Tannic acid inhibits in vitro iron-dependent free radical formation

The antioxidant activity of tannic acid (TA), a plant polyphenol claimed to possess antimutagenic and anticarcinogenic activities, was studied by monitoring (i) 2-deoxyribose degradation (a technique for OH detection), (ii) ascorbate oxidation, (iii) ascorbate radical formation (determined by EPR analysis) and (iv) oxygen uptake induced by the system, which comprised Fe(III) complexes (EDTA, nitrilotriacetic acid (NTA) or citrate as co-chelators), ascorbate and oxygen. TA removes Fe(III) from the co-chelators (in the case of EDTA, this removal is slower than with NTA or citrate), forming an iron-TA complex less capable of oxidizing ascorbate into ascorbate radical or mediating 2-deoxyribose degradation. The effectiveness of TA against 2-deoxyribose degradation, ascorbate oxidation and ascorbate radical formation was substantially higher in the presence of iron-NTA (or iron-citrate) than with iron-EDTA, which is consistent with the known formation constants of the iron complexes with the co-chelators. Oxygen uptake and 2-deoxyribose degradation induced by Fe(II) autoxidation were also inhibited by TA. These results indicate that TA inhibits OH formation induced by Fe(III)/ascorbate/O(2) mainly by arresting Fe(III)-induced ascorbate oxidation and Fe(II) autoxidation (which generates Fe(II) and H(2)O(2), respectively), thus limiting the production of Fenton reagents and OH formation. We also hypothesize that the Fe(II) complex with TA exhibits an OH trapping activity, which explains the effect of TA on the Fenton reaction.     

Enhanced susceptibility of erythrocytes deficient in glucose-6-phosphate dehydrogenase to alloxan/glutathione-induced decrease in red cell deformability

It has been hypothesized that enhanced oxidant sensitivity of glucose-6-phosphate dehydrogenase (G6PD) deficient red cells(RBCs) is the underlying mechanism for drug- or chemical-induced hemolytic crises in G6PD-deficiency. To further test this hypothesis, we used an alloxanglutathione system to mimic oxidative stress and see how oxidative damage might affect RBC deformability. RBC deformability, a major determinant of RBC survival in vivo, was monitored by a laser viscodiffractometer. Under our experimental conditions, GSH alone had very little effect on the deformability of either normal or G6PD-deficient RBCs. In contrast, alloxan alone induced a small but significant decrease in the deformability of either normal or G6PD-deficient RBCs. Interestingly, alloxan and GSH together induced a further decrease in the deformability of either normal or G6PD-deficient RBCs. The decrease in deformability in G6PD-deficient RBCs was much more profound than in normal RBCs. In addition, an alloxan-vitamin C system produced a similar deleterious effect on RBC deformability as that produced by the alloxan-GSH system. Appreciable amount of hydroxyl radicals was generated by both alloxan-GSH and alloxan-vitamin C systems as evidenced by the production of hydroxylated products of salicylate which was used as a radical trap. Moreover, salicylate could ameliorate the deleterious effect of the alloxan system on the deformability of RBCs. Taken together, our results demonstrated that G6PD-deficient RBCs were particularly susceptible to oxidant-induced damage leading to a dramatic decrease in their deformability and thus provided strong support for the hypothesis that enhanced oxidant sensitivity of G6PD-deficient RBCs is the underlying mechanism for accelerated destruction of these RBCs in vivo.     

Oxidative damage to human red cells induced by copper and iron complexes in the presence of ascorbate

The role of trace metals in the generation of free radical mediated oxidative stress in normal human red cells was studied. Ascorbate and either soluble complexes of Cu(II) or Fe(III) provoked changes in red cell morphology, alteration in the polypeptide pattern of membrane proteins, and significant increases in methemoglobin. Neither ascorbate nor the metal complexes alone caused significant changes to the cells. The rate of methemoglobin formation was a function of ascorbate and metal concentrations, and the chemical nature of the chelate. Cu(II) was about 10-times more effective than Fe(III) in the formation of methemoglobin. Several metals were tested for their ability to compete with Cu(II) and Fe(III). Only zinc caused a significant inhibition of methemoglobin formation by Fe(III)-fructose. These observations suggest that site-specific as well as general free radical damage is induced by redox metals when the metals are either bound to membrane proteins or to macromolecules in the cytoplasm. The Cu(II) and Fe(III) function in two catalytic capacities: (1) oxidation of ascorbate by O2 to yield H2O2, and (2) generation of hydroxyl radicals from H2O2 in a Fenton reaction. These mechanisms are different from the known damage to red cells caused by the binding of Fe(III) or Cu(II) to the thiol groups of glucose-6-phosphate dehydrogenase. Our system may be a useful model for understanding the mechanisms for oxidative damage associated with thalassemia and other congenital hemolytic anemias.     

A new glucose 6-phosphate dehydrogenase variant (G6PD Thessaloniki) in a patient with idiopathic myelofibrosis

A new deficient glucose 6-phosphate dehydrogenase (G6PD) variant, G6PD Thessaloniki, which was found in the red blood cells of a 70-year-old woman who had idiopathic myelofibrosis, is described. G6PD Thessaloniki had a low Michaelis constant (Km) for G6P (20 microM), high Km for NADP (10.1 microM), normal pH optimum, reduced heat stability, decreased electrophoretic mobility (96-98% of the normal), increased 2-deoxy-G6P and decreased galactose 6-phosphate utilization. Several other enzymatic activities measured in the patient's red blood cells were normal. Studies of red blood cell survival and glucose utilization gave evidence of haemolysis caused by defective glucose utilization by the pentose phosphate pathway. The only son of the patient had normal G6PD in his red blood cells. In an attempt to investigate the origin of G6PD Thessaloniki, heat stability tests of G6PD extracted from the patient's skin have been performed.     

G6PD Vientiane: A new glucose-6-phosphate dehydrogenase variant with increased stability

Summary A new G6PD variant, called G6PD Vientiane, has been discovered in a patient from Laos.The characteristics of this variant are: mild enzyme deficiency (about 50% of the normal activity) in the granulocytes and the red cells, with normal G6PD-related antigen concentration; increased stability; normal Km glucose 6-phosphate and NADP⁺; increased inhibition constant by NADPH; decreased inhibition by ATP; slightly increased utilization of the substrate analogue; abnormal pH curve, with maximum activity at pH 9.5; slightly reduced starch gel electrophoretic migration. The implications of the molecular stability of a deficient mutant variant are discussed.     

Membrane thiol-disulfide status in glucose-6-phosphate dehydrogenase deficient red cells. Relationship to cellular glutathione

The behavior of glucose-6-phosphate dehydrogenase (G6PD)-deficient red cell membrane proteins upon treatment with diamide, the thiol-oxidizing agent (Kosower, N.S. et al. (1969) Biochem. Biophys. Res. Commun. 37, 593–596), was studied with the aid of monobromobimane, a fluorescent labeling agent (Kosower, N.S., Kosower, E.M., Newton, G.L. and Ranney, H.M. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 3382–3386) convenient for following membrane thiol group status. In diamide-treated G6PD-deficient red cells (and in glucose deprived normal cells), glutathione (GSH) is oxidized to glutathione disulfide (GSSG). When cellular GSH is absent, membrane protein thiols are oxidized with the formation of intrachain and interchain disulfides. Differences in sensitivity to oxidation are found among membrane thiols. In diamidetreated normal red cells, GSH is regenerated in the presence of glucose and membrane disulfides reduced. In G6PD-deficient cells, GSSG is not reduced, and the oxidative damage (disulfide formation) in the membrane not repaired. Reduction of membrane disulfides does occur after the addition of GSH to these membranes. A direct link between the thiol status of the cell membrane and cellular GSH is thereby established. GSH serves as a reductant of membrane protein disulfides, in addition to averting membrane thiol oxidation.     

Deleterious synergistic effects of ascorbate and copper on the development of Plasmodium falciparum: an in vitro study in normal and in G6PD-deficient erythrocytes

The effects of ascorbate and copper on the development of Plasmodium falciparum were studied in two modes: pretreatment of uninfected erythrocytes followed by infection by P. falciparum and treatment of parasitized erythrocytes. Pretreatment of G6PD(+) cells with ascorbate caused a slight enhancement in parasite development, while in G6PD(-) cells a suppressive effect on the plasmodia was demonstrated. Copper alone interfered with parasite growth in both cell types. The combination of copper and ascorbate arrested parasite maturation, an effect which was more pronounced in G6PD(-) cells. Synergism between copper and ascorbate was better demonstrated following the treatment of infected erythrocytes: while ascorbate alone supported parasite development and copper alone had only a marginal suppressive effect, the combination of copper and ascorbate yielded a marked inhibition of parasite growth. Ascorbate proved destructive to the parasites in the presence of adventitious copper, or on the second day of the parasite life cycle. In these cases it acted as a pro-oxidant, while in other systems, in particular in the presence of a chelator, ascorbate acted as an antioxidant and promoted parasite growth. The understanding of the role of transition metals and free radicals in parasite development and injury could shed light on novel approaches to fight malaria.     

Effective NET formation in neutrophils from individuals with G6PD Taiwan-Hakka is associated with enhanced NADP+ biosynthesis

Abstract

In response to infection, neutrophils employ various strategies to defend against the invading microbes. One of such defense mechanisms is the formation of neutrophil extracellular traps (NETs). Recent studies suggest that reactive oxygen species is a signal critical to NET formation. This prompts us to examine whether neutrophils from individuals with glucose-6-phosphate dehydrogenase (G6PD) Taiwan-Hakka variant, which are prone to oxidative stress generation, have altered ability to form NET. We adopted an image-based method to study the NET formation potential in neutrophils from G6PD-deficient patients. Neutrophils from either normal or G6PD-deficient individuals underwent NETosis in response to phorbol 12-myristate 13-acetate (PMA). The extent of NETosis in the former did not significantly differ from that of the latter. Diphenyleneiodonium sulfate (DPI) and 3-methyladenine (MA) inhibited PMA-stimulated NET formation in these cells, suggesting the involvement of NADPH oxidase and autophagy in the process. Glucose oxidase (GO) and xanthine oxidase/xanthine (XO/X) could induce a similar extent of NET formation in normal and G6PD-deficient neutrophils. GO- or XO-induced NETosis was not inhibitable by MA, implying that reactive oxygen species (ROS) can act as an independent signal for activation of NETosis. Mechanistically, enhanced superoxide production in neutrophils was associated with increases in levels of NAD⁺ and NADP⁺, as well as activation of NAD⁺ kinase. Taken together, these findings suggest that G6PD-deficient neutrophils are as equally efficient as normal cells in NET formation, and their deficiency in G6PD-associated NADPH regeneration capacity is largely compensated for by nicotinamide nucleotide biosynthesis.     

In vitro antioxidant properties of the iron chelator pyridoxal isonicotinoyl hydrazone and some of its analogs

Since there are several problems with desferrioxamine (DFO) therapy, pyridoxal isonicotinoyl hydrazone (PIH) has been studied for more than 10 years as a promising new candidate for iron chelation therapy in iron-overload diseases. Iron chelation could also be helpful for experimental treatment of several other pathologies including rheumatoid arthritis and heart ischemia/reperfusion, due to the generation of oxyradicals and lipid peroxidation mediated by delocalized iron. We demonstrate here that sub-millimolar levels of PIH can inhibit the Fe(III)-EDTA/ascorbate-mediated formation of hydroxyl-like radicals as tested by the release of ethylene from 2-keto-4-methylthiobutyric acid (KMB assay) and the formation of malonaldehyde from 2-deoxyribose damage. PIH could also decrease the rates of Fe(III)-EDTA-mediated oxidation of ascorbate and block the peroxidation of liposomes of rat brain phospholipids induced by ferrous iron-EDTA. In all cases the in vitro antioxidant effectiveness of PIH was comparable to its analogs—including salicylaldehyde isonicotinoyl hydrazone—and to DFO. We conclude that PIH and its analogs are effective new candidates against iron-mediated oxidative stress for use in experimental medicine.     

Phytochrome Action in Oryza sativa L: IV. Red and Far Red Reversible Effect on the Production of Ethylene in Excised Coleoptiles

Excised apical segments of etiolated rice (Oryza sativa L.) coleoptiles produced ethylene. Increasing the number of cut sites per coleoptile increased the rate of ethylene formation. Ethylene produced by an etiolated-intact seedling in the dark was about a half of that by the excised coleoptile segment. Red light of low energy as well as of continuous irradiation inhibited the production of ethylene. The inhibition by a low energy dose of red light was partly relieved, if the red light was followed immediately by a small dose of far red light. The effect of red and far red light was repeatedly reversible, indicating that ethylene production was regulated by a phytochrome system. If the exposure to far red light was preceded by a period of darkness, this photoreversibility disappeared; 50% of the initial reversibility was lost within 5 hours. Applied ethylene (10 microliters per liter) significantly promoted the growth of intact coleoptiles of either totally etiolated or red light-treated seedlings, but had no effect on the excised apical segment of coleoptile.     

Altered aggregational properties in a genetic variant of human glucose 6-phosphate dehydrogenase

The Tel Hashomer variant of human G6PD migrates as two prominent components during electrophoresis in several gel systems in which red cell G6PD from other males migrates predominantly as a single band. Since human males normally have but one X-chromosome, the extra band of this variant seemed an exception to earlier biochemical and genetic observations suggesting that human red cell G6PD is determined by a locus on the X chromosome. Results of the present studies indicate that the Tel Hashomer variant is unusually susceptible to the formation of a complex which has a higher molecular weight than normal G6PD and which represents the slow electrophoretic component. The conditions of formation and disruption of this complex in crude and purified Tel Hashomer preparations suggest that it results from the formation of disulfide bridges between molecules of Tel Hashomer G6PD.Supported by U.S. Public Health Service Research Grants AM-11065 and FR-5406 and Research Career Development Award 5 K3 AM 7992.     

G6PD lozere and trinacria-like

Summary Two new G6PD variants have been found in red blood cells of the members of a French family originating from Lozere. The father is hemizygous for an electrophoretically fast variant with mild enzyme deficiency (50–60% of normal). The abnormal paternal G6PD gene is segregating in his daughter who is double heterozygous for maternal and paternal variants. This mutant enzyme, different from previously described variants is designated as Gd Lozère. The mother is heterozygous for another G6PD variant. Two sons are hemizygous for this latter mutant enzyme characterized by a moderate deficiency (25–30% of normal) and slower electrophoretic mobility with some slightly altered kinetic properties. This G6PD has been identified as Gd Trinacria like.These two abnormal enzymes are not associated with any hemolytic problem. Case reported is the first showing the segregation of two new mutant enzymes, distinct from common G6PD variants, among the members of the same family.     

Effect of iron and manganese on hydroxyl radical production by 6-hydroxydopamine: mediation of antioxidants

6-Hydroxydopamine (6-OHDA) neurotoxicity has often been related to the generation of free radicals. Here we examined the effect of the presence of iron (Fe(2+) and Fe(3+)) and manganese and the mediation of ascorbate, L-cysteine (CySH), glutathione (GSH), and N-acetyl-CySH on hydroxyl radical (*OH) production during 6-OHDA autoxidation. In vitro, the presence of 800 nM iron increased (> 100%) the production of *OH by 5 microM 6-OHDA while Mn(2+) caused a significant reduction (72%). The presence of ascorbate (100 microM) induced a continuous generation of *OH while the presence of sulfhydryl reductants (100 microM) limited this production to the first minutes of the reaction. In general, the combined action of metal + antioxidant increased the *OH production, this effect being particularly significant (> 400%) with iron + ascorbate. In vivo, tyrosine hydroxylase immunohistochemistry revealed that intrastriatal injections of rats with 6-OHDA (30 nmol) + ascorbate (600 nmol), 6-OHDA + ascorbate + Fe(2+) (5 nmol), and 6-OHDA + ascorbate + Mn(2+) (5 nmol) caused large striatal lesions, which were markedly reduced (60%) by the substitution of ascorbate by CySH. Injections of Fe(2+) or Mn(2+) alone showed no significant difference to those of saline. These results clearly demonstrate the role of ascorbate as an essential element for the neurotoxicity of 6-OHDA, as well as the diminishing action of sulfhydryl reductants, and the negligible effect of iron and manganese on 6-OHDA neurotoxicity.     

Photooxidative reactions in chloroplast thylakoids. Evidence for a Fenton-type reaction promoted by superoxide or ascorbate

A methyl viologen (MV)^* mediated Mehler reaction was studied using Type C and D chloroplasts (thylakoids) from spinach. The extent of photooxidative reactions were measured as (a) rate of ethylene formation from methional oxidation indicating the production of oxygen radicals, and (b) rate of malondialdehyde (MDA) formation as a measure of lipid peroxidation. Without added ascorbate, 1 M FerricEDTA increased ethylene formation by greater than 4-fold, but had no effect on MDA production. Ascorbate (1 mM) produced a tripling of ethylene while it reduced MDA formation in the presence of iron. Radical scavengers diethyldithiocarbamate (DDTC), formate, 1,4-diazabicyclo (2.2.2octane) (DABCO), inhibited ethylene formation. Using 0,4 M mannitol to scavenge hydroxyl radicals, the rates of ethylene formation were reduced 40 to 60% with or without 1 M Fe(III) EDTA. The strong oxidant(s) not scavenged by mannitol are hypothesized to be either alkoxyl radicals from lipid peroxidation, or site specific formation of hydroxyl radicals in a lipophillic environment not exposed to mannitol. Singlet oxygen does not appear to be a significant factor in this system. Catalase strongly inhibited both ethylene and MDA synthesis under all conditions; 1 mM ascorbate did not reverse this inhibition. However, the strong superoxide dismutase (SOD) inhibition of ethylene and MDA formation was completely reversed by 1 mM ascorbate. This suggests that superoxide was functioning as an iron reducing agent and that in its absence, ascorbate was similarly promoting oxidations. Therefore, these oxidative processes were dependent on the presence of H₂O₂ and a reducing agent, suggesting the involvement of a Fenton-type reaction.Abbreviation DABCO 1,4-diazabicyclo(2.2.2.octane) - DCMU 3-(3,4 Dichlorophenyl). 1,1-dimethyl urea - DDTC diethyldithiocarbamate - EDTA ethylenediamine-tetraacetic acid - MDA malondialdehyde - MV methyl viologen - SOD superoxide dismutase - TBA thiobarbituric acid - TCA trichloroacetic acid Scientific contribution number 1315 from the New Hampshire Agriculture Experiment Station.     

Protective effect of serotonin on 6-hydroxydopamine- and dopamine-induced oxidative damage of brain mitochondria and synaptosomes and PC12 cells

The present study elucidated the effects of indoleamines (serotonin, melatonin, and tryptophan) on oxidative damage of brain mitochondria and synaptosomes induced either by 6-hydroxydopamine (6-OHDA) or by iron plus ascorbate and on viability loss in dopamine-treated PC12 cells. Serotonin (1-100 microM), melatonin (100 microM), and antioxidant enzymes attenuated the effects of 6-OHDA, iron plus ascorbate, or 1-methyl-4-phenylpyridinium on mitochondrial swelling and membrane potential formation. Serotonin and melatonin decreased the attenuation of synaptosomal Ca(2+) uptake induced by either 6-OHDA alone or iron plus ascorbate. Serotonin and melatonin inhibited the production of reactive oxygen species, formation of malondialdehyde and carbonyls, and thiol oxidation in mitochondria and synaptosomes and decreased degradation of 2-deoxy-D-ribose. Unlike serotonin, melatonin did not reduce the iron plus ascorbate-induced thiol oxidation. Tryptophan decreased thiol oxidation and 2-deoxy-D-ribose degradation but did not inhibit the production of reactive oxygen species and formation of oxidation products in the brain tissues. Serotonin and melatonin attenuated the dopamine-induced viability loss, including apoptosis, in PC12 cells. The results suggest that serotonin may attenuate the oxidative damage of mitochondria and synaptosomes and the dopamine-induced viability loss in PC12 cells by a decomposing action on reactive oxygen species and inhibition of thiol oxidation and shows the effect comparable to melatonin. Serotonin may show a prominent protective effect on the iron-mediated neuronal damage.     

Maintaining Specimen Integrity for G6PD Screening by Cytofluorometric Assays

Cytochemical staining remains an efficient way of identifying females who are heterozygous for the X chromosome-linked glucose-6-phosphate dehydrogenase (G6PD) gene. G6PD is highly polymorphic with certain alleles resulting in low intracellular G6PD activity in red blood cells. Low intracellular G6PD activity is associated with a risk of severe hemolysis when exposed to an oxidative stress such as fava beans, certain drugs and infections. Heterozygous females express the enzyme from both X-chromosome alleles resulting in two red blood cell populations each with G6PD enzyme characteristics representative of each allele; for example, normal and deficient. Cytochemical staining is the only way to determine the relative representation of each allele in red blood cells, a feature that is critical when assessing the risk for severe hemolysis when exposed to an oxidant such as the anti-malarial drug primaquine. This letter discusses red blood cell integrity with respect to the cytofluorometric assays for G6PD activity. An approach to making this test more robust is suggested. The approach makes this test more reliable and extends its use to a broader range of blood specimens.     

Ethylene formation in sugar beet leaves: evidence for the involvement of 3-hydroxytyramine and phenoloxidase after wounding

Ethylene production by sugar beet (Beta vulgaris L.) leaf discs is inhibited by white (or red, >610 nm) light or by wounding. In contrast, in wounded leaf discs, ethylene production is stimulated by light. The effect of light on wounded leaf discs has been studied by using an in vitro system which mimics the loss of compartmentation in the wounded leaf. Chlorophyll-free extracts from sugar beet leaves stimulate the production of the superoxide free radical ion (as a prerequisite for ethylene formation) by illuminated chloroplast lamellae. The substance from the crude leaf extracts which is active in stimulating the production of the superoxide free radical ion has been identified as 3-hydroxytyramine (dopamine). Exogenous dopamine between 5 mum and 100 mum stimulates ethylene formation by illuminated chloroplast lamellae from methional. It also stimulates the production of the superoxide free radical ion, the formation of which apparently involves both a lamellar phenoloxidase and photosynthetic electron transport as a 1-electron donor, and is cyanide-sensitive.     

Glucose-6-phosphate dehydrogenase modulates vascular endothelial growth factor-mediated angiogenesis

Glucose-6-phosphate dehydrogenase (G6PD), the first enzyme of the pentose phosphate pathway, is the principal intracellular source of NADPH. NADPH is utilized as a cofactor by vascular endothelial cell nitric-oxide synthase (eNOS) to generate nitric oxide (NO*). To determine whether G6PD modulates NO*-mediated angiogenesis, we decreased G6PD expression in bovine aortic endothelial cells using an antisense oligodeoxynucleotide to G6PD or increased G6PD expression by adenoviral gene transfer, and we examined vascular endothelial growth factor (VEGF)-stimulated endothelial cell proliferation, migration, and capillary-like tube formation. Deficient G6PD activity was associated with a significant decrease in endothelial cell proliferation, migration, and tube formation, whereas increased G6PD activity promoted these processes. VEGF-stimulated eNOS activity and NO* production were decreased significantly in endothelial cells with deficient G6PD activity and enhanced in G6PD-overexpressing cells. In addition, G6PD-deficient cells demonstrated decreased tyrosine phosphorylation of the VEGF receptor Flk-1/KDR, Akt, and eNOS compared with cells with normal G6PD activity, whereas overexpression of G6PD enhanced phosphorylation of Flk-1/KDR, Akt, and eNOS. In the Pretsch mouse, a murine model of G6PD deficiency, vessel outgrowth from thoracic aorta segments was impaired compared with C3H wild-type mice. In an in vivo Matrigel angiogenesis assay, cell migration into the plugs was inhibited significantly in G6PD-deficient mice compared with wild-type mice, and gene transfer of G6PD restored the wild-type phenotype in G6PD-deficient mice. These findings demonstrate that G6PD modulates angiogenesis and may represent a novel angiogenic regulator.     

Hydroxyl radical attack on dopamine

Hydroxyl radicals were generated in the presence of 1 mM dopamine (3,4-dihydroxyphenylethylamine) at pH 7.2 (50 mM phosphate buffer) by the following two mechanisms: 1) a classic Fenton-type reaction between hydrogen peroxide and a ferrous chelate (ferrous diethylenetriaminepentaacetate) and 2) the cyclical redox reactions of iron-EDTA/ascorbate. Three ring-monohydroxylated products of dopamine were detected by high performance liquid chromatography with electrochemical detection: 2-hydroxydopamine, 5-hydroxydopamine, and 6-hydroxydopamine in an approximate ratio of 3:2:1. Scavengers of hydroxyl radicals (dimethyl sulfoxide, mannitol, ethanol) suppressed the yields of products in a concentration-dependent manner. The formation of nonphysiologic hydroxylated forms of dopamine can provide a probe for the formation of hydroxyl radicals in dopamine neurons.     

Glycolytic cancer cells lacking 6-phosphogluconate dehydrogenase metabolize glucose to induce senescence

We show that knockdown of 6-phosphogluconate dehydrogenase (6PGD) of the pentose phosphate pathway (PPP) inhibits growth of lung cancer cells by senescence induction. This inhibition is not due to a defect in the oxidative PPP per se. NADPH and ribose phosphate production are normal in 6PGD knockdown cells and shutdown of PPP by knockdown of glucose-6-phosphate dehydrogenase (G6PD) has little effect on cell growth. Moreover, 6PGD knockdown cells can proliferate when the PPP is bypassed by using fructose instead of glucose in medium. Significantly, G6PD knockdown rescues proliferation of cells lacking 6PGD, suggesting an accumulation of growth inhibitory glucose metabolics in cells lacking 6PGD. Therefore, 6PGD inhibition may provide a novel strategy to treat glycolyic tumors such as lung cancer.