Inhibition of cancer growth and selective glutathione depletion in Ehrlich tumour cells <i>in vivo</i> by extracellular ATP

Vega, Maribel; Terradez, P; Obrador, Elena; Navarro, José; Pellicer, José A.; Estrela, José M.

doi:10.1042/bj2980099

Cited by 37 publications

(26 citation statements)

References 36 publications

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“…We assume that the changes in intracellular nucleotide levels observed after incubation with ADP might have been, in fact, related to adenosine, originating from hydrolysis of ADP. This hypothesis is supported by published data, 45,46 showing that inhibitor of adenosine transporter, dipyridamole, entirely blocks changes in the intracellular nucleotide levels in response to extracellular nucleotides.…”

Section: Discussionsupporting

confidence: 79%

“…Such increase in the concentration of intracellular ATP was observed earlier in cells treated with extracellular nucleotides or adenosine. 45,46 Therefore, the ratio of AMP to ATP did not change significantly, in contrast to other studies showing 6-or 25-fold increases in the AMP:ATP ratio. 47,48 We propose that this increasing AMP level may affect AMPK activation and that the actual level of AMP is more important for activation of da Silva et al AMPK Activation by Nucleotides and Adenosine e45 AMPK than the AMP:ATP ratio.…”

Section: Discussioncontrasting

confidence: 48%

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Extracellular Nucleotides and Adenosine Independently Activate AMP-Activated Protein Kinase in Endothelial Cells

Silva¹,

Jarzyna²,

Specht³

et al. 2006

Circulation Research

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Abstract-AMP-activated protein kinase (AMPK) plays a key role in the regulation of energy homeostasis and is activated in response to cellular stress, including hypoxia/ischemia and hyperglycemia. The stress events are accompanied by rapid release of extracellular nucleotides from damaged tissues or activated endothelial cells (EC) and platelets. We demonstrate that extracellular nucleotides (ATP, ADP, and UTP, but not UDP) and adenosine independently induce phosphorylation and activation of AMPK in human umbilical vein EC (HUVEC) by the mechanism that is not linked to changes in AMP:ATP ratio. HUVEC express NTPDases, as well as 5Ј-nucleotidase; hence, nucleotides can be metabolized to adenosine. However, inhibition of 5Ј-nucleotidase had no effect on ATP/ADP/UTP-induced phosphorylation of AMPK, indicating that AMPK activation occurred as a direct response to nucleotides. Nucleotide-evoked phosphorylation of AMPK in HUVEC was mediated by P2Y1, P2Y2, and/or P2Y4 receptors, whereas P2Y6, P2Y11, and P2X receptors were not involved. The nucleotide-induced phosphorylation of AMPK was affected by changes in the concentration of intracellular Ca 2ϩ and by Ca 2ϩ /calmodulin-dependent kinase kinase (CaMKK), although most likely it was not dependent on LKB1 kinase. Adenosine-induced phosphorylation of AMPK was not mediated by P1 receptors but required adenosine uptake by equilibrative nucleoside transporters followed by its (intracellular) metabolism to AMP. Moreover, adenosine effect was Ca 2ϩ and CaMKK independent, although probably associated with upstream LKB1. We hypothesize that P2 receptors and adenosine transporters could be novel targets for the pharmacological regulation of AMPK activity and its downstream effects on EC function. is an evolutionarily conserved enzyme that acts as an ultrasensitive energy charge sensor and regulates cell energy metabolism. 1 AMPK is a heterotrimeric Ser/Thr kinase consisting of a catalytic ␣ subunit and regulatory ␤ and ␥ subunits. AMPK is activated in response to ATP depletion associated with an increase in the intracellular AMP:ATP ratio. This ATP consumption can originate from pathological cellular stress, such as heat shock, hypoxia, or ischemia, and from physiological exerciseinduced skeletal muscle contraction. 2 AMPK can also be phosphorylated and activated by the mechanism independent of changes in AMP:ATP levels. 3 AMPK is activated allosterically by AMP (up to 5-fold) and by phosphorylation of Thr172 on the ␣ subunit (50-to 100-fold), which is catalyzed by upstream kinases, including LKB1 and CaMKK. 4,5 Activated AMPK turns on catabolic pathways that generate ATP and turns off pathways that consume ATP by phosphorylation of multiple targets. These include glycogen synthase, acetylcoenzyme A carboxylase (ACC), and hydroxymethylglutarylcoenzyme A (HMG-CoA) reductase. Because AMPK is central in controlling the metabolism of glucose and fatty acids, its role in obesity and type 2 diabetes is of major importance. AMPK is expressed in skeletal muscle, brain, liver, and pancre...

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Section: Discussionsupporting

confidence: 79%

Section: Discussioncontrasting

confidence: 48%

Extracellular Nucleotides and Adenosine Independently Activate AMP-Activated Protein Kinase in Endothelial Cells

Silva¹,

Jarzyna²,

Specht³

et al. 2006

Circulation Research

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“…7 ␥-Glutamylcysteine synthetase (␥-GCS) and GSH synthetase (GSH-S) activities were measured as described elsewhere. 30 Expression of Results and Statistical Analyses. Data are presented as mean Ϯ SD.…”

Section: Methodsmentioning

confidence: 99%

γ-Glutamyl transpeptidase overexpression increases metastatic growth of B16 melanoma cells in the mouse liver

et al. 2002

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B16 melanoma (B16M) cells with high glutathione (GSH) content show rapid proliferation in vitro and high metastatic activity in the liver in vivo. ␥-Glutamyl transpeptidase (GGT)-mediated extracellular GSH cleavage and intracellular GSH synthesis were studied in vitro in B16M cells with high (F10) and low (F1) metastatic potential. GGT activity was modified by transfection with the human GGT gene (B16MF1/Tet-GGT cells) or by acivicin-induced inhibition. B16MF1/Tet-GGT and B16MF10 cells exhibited higher GSH content (35 ؎ 6 and 40 ؎ 5 nmol/10 6 cells, respectively) and GGT activity (89 ؎ 9 and 37 ؎ 7 mU/10 6 cells, respectively) as compared (P < .05) with B16MF1 cells (10 ؎ 3 nmol GSH and 4 mU GGT/10 6 cells). Metastasis (number of foci/100 mm 3 of liver) increased in B16MF1 cells pretreated with GSH ester (ϳ3-fold, P < .01), and decreased in B16MF1/Tet-GGT and B16MF10 cells pretreated with the GSH synthesis inhibitor L-buthionine (S,R)-sulphoximine (ϳ5-fold and 2-fold, respectively, P < .01). Liver, kidney, brain, lung, and erythrocyte GSH content in B16MF1/Tet-GGT-or B16MF10-bearing mice decreased as compared with B16MF1-and non-tumor-bearing mice. Organic anion transporting polypeptide 1-independent sinusoidal GSH efflux from hepatocytes increased in B16MF1/Tet-GGT-or B16MF10-bearing mice (ϳ2-fold, P < . ␥ -Glutamyl-cysteinyl-glycine (GSH) is involved in many cellular functions, e.g., bioreductive reactions, maintenance of enzyme activity, amino acid transport, protection against oxidative/nitrosative stress, and detoxification of xenobiotics. 1 Induction of GSH deficiency in tumors was shown to be potentially useful in cancer therapy. 2 Elevation of intracellular GSH levels is associated with mitogenic stimulation 3 and regulation of DNA synthesis. 4 We found that GSH controls the onset of tumor-cell proliferation by regulating protein kinase C activity and intracellular pH, 5 and that a direct increase of intracellular GSH levels promotes survival and metastatic growth of B16M cells in the liver. 6,7 In rapidly growing tumors, cyst(e)ine, whose concentration in blood is low, may become limiting for GSH synthesis and cell growth. 8,9 Thus, malignant cells might require alternative pathways to ensure free cyst(e)ine availability. Recently, as it was shown in 3-methylcholantrene-induced sarcomas growing in rats, blood GSH can be used by solid tumors. 10 ␥-glutamyl transpeptidase (GGT) is the only known enzyme that cleaves the ␥-glutamyl-cysteine peptide bond in GSH and other ␥-glutamyl compounds. 11 GGT contains a heavy subunit (41.6 kd) that anchors the enzyme to the plasma membrane, and a light subunit (19.7 kd) orientated towards the outer surface of the cell membrane, which contains the catalytic site. 12 GGT cleaves GSH releasing ␥-glutamyl amino acids and cysteinylglycine, which is further cleaved by membrane-bound dipeptidases into cysteine and glycine. 13 Free ␥-glutamyl-amino acids, cysteine, and glycine entering the cell serve as GSH precursors. 8 Hence GGT expression may provide tumor cells with a gro...

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“…It is known that inhibition of GR can lead to the depletion of GSH (Kassahun et al, 1994). Depletion of intracellular GSH has been demonstrated to be an effective approach in decreasing cellular proliferation rates and cancer cell growth (Terradez et al, 1993;Lasso de la Vega et al, 1994;Estrela et al, 1995). Recently, GSH depletion has been shown to effectively cause apoptosis in prostate carcinoma cells (Coffey et al, 2000).…”

Section: Discussionmentioning

confidence: 99%

Glutathione and Mercapturic Acid Conjugates of Sulofenur and Their Activity against a Human Colon Cancer Cell Line

Guan¹,

Hoffman²,

McFarland³

et al. 2002

Drug Metab Dispos

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ABSTRACT:Sulofenur is one of the diarylsulfonylureas developed as an anticancer agent. Sulofenur possesses a broad spectrum of activity in several solid tumor models and has undergone extensive clinical trials based on its impressive preclinical activity. However, the clinical response of sulofenur has been disappointing because of the side effect of anemia. Furthermore, the anticancer mechanism of sulofenur and its diarylsulfonylurea analogs still remains unknown. Elucidation of the metabolic fates of sulofenur may help to delineate the mechanism and provide information to guide the structural modification for more potent anticancer agents with less side effects. We have identified a glutathione conjugate and a mercapturic acid conjugate from sulofenur-dosed rats with the aid of liquid chromatography/mass spectrometry. The fraction of the dose of sulofenur as the glutathione conjugate in the dosed-rat bile over 5 h was 0.12 ؎ 0.03%, and the mercapturic acid conjugate in urine over 24 h was 1.4 ؎ 0.7%. Protein binding of the glutathione conjugate and mercapturic acid conjugate was determined to be 20 ؎ 3 and 84 ؎ 2%, respectively, as opposed to >99% of sulofenur. The high protein binding of sulofenur requires a higher than in vitro dose, which is believed to cause the side effect of anemia. The significance of this metabolic pathway is that both conjugates were found to be glutathione reductase inhibitors and to possess anticancer activity comparable to sulofenur against human colon adenocarcinoma GC 3 /c1 cells, a sulofenur-sensitive cell line. These conjugates may serve as new leads for the development of novel anticancer agents.

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Inhibition of cancer growth and selective glutathione depletion in Ehrlich tumour cells in vivo by extracellular ATP

Cited by 37 publications

References 36 publications

Extracellular Nucleotides and Adenosine Independently Activate AMP-Activated Protein Kinase in Endothelial Cells

Extracellular Nucleotides and Adenosine Independently Activate AMP-Activated Protein Kinase in Endothelial Cells

γ-Glutamyl transpeptidase overexpression increases metastatic growth of B16 melanoma cells in the mouse liver

Glutathione and Mercapturic Acid Conjugates of Sulofenur and Their Activity against a Human Colon Cancer Cell Line

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