6-Phosphogluconate dehydrogenase regulates tumor cell migration in vitro by regulating receptor tyrosine kinase c-Met

6-Phosphogluconate dehydrogenase (6PGD) is the third enzyme in the oxidative pentose phosphate pathway (PPP). Recently, we reported that knockdown of 6PGD inhibited lung tumor growth in vitro and in a xenograft model in mice. In this study, we continued to examine the functional role of 6PGD in cancer. We show that 6PGD expression positively correlates with advancing stage of lung carcinoma. In search of functional signals related to 6PGD, we discovered that knockdown of 6PGD significantly inhibited phosphorylation of c-Met at tyrosine residues known to be critical for activity. This downregulation of c-Met phosphorylation correlated with inhibition of cell migration in vitro. Overexpression of a constitutively active c-Met specifically rescued the migration but not proliferation phenotype of 6PGD knockdown. Therefore, 6PGD appears to be required for efficient c-Met signaling and migration of tumor cells in vitro.     

Identification of Macrolepiota procera extract as a novel G6PD inhibitor for the treatment of lung cancer

Tumor metabolism, an emerging hallmark of cancer, is characterized by aberrant expression of enzymes from various metabolic pathways including glycolysis and PPP (pentose phosphate pathway). Glucose 6 phosphate dehydrogenase (G6PD) and 6-phosphogluconate dehydrogenase (6PGD), oxidative carboxylases of PPP, have been reported to accomplish different biosynthetic and energy requirements of cancer cells. G6PD and 6PGD have been proposed as potential therapeutic targets for cancer therapy during recent years due to their overexpression in various cancers. Here, we have employed enzymatic assay based screening using in-house G6PD and 6PGD assay protocols for the identification of mushroom extracts which could inhibit G6PD or 6PGD enzymatic activity for implications in cancer therapy. For the fulfillment of the objectives of present study, nine edible mushrooms were subjected to green extraction for preparation of ethanolic extracts. 6xhis-G6PD and pET-28a-h6PGD plasmids were expressed in BL21-DE3 E. coli cells for the expression and purification of protein of interests. Using purified proteins, in house enzymatic assay protocols were established. The preliminary screening identified two extracts (Macrolepiota procera and Terfezia boudieri) as potent and selective G6PD inhibitors, while no extract was found highly active against 6PGD. Further, evaluation of anticancer potential of mushroom extracts against lung cancer cells revealed Macrolepiota procera as potential inhibitor of cancer cell proliferation with IC₅₀ value of 6.18 μg/ml. Finally, screening of M. procera-derived compounds against G6PD via molecular docking has identified paraben, quercetin and syringic acid as virtual hit compounds possessing good binding affinity with G6PD. The result of present study provides novel findings for possible mechanism of action of M. procera extract against A549 via G6PD inhibition suggesting that M. procera might be of therapeutic interest for lung cancer treatment.     

A critical role of glucose-6-phosphate dehydrogenase in TAp73-mediated cell proliferation

The pentose phosphate pathway (PPP) provides ribose and NADPH that support biosynthesis and antioxidant defense. Our recent findings suggest that the p53-related protein TAp73 enhances the PPP flux. TAp73 stimulates the expression of glucose-6-phophate dehydrogenase (G6PD), the rate-limiting enzymes of the PPP. Through this regulation, TAp73 promotes the accumulation of macromolecules and increases cellular capability to withstand oxidative stresses. TAp73 also regulates other metabolic enzymes, and the relative importance of these targets in TAp73-mediated cell growth is not well understood. Here we show that, like in other cell lines, TAp73 is required for supporting proliferation and maintaining the expression of G6PD in the human lung cancer H1299 cells. Restoration of G6PD expression almost fully rescues the defects in cell growth caused by TAp73 knockdown, suggesting that G6PD is the major proliferative target of TAp73 in these cells. G6PD expression is elevated in various tumors, correlating with the upregulation of TAp73. These results indicate that TAp73 may function as an oncogene, and that G6PD is likely a focal point of regulation in oncogenic growth.     

Crocidolite asbestos inhibits pentose phosphate oxidative pathway and glucose 6-phosphate dehydrogenase activity in human lung epithelial cells

The cytotoxicity of asbestos has been related to its ability to increase the production of reactive oxygen species (ROS), via the iron-catalyzed reduction of oxygen and/or the activation of NADPH oxidase. The pentose phosphate pathway (PPP) is generally activated by the cell exposure to oxidant molecules. Contrary to our expectations, asbestos (crocidolite) fibers caused a dose- and time-dependent inhibition of PPP and decreased its activation by an oxidative stress in human lung epithelial cells A549. In parallel, the intracellular activity of the PPP rate-limiting enzyme, glucose 6-phosphate dehydrogenase (G6PD), was significantly diminished by crocidolite exposure. This inhibition was selective, as the activity of other PPP and glycolysis enzymes was not modified, and was not attributable to a decreased expression of G6PD. On the opposite, the incubation with glass fibers MMVF10 did not modify PPP and G6PD activity. PPP and G6PD inhibition did not correlate with the increased nitric oxide (NO) production elicited by crocidolite in A549 cells. Experiments with the purified enzyme suggest that crocidolite inhibits G6PD by directly interacting with the protein. We propose here a new mechanism of asbestos-evoked oxidative stress, wherein fibers increase the intracellular ROS levels also by inhibiting the main antioxidant pathway of the cell.     

二氢硫辛酰转乙酰基酶通过乙酰化磷酸葡糖酸脱氢酶促进核酸合成

丙酮酸脱氢酶复合物(pyruvate dehydrogenase complex,PDC)是位于线粒体内的多酶复合物,催化丙酮酸不可逆地氧化脱羧转为乙酰辅酶A,二氢硫辛酰转乙酰基酶(dihydrolipoyl acetyltransferase,DLAT)是PDC的1个亚基.PDC在细胞线粒体呼吸中发挥关键作用.但是D...     

p53 regulates biosynthesis through direct inactivation of glucose-6-phosphate dehydrogenase

Cancer cells consume large quantities of glucose and primarily use glycolysis for ATP production, even in the presence of adequate oxygen. This metabolic signature (aerobic glycolysis or the Warburg effect) enables cancer cells to direct glucose to biosynthesis, supporting their rapid growth and proliferation. However, both causes of the Warburg effect and its connection to biosynthesis are not well understood. Here we show that the tumour suppressor p53, the most frequently mutated gene in human tumours, inhibits the pentose phosphate pathway (PPP). Through the PPP, p53 suppresses glucose consumption, NADPH production and biosynthesis. The p53 protein binds to glucose-6-phosphate dehydrogenase (G6PD), the first and rate-limiting enzyme of the PPP, and prevents the formation of the active dimer. Tumour-associated p53 mutants lack the G6PD-inhibitory activity. Therefore, enhanced PPP glucose flux due to p53 inactivation may increase glucose consumption and direct glucose towards biosynthesis in tumour cells.     

Neuroprotection by glucose-6-phosphate dehydrogenase and the pentose phosphate pathway

Glucose-6-phosphate dehydrogenase (G6PD), the rate limiting enzyme that channels glucose catabolism from glycolysis into the pentose phosphate pathway (PPP), is vital for the production of reduced nicotinamide adenine dinucleotide phosphate (NADPH) in cells. NADPH is in turn a substrate for glutathione reductase, which reduces oxidized glutathione disulfide to sulfhydryl glutathione. Best known for inherited deficiencies underlying acute hemolytic anemia due to elevated oxidative stress by food or medication, G6PD, and PPP activation have been associated with neuroprotection. Recent works have now provided more definitive evidence for G6PD's protective role in ischemic brain injury and strengthened its links to neurodegeneration. In Drosophila models, improved proteostasis and lifespan extension result from an increased PPP flux due to G6PD induction, which is phenocopied by transgenic overexpression of G6PD in neurons. Moderate transgenic expression of G6PD was also shown to improve healthspan in mouse. Here, the deciphered and implicated roles of G6PD and PPP in protection against brain injury, neurodegenerative diseases, and in healthspan/lifespan extensions are discussed together with an important caveat, namely NADPH oxidase (NOX) activity and the oxidative stress generated by the latter. Activation of G6PD with selective inhibition of NOX activity could be a viable neuroprotective strategy for brain injury, disease, and aging.     

NADPH production by the pentose phosphate pathway in the zona fasciculata of rat adrenal gland.

Biosynthesis of steroid hormones in the cortex of the adrenal gland takes place in smooth endoplasmic reticulum and mitochondria and requires NADPH. Four enzymes produce NADPH: glucose-6-phosphate dehydrogenase (G6PD), the key regulatory enzyme of the pentose phosphate pathway, phosphogluconate dehydrogenase (PGD), the third enzyme of that pathway, malate dehydrogenase (MDH), and isocitrate dehydrogenase (ICDH). However, the contribution of each enzyme to NADPH production in the cortex of adrenal gland has not been established. Therefore, activity of G6PD, PGD, MDH, and ICDH was localized and quantified in rat adrenocortical tissue using metabolic mapping, image analysis, and electron microscopy. The four enzymes have similar localization patterns in adrenal gland with highest activities in the zona fasciculata of the cortex. G6PD activity was strongest, PGD, MDH, and ICDH activity was approximately 60%, 15%, and 7% of G6PD activity, respectively. The K(m) value of G6PD for glucose-6-phosphate was two times higher than the K(m) value of PGD for phosphogluconate. As a consequence, virtual flux rates through G6PD and PGD are largely similar. It is concluded that G6PD and PGD provide the major part of NADPH in adrenocortical cells. Their activity is localized in the cytoplasm associated with free ribosomes and membranes of the smooth endoplasmic reticulum, indicating that NADPH-demanding processes related to biosynthesis of steroid hormones take place at these sites. Complete inhibition of G6PD by androsterones suggests that there is feedback regulation of steroid hormone biosynthesis via G6PD.     

Peroxynitrite protects neurons against nitric oxide-mediated apoptosis. A key role for glucose-6-phosphate dehydrogenase activity in neuroprotection

Peroxynitrite is thought to be a nitric oxide-derived neurotoxic effector molecule involved in the disruption of key energy-related metabolic targets. To assess the consequences of such interference in cellular glucose metabolism and viability, we studied the possible modulatory role played by peroxynitrite in glucose oxidation in neurons and astrocytes in primary culture. Here, we report that peroxynitrite triggered rapid stimulation of pentose phosphate pathway (PPP) activity and the accumulation of NADPH, an essential cofactor for glutathione regeneration. In contrast to peroxynitrite, nitric oxide elicited NADPH depletion, glutathione oxidation, and apoptotic cell death in neurons, but not in astrocytes. These events were noticeably counteracted by pretreatment of neurons with peroxynitrite. In an attempt to elucidate the mechanism responsible for this PPP stimulation and neuroprotection, we found evidence consistent with both exogenous and endogenous peroxynitrite-mediated activation of glucose-6-phosphate dehydrogenase (G6PD), an enzyme that catalyzes the first rate-limiting step in the PPP. Moreover, functional overexpression of the G6PD gene in stably transformed PC12 cells induced NADPH accumulation and offered remarkable resistance against nitric oxide-mediated apoptosis, whereas G6PD gene-targeted antisense inhibition depleted NADPH levels and exacerbated cellular vulnerability. In light of these results, we suggest that G6PD activation represents a novel role for peroxynitrite in neuroprotection against nitric oxide-mediated apoptosis.     

SIRT2 controls the pentose phosphate switch

The most common enzyme defect in humans is glucose‐6‐phosphate dehydrogenase (G6PD) deficiency, which affects more than 400 million people. G6PD shunts glucose into the pentose phosphate pathway (PPP) to generate nucleotides and reducing potential in the form of NADPH. In this issue, Wang et al ( 2014 ) show that G6PD activity is post‐translationally regulated by SIRT2, a cytoplasmic NAD⁺‐dependent deacetylase, thereby linking NAD⁺ levels to DNA repair and oxidative defences, and identifying potential new approaches to treating this common genetic disease.     

Modulation of doxorubicin resistance by the glucose-6-phosphate dehydrogenase activity

How anti-neoplastic agents induce MDR (multidrug resistance) in cancer cells and the role of GSH (glutathione) in the activation of pumps such as the MRPs (MDR-associated proteins) are still open questions. In the present paper we illustrate that a doxorubicin-resistant human colon cancer cell line (HT29-DX), exhibiting decreased doxorubicin accumulation, increased intracellular GSH content, and increased MRP1 and MRP2 expression in comparison with doxorubicin-sensitive HT29 cells, shows increased activity of the PPP (pentose phosphate pathway) and of G6PD (glucose-6-phosphate dehydrogenase). We observed the onset of MDR in HT29 cells overexpressing G6PD which was accompanied by an increase in GSH. The G6PD inhibitors DHEA (dehydroepiandrosterone) and 6-AN (6-aminonicotinamide) reversed the increase of G6PD and GSH and inhibited MDR both in HT29-DX cells and in HT29 cells overexpressing G6PD. In our opinion, these results suggest that the activation of the PPP and an increased activity of G6PD are necessary to some MDR cells to keep the GSH content high, which is in turn necessary to extrude anticancer drugs out of the cell. We think that our data provide a new further mechanism for GSH increase and its effects on MDR acquisition.     

Investigation of the Effects of Some Phenolic Compounds on the Activities of Glucose‐6‐Phosphate Dehydrogenase and 6‐Phosphogluconate Dehydrogenase from Human Erythrocytes

Polyphenols are the important compounds that have various bioactivities. They constitute vital active agents of not only daily diet but also natural medicines that are used traditionally. It is generally considered that they are safe because they are natural. In some conducted studies, different negative effects of these compounds were mentioned. Twelve phenolic compounds have been assayed to determine the effect of inhibition on glucose‐6‐phosphate dehydrogenase (G6PD) and 6‐phosphogluconate dehydrogenase (6PGD) enzymes activity. For in vitro studies, the enzymes were purified from human erythrocytes using 2′,5′‐ADP Sepharose 4B affinity chromatography. Naringenin, caffeic acid, ellagic acid, ferulic acid, and sinapic acid against two enzymes, hesperidin and polydatin, only on G6PD activity and chrysin solely against 6PGD showed inhibitory effect. Chlorogenic acid, p‐coumaric acid, and syringic acid did not exhibit an effect on the activity of the two enzymes.     

The synthesis of N‐benzoylindoles as inhibitors of rat erythrocyte glucose‐6‐phosphate dehydrogenase and 6‐phosphogluconate dehydrogenase

Glucose‐6‐phosphate dehydrogenase (G6PD) and 6‐phosphogluconate dehydrogenase (6PGD) play an important function in various biochemical processes as they generate reducing power of the cell. Thus, metabolic reprogramming of reduced nicotinamide adenine dinucleotide phosphate (NADPH) homeostasis is reported to be a vital step in cancer progression as well as in combinational therapeutic approaches. In this study, N‐benzoylindoles 9a‐ ‐ 9d , which form the main framework of many natural indole derivatives such as indomethacin and N‐benzoylindoylbarbituric acid, were synthesized through three easy and effective steps as an in vitro inhibitor effect of G6PD and 6PGD. The N‐benzoylindoles inhibited the enzymatic activity with IC₅₀ in the range of 3.391505 μM for G6PD and 2.19–990 μM for 6PGD.     

Rewiring of purine metabolism in response to acidosis stress in glioma stem cells

Glioma stem cells (GSCs) contribute to therapy resistance and poor outcomes for glioma patients. A significant feature of GSCs is their ability to grow in an acidic microenvironment. However, the mechanism underlying the rewiring of their metabolism in low pH remains elusive. Here, using metabolomics and metabolic flux approaches, we cultured GSCs at pH 6.8 and pH 7.4 and found that cells cultured in low pH exhibited increased de novo purine nucleotide biosynthesis activity. The overexpression of glucose-6-phosphate dehydrogenase, encoded by G6PD or H6PD, supports the metabolic dependency of GSCs on nucleotides when cultured under acidic conditions, by enhancing the pentose phosphate pathway (PPP). The high level of reduced glutathione (GSH) under acidic conditions also causes demand for the PPP to provide NADPH. Taken together, upregulation of G6PD/H6PD in the PPP plays an important role in acidic-driven purine metabolic reprogramming and confers a predilection toward glioma progression. Our findings indicate that targeting G6PD/H6PD, which are closely related to glioma patient survival, may serve as a promising therapeutic target for improved glioblastoma therapeutics. An integrated metabolomics and metabolic flux analysis, as well as considering microenvironment and cancer stem cells, provide a precise insight into understanding cancer metabolic reprogramming.     

Regulation of G6PD acetylation by SIRT2 and KAT9 modulates NADPH homeostasis and cell survival during oxidative stress

Glucose‐6‐phosphate dehydrogenase (G6PD) is a key enzyme in the pentose phosphate pathway (PPP) and plays an essential role in the oxidative stress response by producing NADPH, the main intracellular reductant. G6PD deficiency is the most common human enzyme defect, affecting more than 400 million people worldwide. Here, we show that G6PD is negatively regulated by acetylation on lysine 403 (K403), an evolutionarily conserved residue. The K403 acetylated G6PD is incapable of forming active dimers and displays a complete loss of activity. Knockdown of G6PD sensitizes cells to oxidative stress, and re‐expression of wild‐type G6PD, but not the K403 acetylation mimetic mutant, rescues cells from oxidative injury. Moreover, we show that cells sense extracellular oxidative stimuli to decrease G6PD acetylation in a SIRT2‐dependent manner. The SIRT2‐mediated deacetylation and activation of G6PD stimulates PPP to supply cytosolic NADPH to counteract oxidative damage and protect mouse erythrocytes. We also identified KAT9/ELP3 as a potential acetyltransferase of G6PD. Our study uncovers a previously unknown mechanism by which acetylation negatively regulates G6PD activity to maintain cellular NADPH homeostasis during oxidative stress.     

Glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase from Lactobacillus casei: responses with different modulators

Glucose 6-phosphate dehydrogenase (G6PD) and 6-phosphogluconate dehydrogenase (6PGD) were separated and partially purified from glucose-grown cells of Lactobacillus casei. The enzymes had similar pH optima, thermosensitivity and molecular weights. They had different net charges and their pI values were 5.38 and 4.52, respectively. Histidine, arginine, lysine and cysteine residues were essential for the activity of G6PD, and all the above amino acids with the exception of lysine were required for 6PGD activity. Mg2+ activated 6PGD up to 15 mM concentration, above which it was inhibitory. It had no effect on G6PD activity. G6PD was specific for NADP+, but 6PGD showed some activity with NAD+ as the cofactor, although it was essentially NADP(+)-preferring. Both the enzymes, were inhibited by NADPH. 6PGD was also inhibited by its product, ribulose 5-phosphate. ATP inhibited 6PGD only at subsaturating concentrations of NADP+. The inhibition was sigmoidal in the absence of Mg2+ and hyperbolic in its presence.     

ATM activates the pentose phosphate pathway promoting anti-oxidant defence and DNA repair

Ataxia telangiectasia (A-T) is a human disease caused by ATM deficiency characterized among other symptoms by radiosensitivity, cancer, sterility, immunodeficiency and neurological defects. ATM controls several aspects of cell cycle and promotes repair of double strand breaks (DSBs). This probably accounts for most of A-T clinical manifestations. However, an impaired response to reactive oxygen species (ROS) might also contribute to A-T pathogenesis. Here, we show that ATM promotes an anti-oxidant response by regulating the pentose phosphate pathway (PPP). ATM activation induces glucose-6-phosphate dehydrogenase (G6PD) activity, the limiting enzyme of the PPP responsible for the production of NADPH, an essential anti-oxidant cofactor. ATM promotes Hsp27 phosphorylation and binding to G6PD, stimulating its activity. We also show that ATM-dependent PPP stimulation increases nucleotide production and that G6PD-deficient cells are impaired for DSB repair. These data suggest that ATM protects cells from ROS accumulation by stimulating NADPH production and promoting the synthesis of nucleotides required for the repair of DSBs.