentrations that is directly proportional to the proliferative status of the cells. Intracellular ATP is then released after lysing the cells and drives a luciferin/luciferase reaction to produce luminescence that is detected in a plate luminometer. The results indicate a variety of responses from no effect on any of the populations over the dose range measured to toxicity for every population with IC50 values in the pM range. From these results and the form of the dose response curves produced, a 5-point prediction model has been established that, when used in conjunction with the 7-population assay, can describe the global response of the lympho-hematopoietic system to virtually any compound. For cadmium chloride, ibuprofen, propanolol HCl, sodium dichromate, 5-fluorouracil, busulfan, chloramphenicol, doxorubicin and donaurubicin, the IC50 values were plotted against the LD50 values provide in the RC list. A positive correlation coefficient of R=0.91 with a probability (P) of <0.001 was obtained. The strong correlation between the 7-population assay and the RC provides validation for the assay and even allows it to be used to predict initial dosing for animal experiments by converting IC50 to in vivo values. In conclusion, a rapid, non-subjective and powerful tool is described that can provide response, regulatory and predictive information on the proliferative status of the lympho-hematopoietic system.