Stimulation Of Pentose Phosphate Pathway Research Articles

PBX3 promotes pentose phosphate pathway and colorectal cancer progression by enhancing G6PD expression.

Metabolic reprogramming is a hallmark of cancers crucial for fulfilling the needs of energy, building blocks, and antioxidants to support tumor cells' rapid proliferation and to cope with the harsh microenvironment. Pre-B-cell leukemia transcription factor 3 (PBX3) is a member of the PBX family whose expression is up-regulated in various tumors, however, whether it is involved in tumor cell metabolic reprogramming remains unclear. Herein, we report that PBX3 is a positive regulator of glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme in the pentose phosphate pathway (PPP). PBX3 promoted G6PD transcriptional activity in tumor cells by binding directly to its promoter, leading to PPP stimulation and enhancing the production of nucleotides and NADPH, a crucial reductant, thereby promoting nucleic acid and lipid biosynthesis while decreasing intracellular reactive oxygen species levels. The PBX3/G6PD axis also promoted tumorigenic potential in vitro and in vivo. Collectively, these findings reveal a novel function of PBX3 as a regulator of G6PD, linking its oncogenic activity with tumor cell metabolic reprogramming, especially PPP. Furthermore, our results suggested that PBX3 is a potential target for metabolic-based anti-tumor therapeutic strategies.

Improvement of neuronal differentiation by carbon monoxide: Role of pentose phosphate pathway

Over the last decades, the silent-killer carbon monoxide (CO) has been shown to also be an endogenous cytoprotective molecule able to inhibit cell death and modulate mitochondrial metabolism. Neuronal metabolism is mostly oxidative and neurons also use glucose for maintaining their anti-oxidant status by generation of reduced glutathione (GSH) via the pentose-phosphate pathway (PPP). It is established that neuronal differentiation depends on reactive oxygen species (ROS) generation and signalling, however there is a lack of information about modulation of the PPP during adult neurogenesis. Thus, the main goal of this study was to unravel the role of CO on cell metabolism during neuronal differentiation, particularly by targeting PPP flux and GSH levels as anti-oxidant system.A human neuroblastoma SH-S5Y5 cell line was used, which differentiates into post-mitotic neurons by treatment with retinoic acid (RA), supplemented or not with CO-releasing molecule-A1 (CORM-A1). SH-SY5Y cell differentiation supplemented with CORM-A1 prompted an increase in neuronal yield production. It did, however, not alter glycolytic metabolism, but increased the PPP. In fact, CORM-A1 treatment stimulated (i) mRNA expression of 6-phosphogluconate dehydrogenase (PGDH) and transketolase (TKT), which are enzymes for oxidative and non-oxidative phases of the PPP, respectively and (ii) protein expression and activity of glucose 6-phosphate dehydrogenase (G6PD) the rate-limiting enzyme of the PPP. Likewise, whenever G6PD was knocked-down CO-induced improvement on neuronal differentiation was reverted, while pharmacological inhibition of GSH synthesis did not change CO's effect on the improvement of neuronal differentiation. Both results indicate the key role of PPP in CO-modulation of neuronal differentiation. Furthermore, at the end of SH-SY5Y neuronal differentiation process, CORM-A1 supplementation increased the ratio of reduced and oxidized glutathione (GSH/GSSG) without alteration of GSH metabolism. These data corroborate with PPP stimulation. In conclusion, CO improves neuronal differentiation of SH-S5Y5 cells by stimulating the PPP and modulating the GSH system.

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.

Antioxidant Enzyme Response of Creeping Bentgrass Clonal Lines with Marine Peptide and Chitosan Oligosaccharide

The effect of antioxidant stimulators on low phenolic and high phenolic single seed originating creeping bentgrass [Agrostis stolonifera var. palustris (Huds.) Farw.] clonal lines were investigated to understand whether these natural compounds could improve the physiological adjustments of creeping bentgrass under low temperature exposure. A marine peptide hydrolysate, and chitosan oligosaccharide (COS) derived from marine chitin were applied to creeping bentgrass foliage and then subjected to cold temperature (4°C, day/night temperature) for 3 wk. Significant stimulation of total phenolic content (8%‐CB 27 to 75%‐CB 15) and antioxidant enzyme response (16%‐CB 27 to 44% CB 13) was observed in creeping bentgrass clonal lines following application of marine peptide + COS. Superoxide dismutase (SOD) (13–50%), and guaiacol peroxidase (GPX) activity (58–99%) increased significantly after treatments in all creeping bentgrass clonal lines, and stimulation of antioxidant enzyme activity was more profound in low phenolic creeping bentgrass clonal lines. Higher glucose‐6‐phosphate dehydrogenase (G6PDH) and higher proline dehydrogenase (PDH) activity was also observed in these lines following application of marine peptide + COS. Stimulation of pentose phosphate pathway (PPP) and a probable proline oxidation in creeping bentgrass clonal lines after foliar application of marine peptide ± COS suggest a role for the enzymes in the improvement of these lines.

CRANBERRY PHENOLICS-MEDIATED ELICITATION OF ANTIOXIDANT ENZYME RESPONSE IN FAVA BEAN (VICIA FABA) SPROUTS

The antioxidant response mechanism by which phenolic phytochemicals show their positive benefits in plants and animals is not very well understood. The ability of cranberry juice powder (CP), ellagic acid (EA), rosmarinic acid (RA) and their synergies to elicit a phenolic response in germinating fava bean was investigated. Results indicated that elicitation with CP, EA, RA and their synergies resulted in increased endogenous phenolic synthesis linked to the stimulation of pentose phosphate pathway (PPP). Further, the stimulation of PPP was linked to the accumulation of free proline, suggesting a possible coupling of proline biosynthesis with PPP. Elicitation also resulted in the stimulation of guaiaicol peroxidase, suggesting a possible involvement in modulating structural development of the germinating sprout. Exogenous phenolic elicitation also resulted in the formation of malondialdehyde, which was gradually reduced because of the activation of antioxidant enzyme systems superoxide dismutase and catalase. The results indicate that the possible mode of action of exogenous phenolic phytochemicals in dark germinating fava bean could be by stimulating the PPP linked to proline biosynthesis and by the activation of the antioxidant enzyme system. The results also suggest that pure exogenous phenolics, EA and RA appeared to be effective when they were present in a cranberry phenolic background, suggesting a possible synergistic mode of action between EA, RA and cranberry phenolics in generating an endogenous fava bean antioxidant enzyme response.

Antioxidant enzyme response studies in H 2O 2-stressed porcine muscle tissue following treatment with oregano phenolic extracts

The effect of oregano ( Origanum vulgare) extracts rich in phenolics in ameliorating the adverse effects of oxidative stress caused by H 2O 2 by mediating an antioxidant enzyme response in porcine muscle tissue was investigated. The changes in the total phenolic content, antioxidant activity, proline content, malondialdehyde (MDA) content, activity of glucose-6-phosphate dehydrogenase (G6PDH), which is the first commited step of pentose phosphate pathway, antioxidant enzymes including total peroxidase (TPX), superoxide dismutase (SOD), and catalase (CAT) activity in stressed porcine muscle tissue was determined. The total phenolic content of control and H 2O 2 treated porcine tissue remained constantly low throughout the study. In the oregano treated tissues the phenolic content increased four-fold immediately indicating that the porcine cells had imbibed the oregano phenolics efficiently. In the case of H 2O 2 + oregano treatment the values were higher than control and H 2O 2 alone but less than oregano alone. This indicates that the oregano phenolics inside the porcine cell were potentially reactive and likely being utilized for counteracting the harmful effects of H 2O 2. In the oregano and H 2O 2 + oregano treated tissues the 1,1-diphenyl-2-picrylhydrazyl (DPPH) scavenging antioxidant activity almost doubled from 35% to 75% by 8 h. This also further indicates the high free radical scavenging, antioxidant potential of the oregano phytochemicals in reducing the harmful effects of H 2O 2. In the oregano and H 2O 2 + oregano treatments the G6PDH activity increased seven-fold compared to stressed and control tissue, which coincides with stimulated TPX activity. The H 2O 2 stress alone enhanced G6PDH activity slightly and this response occurred concomitantly with an increase in SOD and CAT activity. The oregano phenolics were able to counter the H 2O 2 effects with reduced need for higher SOD expression. In the present research the highest antioxidant enzyme activity was observed in the stressed H 2O 2 treatment at 8 h incubation indicating the active role of CAT in modulating the harmful effects of H 2O 2, which directly correlated to higher SOD activity. In the case of oregano and oregano + H 2O 2, proline levels were higher compared to H 2O 2 alone, again suggesting the ameliorating effect of oregano on oxidative stress and this may involve proline-linked stimulation of pentose phosphate pathway. The MDA content in the case of oregano + H 2O 2 treatment was comparatively lower than H 2O 2 alone showing the protective effects of oregano against reactive oxygen species and eventually from membrane damage. The major implication from this study is evidence that oregano being rich in phenolics is an effective direct quencher of free radicals. Further, the same phenolics play a beneficial role in protecting porcine muscle tissue from the harmful effects of reactive oxygen species through regulation of antioxidant enzyme response through the phenolic-dependent peroxidases with dependency on pentose phosphate pathway but with reduced dependency on SOD and CAT.

The secondary alcohol and aglycone metabolites of doxorubicin alter metabolism of human erythrocytes.

Anthracyclines, a class of antitumor drugs widely used for the treatment of solid and hematological malignancies, cause a cumulative dose-dependent cardiac toxicity whose biochemical basis is unclear. Recent studies of the role of the metabolites of anthracyclines, i.e., the alcohol metabolite doxorubicinol and aglycone metabolites, have suggested new hypotheses about the mechanisms of anthracycline cardiotoxicity. In the present study, human red blood cells were used as a cell model. Exposure (1 h at 37 C) of intact human red blood cells to doxorubicinol (40 M) and to aglycone derivatives of doxorubicin (40 M) induced, compared with untreated red cells: i) a ~2-fold stimulation of the pentose phosphate pathway (PPP) and ii) a marked inhibition of the red cell antioxidant enzymes, glutathione peroxidase (~20%) and superoxide dismutase (~60%). In contrast to doxorubicin-derived metabolites, doxorubicin itself induced a slighter PPP stimulation (~35%) and this metabolic event was not associated with any alteration in glutathione reductase, glutathione peroxidase, catalase or superoxide dismutase activity. Furthermore, the interaction of hemoglobin with doxorubicin and its metabolites induced a significant increase (~22%) in oxygen affinity compared with hemoglobin incubated without drugs. On the basis of the results obtained in the present study, a new hypothesis, involving doxorubicinol and aglycone metabolites, has been proposed to clarify the mechanisms responsible for the doxorubicin-induced red blood cell toxicity.

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.

O 2-dependent stimulation of the pentose phosphate pathway by S-nitrosocysteine in human erythrocytes

In the present study we analysed the effects of S-nitrosocysteine (CysNO) on adult human red blood cell metabolism and observed that metabolic response depended on the degree of cell oxygenation. In particular, glucose metabolised through the pentose phosphate pathway (PPP) was higher in treated erythrocytes than in untreated cells only at high O 2 pressure. Since, following the treatment of intact cells with CysNO, glucose-6-phosphate dehydrogenase (G6PD) and phosphofructokinase (PFK) activities did not evidence any significant alteration, the possibility that the stimulation of PPP was triggered by a CysNO mediated modification of these enzymes was excluded. Intracellular S-nitrosoglutathione (GSNO), detected only in treated red blood cells, may be linked solely to the exposition to the NO donor. A possible rationalisation of the different metabolic behaviour shown by erythrocytes as a function of their oxygenation state is proposed. It takes into account the different route of catabolic degradation observed in vitro for GSNO under aerobic and anaerobic condition.

Regulation of Benzyladenine-Induced in Vitro Shoot Organogenesis and Endogenous Proline in Melon (Cucumis melo L.) by Exogenous Proline, Ornithine, and Proline Analogues

Melon (Cucumis melo L.) is an excellent system to study the role of proline in regulating benzyladenine-induced shoot organogenesis, which is also an important system in our laboratory for investigating flavor and antioxidant metabolite synthesis in differentiated tissues. We have hypothesized that proline stimulated in vitro shoot organogenesis through the activation of the proposed proline-linked pentose phosphate pathway, which may regulate endogenous cytokinin and auxin biosynthesis. To further investigate this hypothesis, it is essential to first confirm whether benzyladenine-induced shoot organogenesis can be stimulated by enhancing proline synthesis through the use of proline analogues and ornithine (proline precursor). In this study, benzyladenine-induced shoot organogenesis was substantially stimulated by exogenous proline and/or ornithine in combination with proline analogues such as azetidine-2-carboxylate (A2C) and hydroxyproline (HP). Among these treatments, proline in combination with A2C or HP showed the highest stimulation. The stimulation of organogenesis correlated closely with increased levels of endogenous proline content, thereby strengthening the hypothesis that proline-linked activation of purine and aromatic metabolism via the stimulation of pentose phosphate pathway may be important for in vitro organogenesis in melon.

Assessment of the role of the glutathione and pentose phosphate pathways in the protection of primary cerebrocortical cultures from oxidative stress.

Reactive oxygen species have been implicated in neuronal injury associated with various neuropathological disorders. However, little is known regarding the relationship between antioxidant enzyme capacity and resultant toxicity. The antioxidant pathways of primary cerebrocortical cultures were directly examined using a novel technique that measures pentose phosphate pathway (PPP) activity, which is enzymatically coupled to glutathione peroxidase (GPx) detoxification of hydrogen peroxide (H2O2). PPP activity was quantified from data obtained by gas chromatography/mass spectrometry analysis of released labeled lactate following metabolic degradation of [1,6-(13)C2, 6,6-(2)H2] glucose by cerebrocortical cultures. The antioxidant capacity of these cultures was systematically evaluated using H2O2, and the resultant toxicity was quantified by lactate dehydrogenase release. Exposure of primary mixed and purified astrocytic cultures to H2O2 caused stimulation of PPP activity in a concentration-dependent fashion from 0.25 to 22.2% and from 6.9 to 66.7% of glucose metabolized to lactate through the PPP, respectively. In the mixed cultures, chelation of iron before H2O2 exposure was protective and resulted in a correlation between PPP saturation and toxicity. Conversely, addition of iron, inhibition of GPx, or depletion of glutathione decreased H2O2-induced PPP stimulation and increased toxicity. These results implicate the Fenton reaction, reflect the pivotal role of GPx in H2O2 detoxification, and contribute to our understanding of the etiological role of free radicals in neuropathological conditions.

Effects of pneumotoxic trialkylphosphorothioates on the pentose phosphate pathway in rat lung slices

The extent of glucose oxidation via the pentose phosphate pathway (PPP), a possible indicator of oxidative stress, was assessed in rat lung slices by measuring the production of 14CO 2 on incubation with either [6- 14C]glucose or [1- 14C]glucose. Incubation in the presence of the pneumotoxins O, S, S-trimethyl phosphorodithioate (OSSMe, 10 −5−10 −3 M), O, O, S-triethyl phosphorothioate (10 −4−10 −2 M) or S, S, S-trimethyl phosphorotrithioate (10 −4−10 −2 M) resulted in a significant, but small stimulation of PPP (max. 216%), compared to that found in the presence of the model oxidant, paraquat (1089%, 10 −5 M). Following in vivo treatment with an LD 50 dose of OSSMe, PPP was unchanged (after 6 h) or decreased (after 24 h) compared to saline-treated rats. PPP was also decreased in slices which had been preincubated with OSSMe (10 −3 M) and then transferred to fresh medium. Following treatment with phorone (250 mg/kg i.p.) pulmonary levels of non-protein sulfhydryls were first reduced (20% of control at 3 h) and then increased (288% of control at 24 h), but at neither time was lung slice PPP activity affected, thus suggesting that in the rat lung PPP activity does not directly depend on pulmonary glutathione content.

The relevance of pentose phosphate pathway stimulation in rat lung to the mechanism of paraquat toxicity

Paraquat and diquat have been shown to stimulate the production of 14CO 2 from [1- 14C]-glucose by slices of rat lung, but not the production of 14CO 2 from [6- 14C]glucose. This indicates stimulation of the pentose phosphate pathway. Paraquat was effective at concentrations as low as 7.5 × 10 −7 M whilst a concentration of diquat of 10 −5 M was required for comparable stimulation. Maximal stimulation occurred with approximately 10 −5 M paraquat and approximately 10 −4 M diquat. The stimulation of pentose phosphate pathway in lung slices by paraquat has been shown to be related to paraquat accumulation. Lung slices from rats dosed intravenously with 65 μmoles of either paraquat or diquat/kg body wt had increased pentose phosphate pathway activity compared with slices from saline injected controls. At all times studied from 0.5 to 18 hr after injection, pentose phosphate pathway activity in slices from diquat poisoned rats was equal to or greater than that observed in slices from paraquat poisoned rats. Since only rats dosed intravenously with paraquat subsequently develop lung damage, it is concluded that there is no simple relationship between stimulation of the pentose phosphate pathway in lung and the production of lung damage.