Inhibition Of Pentose Phosphate Pathway Research Articles

Analysis of the Effects of Pentose Phosphate Pathway Inhibition on the Generation of Reactive Oxygen Species and Epileptiform Activity in Hippocampal Slices.

The pentose phosphate pathway (PPP) is one of three major pathways involved in glucose metabolism, which is regulated by glucose-6-phosphate dehydrogenase (G6PD) controls NADPH formation. NADPH, in turn, regulates the balance of oxidative stress and reactive oxygen species (ROS) levels. G6PD dysfunction, affecting the PPP, is implicated in neurological disorders, including epilepsy. However, PPP's role in epileptogenesis and ROS production during epileptic activity remains unclear. To clarify these points, we conducted electrophysiological and imaging analyses on mouse hippocampal brain slices. Using the specific G6PD inhibitor G6PDi-1, we assessed its effects on mouse hippocampal slices, examining intracellular ROS, glucose/oxygen consumption, the NAD(P)H level and ROS production during synaptic stimulation and in the 4AP epilepsy model. G6PDi-1 increased basal intracellular ROS levels and reduced synaptically induced glucose consumption but had no impact on baselevel of NAD(P)H and ROS production from synaptic stimulation. In the 4AP model, G6PDi-1 did not significantly alter spontaneous seizure frequency or H2O2 release amplitude but increased the frequency and peak amplitude of interictal events. These findings suggest that short-term PPP inhibition has a minimal impact on synaptic circuit activity.

Abstract WMP83: The Pentose Phosphate Pathway is Crucial for Combating Heme-Induced Ferroptosis in Red Blood Cells After ICH

Intracerebral hemorrhage (ICH) occurs upon blood vessel rupture, allowing blood into brain tissue. Red blood cells (RBCs) make up 99%+ of blood cells. Brain-trapped RBCs undergo phagocytosis; however, many RBCs lyse prior to being phagocytosed due to their abundance. RBC lysis releases hemoglobin and toxic heme, driving oxidative stress especially in lipid-rich plasma membranes (PM), causing tissue damage and ferroptosis. Few studies have explored RBC fate in ICH, but unraveling the factors governing RBC lysis may yield ICH therapies.In in vitro human and murine RBC cultures, we observed RBCs are exquisitely sensitive to heme-induced death with increased oxidized fatty acids (FAs), a ferroptosis marker (Fig 1A-B). The ferroptosis inducer Erastin validated RBC susceptibility to ferroptosis (Fig 1C). Heme-induced cell death was countered by β-mercaptoethanol (β-ME), N-acetylcysteine (NAC), ferrostatin-1 (Fer-1) and Vitamin E (Vit. E), ferroptosis inhibitors, confirming RBC death was due to ferroptosis (Fig 1D-G). Heme and Erastin induced RBC ferroptosis differs from eryptosis, RBC specific programmed cell death, as it isn't Ca++ dependent (Fig1H&I). Unlike other cells, RBCs solely rely on the Pentose Phosphate Pathway (PPP) for NADPH production for oxidation defense. PPP inhibition with Dehydroisoandrosterone (DHEA) and treatment with ThioNa, an NADP+ synthesis inhibitor, both heightened heme-induced RBC ferroptosis (Fig 1J-L). These data support the PPP's role against heme-driven ferroptosis. Heme induced RBC ferroptosis may trigger a chain reaction of RBC lysis and ongoing heme release. Notably, 400 million people face G6PD deficiency, the rate-limiting PPP enzyme, leading to NADPH depletion in RBCs with enhanced susceptibility to oxidation. Hence, in addition to identifying the PPP in RBCs resistance to heme toxicity, our findings could aid in identifying a population that is particularly vulnerable to the devastating effects of ICH.

Leishmania mexicana centrin knockout parasites promote M1-polarizing metabolic changes

Leishmaniasis is a tropical disease prevalent in 90 countries. Presently, there is no approved vaccine for human use. We developed a live attenuated L.mexicana Cen-/-(LmexCen-/-) strain as a vaccine candidate that showed excellent efficacy, characterized by reduced Th2 and enhanced Th1 responses in C57BL/6 and BALB/c mice, respectively, compared to wild-type L.mexicana (LmexWT) infection. Toward understanding the immune mechanisms of protection, we applied untargeted mass spectrometric analysis to LmexCen-/- and LmexWT infections. Data showed enrichment of the pentose phosphate pathway (PPP) in ears immunized with LmexCen-/-versus naive and LmexWT infection. PPP promotes M1 polarization in macrophages, suggesting a switch to a pro-inflammatory phenotype following LmexCen-/- inoculation. Accordingly, PPP inhibition in macrophages infected with LmexCen-/- reduced the production of nitric oxide and interleukin (IL)-1β, hallmarks of classical activation. Overall, our study revealed the immune regulatory mechanisms that may be critical for the induction of protective immunity.

The pentose phosphate pathway in health and disease.

The pentose phosphate pathway (PPP) is a glucose-oxidizing pathway that runs in parallel to upper glycolysis to produce ribose 5-phosphate and nicotinamide adenine dinucleotide phosphate (NADPH). Ribose 5-phosphate is used for nucleotide synthesis, while NADPH is involved in redox homoeostasis as well as in promoting biosynthetic processes, such as the synthesis of tetrahydrofolate, deoxyribonucleotides, proline, fatty acids and cholesterol. Through NADPH, the PPP plays a critical role in suppressing oxidative stress, including in certain cancers, in which PPP inhibition may be therapeutically useful. Conversely, PPP-derived NADPH also supports purposeful cellular generation of reactive oxygen species (ROS) and reactive nitrogen species (RNS) for signalling and pathogen killing. Genetic deficiencies in the PPP occur relatively commonly in the committed pathway enzyme glucose-6-phosphate dehydrogenase (G6PD). G6PD deficiency typically manifests as haemolytic anaemia due to red cell oxidative damage but, in severe cases, also results in infections due to lack of leucocyte oxidative burst, highlighting the dual redox roles of the pathway in free radical production and detoxification. This Review discusses the PPP in mammals, covering its roles in biochemistry, physiology and disease.

Lipidomics and Transcriptomics Differ Liposarcoma Differentiation Characteristics That Can Be Altered by Pentose Phosphate Pathway Intervention.

Liposarcoma (LPS) is a rare and heterogeneous malignancy of adipocytic origin. Well-differentiated liposarcoma (WDLPS) and dedifferentiated liposarcoma (DDLPS) are two of the most common subtypes, showing similar genetic characterizations but distinct biological behaviors and clinical prognosis. Compared to WDLPS, DDLPS is more aggressive and has the potential of metastasis, as the malignant adipocytic tumor's metabolic changes may have taken place during the tumorigenesis of LPSs. Therefore, to investigate the lipid alterations between the two subtypes, high-resolution liquid chromatography tandem mass spectrometry (LC-MS/MS) based untargeted lipidomic analysis was performed onto LPS tissues from 6 WDLPS and 7 DDLPS patients. The lipidomic analysis showed the upregulated phosphatidylcholines and phosphoethanolamines in DDLPS, and the upregulated triglycerides and diglycerides in WDLPS, which might be due to the uncompleted adipocytic dedifferentiation leading to such tumorigenesis. Such a finding was also confirmed by the similarity comparison of two LPS subtypes to the transcriptome of stromal vascular fraction at different differentiation stages. Transcriptomic analysis also demonstrated that metabolic pathways including the pentose phosphate pathway (PPP) were upregulated in WDLPS compared to DDLPS. Therefore, the cell line LPS853 was treated with the PPP inhibitor 6-aminonicotinamide ex vivo and the proliferation and invasion of LPS853 was significantly promoted by PPP inhibition, suggesting the potential role of PPP in the development and differentiation of LPS. In conclusion, this study described the altered lipid profiles of WDLPS and DDLPS for the first time, revealing the different differentiation stages of the two subtypes and providing a potential metabolic target for LPS treatment.

Elevated pentose phosphate pathway flux supports appendage regeneration

A fundamental step in regeneration is rapid growth to replace lost tissue. Cells must generate sufficient lipids, nucleotides, and proteins to fuel rapid cell division. To define metabolic pathways underlying regenerative growth, we undertake a multimodal investigation of metabolic reprogramming in Xenopus tropicalis appendage regeneration. Regenerating tissues have increased glucose uptake; however, inhibition of glycolysis does not decrease regeneration. Instead, glucose is funneled to the pentose phosphate pathway (PPP), which is essential for full tail regeneration. Liquid chromatography-mass spectrometry (LC-MS) metabolite profiling reveals increased nucleotide and nicotinamide intermediates required for cell division. Using single-cell RNA sequencing (scRNA-seq), we find that highly proliferative cells have increased transcription of PPP enzymes and not glycolytic enzymes. Further, PPP inhibition results in decreased cell division specifically in regenerating tissue. Our results inform a model wherein regenerating tissues direct glucose toward the PPP, yielding nucleotide precursors to drive regenerative cell proliferation.

Ketogenic diet attenuates post-cardiac arrest brain injury by upregulation of pentose phosphate pathway–mediated antioxidant defense in a mouse model of cardiac arrest

ObjectiveThe aim of this study was to investigate the effect of the ketogenic diet (KD) on post-cardiac arrest brain injury in a mouse model of cardiac arrest and cardiopulmonary resuscitation. MethodsMice were fed a KD for 4 wk and then subjected to cardiac arrest and cardiopulmonary resuscitation. The HT22 cells after β-hydroxybutyrate (β-OHB) treatment were exposed to oxygen-glucose deprivation/reoxygenation. Survival and neurologic function were measured after return of spontaneous circulation. Positron emission tomography/computed tomography scanning, 13C-magnetic resonance spectroscopy analysis, and seahorse assay were performed to explore the mechanism underlying the phenotype. ResultsResults of this study demonstrated that KD improved neurologic function and reduced apoptotic neurons in cardiac arrest and cardiopulmonary resuscitation mice. With no alteration of glucose uptake, KD suppressed glucose oxidation in mouse brain. Consistently, the glycolytic capacity of the HT22 cells was also downregulated by β-OHB treatment. Moreover, KD increased nicotinamide adenine dinucleotide phosphate/oxidized nicotinamide adenine dinucleotide phosphate and reduced glutathione/oxidized glutathione couples and reduced reactive oxygen species in the brain, probably due to activation of glucose-6-phosphate dehydrogenase, the rate-limiting enzyme in the pentose phosphate pathway. Pharmacologic inhibition of pentose phosphate pathway totally abolished the influence of β-OHB on glycolysis, and post–oxygen-glucose deprivation/reoxygenation cell viability and reactive oxygen species production in HT22 cells. ConclusionKD improved survival and attenuated post-cardiac arrest brain injury, which was mediated by upregulation of pentose phosphate pathway and related antioxidant defense system.

P1249: INHIBITION OF THE PENTOSE-PHOSPHATE-PATHWAY (PPP) MEDIATES MACROPHAGE REPROGRAMMING FROM SUPPORTIVE BYSTANDER FUNCTION TOWARDS ANTIBODY-DEPENDENT LYMPHOMA CELL CLEARANCE AND LYMPHOMA SUPPRESSION

Background: Treatment response in lymphoma depends on the interaction and functional status of macrophages as important effector cells for tumor cell clearance by antibody-dependent cellular phagocytosis (ADCP). In the tumor microenvironment (TME) there is a limited supply of nutrients which implicates metabolic reprograming of immune cells such as changed phagocytic capacity of macrophages. Aims: We aimed to identify metabolic pathways regulating macrophages in the TME to improve macrophage effector cell function in lymphoma therapy. Methods: We addressed macrophage lymphoma cell co-cultures using macrophage cell lines, primary murine macrophages and human monocyte-derived macrophages. For pathway inhibition specific compounds as well as shRNA-mediated knockdown were used for functional read-out of ADCP and viability in lymphoma co-culture. Macrophage reprogramming was assessed by microscopy, FACS-immunophenotyping, ELISA, (phospho)-proteomic and metabolomic assessment. C57BL/6 mice and the humanized aggressive lymphoma model hMB were used for in vivo treatment with the pentose phosphate pathway (PPP) inhibitor S3 and analysis of macrophage programming and overall survival. Results: Inhibition of AMP kinase, glycolysis or mitochondrial ATP-production did not increase the phagocytosis rate in ADCP co-cultures. Inhibiting the PPP induced an increased lymphoma cell phagocytosis by macrophages. Under PPP-inhibition, the oxygen consumption and the glycolysis of macrophages was increased and the cells developed a more activated morphology with formation of filopodia, which are needed for phagocytosis initiation. Moreover, a down-regulation of immune-inhibitory surface proteins PD-L1 and SIRP1α was seen and cytokine secretion was shifted from anti-inflammatory IL10 to pro-inflammatory IL6 secretion. PPP-inhibition of macrophages significantly reduced CLL-cell support in vitro abrogating the “nurse-like” effect of macrophages. The increased lymphoma cell clearance and phenotypic alterations of macrophages were also observed in PPP-enzyme knockdown of transketolase (TKT) and 6-phosphogluconatedehydrogenase (6PGD) in macrophages and by inhibiting the PPP in primary murine and human macrophages. In a multiomics assessment of PPP-inhibition on proteome, phosphoproteome and metabolome level, we found protein expression alterations regulating metabolism and immunity. A connection between PPP inhibition and changed immune profile by modulation of the Stat1-Irg1-itaconate axis was identified and validated. In a humanized lymphoma mouse model the addition of PPP-inhibitor S3 led to significant prolonged survival of lymphoma bearing mice and an increased maturation and pro-inflammatory polarization of macrophages. Summary/Conclusion: An increased maturation, activation and repolarization of macrophages towards a pro-inflammatory and phagocytic active phenotype was observed by inhibiting the pentose-phosphate-pathway (PPP). This led to a significant survival benefit in in vivo lymphoma mouse model. We were able to point out the underlying Stat1-Irg1-itaconate signalling axis, connecting metabolic regulation and immune-phenotype of macrophages. We hypothesize the PPP as a key regulator of macrophage function determining the support of malignant B cell growth versus the capacity of lymphoma cell clearance and thereby therapy outcome. By that, we have identified the PPP as a targetable modulator of macrophage polarization to improve efficacy of B cell lymphoma and CLL therapy.

Metabolic plasticity imparts erlotinib-resistance in pancreatic cancer by upregulating glucose-6-phosphate dehydrogenase

Pancreatic ductal adenocarcinoma (PDAC) is one of the most malignant forms of cancer. Lack of effective treatment options and drug resistance contributes to the low survival among PDAC patients. In this study, we investigated the metabolic alterations in pancreatic cancer cells that do not respond to the EGFR inhibitor erlotinib. We selected erlotinib-resistant pancreatic cancer cells from MiaPaCa2 and AsPC1 cell lines. Metabolic profiling of erlotinib-resistant cells revealed a significant downregulation of glycolytic activity and reduced level of glycolytic metabolites compared to the sensitive cells. The resistant cells displayed elevated expression of the pentose phosphate pathway (PPP) enzymes involved in ROS regulation and nucleotide biosynthesis. The enhanced PPP elevated cellular NADPH/NADP+ ratio and protected the cells from reactive oxygen species (ROS)-induced damage. Inhibition of PPP using 6-aminonicotinamide (6AN) elevated ROS levels, induced G1 cell cycle arrest, and sensitized resistant cells to erlotinib. Genetic studies identified elevated PPP enzyme glucose-6-phosphate dehydrogenase (G6PD) as an important contributor to erlotinib resistance. Mechanistically, our data showed that upregulation of inhibitor of differentiation (ID1) regulates G6PD expression in resistant cells thus contributing to altered metabolic phenotype and reduced response to erlotinib. Together, our results highlight an underlying role of tumor metabolism in PDAC drug response and identify G6PD as a target to overcome drug resistance.

Abstract 2548: Pentose phosphate pathway inhibition potentiates metformin cytotoxic effects on HNSCC cells

Abstract The pentose phosphate pathway (PPP) enables cancer cells to adapt to anabolic demands that require rapid DNA, RNA, and lipid synthesis and to oxidative cellular stress. Metformin can efficiently inhibit the malignant progression of oral premalignant lesions by indirectly inhibiting the mammalian target of rapamycin (mTORC1). Frequently activated in cancer cells, mTORC1 may upregulate the oxidative branch of the pentose phosphate pathway (PPP) through the sterol regulatory element binding protein (SREBP1). Our previous results showed that the combination of metformin and 6-AN (a PPP inhibitor) strongly potentiates their individual cytotoxic effects on melanoma, glioblastoma, mammary and colon carcinoma cell lines. However, the mechanism of this synergism remains unclear. In this context, the aim of this project was to determine the cell signaling events underlying the therapeutic potential of this combinatory approach, against HNSCC cell lines. Then, we study the effect of metformin alone or in combination with 6-AN on AMPK/mTOR/S6K/S6 and SREBP1 pathway. We found that the inhibition of PPP strongly potentiated metformin cytotoxic effect on 2D and 3D cultures of CAL33 and HN12, both HNSCC cell lines. Furthermore, the combination synergized to increase the activation of AMPK, concomitant with decreased phosphorylation of mTOR, decreased phosphorylation of S6K and decreased phosphorylation of S6. Interestingly, activation of SREBP1 was also synergistically decreased by this combinatory approach. We conclude that the inhibition of PPP and metformin converges to inhibit mTOR pathway. This interaction deserves to be further investigated as a potential strategy to potentiate metformin therapy. Citation Format: Marcela Solange Villaverde, Jorge Silvio Gutkind. Pentose phosphate pathway inhibition potentiates metformin cytotoxic effects on HNSCC cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2548.

IsoSearch: An Untargeted and Unbiased Metabolite and Lipid Isotopomer Tracing Strategy from HR-LC-MS/MS Datasets.

Stable isotopic tracer analysis is a technique used to determine carbon or nitrogen atom incorporation into biological systems. A number of mass spectrometry based approaches have been developed for this purpose, including high-resolution tandem mass spectrometry (HR-LC-MS/MS), selected reaction monitoring (SRM) and parallel reaction monitoring (PRM). We have developed an approach for analyzing untargeted metabolomic and lipidomic datasets using high-resolution mass spectrometry with polarity switching and implemented our approach in the open-source R script IsoSearch and in Scaffold Elements software. Using our strategy, which requires an unlabeled reference dataset and isotope labeled datasets across various biological conditions, we traced metabolic isotopomer alterations in breast cancer cells (MCF-7) treated with the metabolic drugs 2-deoxy-glucose, 6-aminonicotinamide, compound 968, and rapamycin. Metabolites and lipids were first identified by the commercial software Scaffold Elements and LipidSearch, then IsoSearch successfully profiled the 13C-isotopomers extracted metabolites and lipids from 13C-glucose labeled MCF-7 cells. The results interpreted known models, such as glycolysis and pentose phosphate pathway inhibition, but also helped to discover new metabolic/lipid flux patterns, including a reactive oxygen species (ROS) defense mechanism induced by 6AN and triglyceride accumulation in rapamycin treated cells. The results suggest the IsoSearch/Scaffold Elements platform is effective for studying metabolic tracer analysis in diseases, drug metabolism, and metabolic engineering for both polar metabolites and non-polar lipids.

Snail augments fatty acid oxidation by suppression of mitochondrial ACC2 during cancer progression.

Despite the importance of mitochondrial fatty acid oxidation (FAO) in cancer metabolism, the biological mechanisms responsible for the FAO in cancer and therapeutic intervention based on catabolic metabolism are not well defined. In this study, we observe that Snail (SNAI1), a key transcriptional repressor of epithelial-mesenchymal transition, enhances catabolic FAO, allowing pro-survival of breast cancer cells in a starved environment. Mechanistically, Snail suppresses mitochondrial ACC2 (ACACB) by binding to a series of E-boxes located in its proximal promoter, resulting in decreased malonyl-CoA level. Malonyl-CoA being a well-known endogenous inhibitor of fatty acid transporter carnitine palmitoyltransferase 1 (CPT1), the suppression of ACC2 by Snail activates CPT1-dependent FAO, generating ATP and decreasing NADPH consumption. Importantly, combinatorial pharmacologic inhibition of pentose phosphate pathway and FAO with clinically available drugs efficiently reverts Snail-mediated metabolic reprogramming and suppresses in vivo metastatic progression of breast cancer cells. Our observations provide not only a mechanistic link between epithelial-mesenchymal transition and catabolic rewiring but also a novel catabolism-based therapeutic approach for inhibition of cancer progression.

A small molecule G6PD inhibitor reveals immune dependence on pentose phosphate pathway

Glucose is catabolized by two fundamental pathways, glycolysis to make ATP and the oxidative pentose phosphate pathway to make NADPH. The first step of the oxidative pentose phosphate pathway is catalyzed by the enzyme glucose-6-phosphate dehydrogenase (G6PD). Here we develop metabolite reporter and deuterium tracer assays to monitor cellular G6PD activity. Using these, we show that the most widely cited G6PD antagonist, dehydroepiandosterone (DHEA), does not robustly inhibit G6PD in cells. We then identify a small molecule (G6PDi-1) that more effectively inhibits G6PD. Across a range of cultured cells, G6PDi-1 depletes NADPH most strongly in lymphocytes. In T cells but not macrophages, G6PDi-1 markedly decreases inflammatory cytokine production. In neutrophils, it suppresses respiratory burst. Thus, we provide a cell-active small molecule tool for oxidative pentose phosphate pathway inhibition, and use it to identify G6PD as a pharmacological target for modulating immune response.

The role of miR-122 in the dysregulation of glucose-6-phosphate dehydrogenase (G6PD) expression in hepatocellular cancer

Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related deaths worldwide. Thus, a better understanding of molecular aberrations involved in HCC pathogenesis is necessary for developing effective therapy. It is well established that cancer cells metabolize energy sources differently to rapidly generate biomass. Glucose-6-phosphate-dehydrogenase (G6PD), the rate-limiting enzyme of the Pentose Phosphate Pathway (PPP), is often activated in human malignancies to generate precursors for nucleotide and lipid synthesis. Here, we determined the clinical significance of G6PD in primary human HCC by analyzing RNA-seq and clinical data in The Cancer Genome Atlas. We found that the upregulation of G6PD correlates with higher tumor grade, increased tumor recurrence, and poor patient survival. Notably, liver-specific miR-122, which is essential for metabolic homeostasis, suppresses G6PD expression by directly interacting with its 3′UTR. Luciferase reporter assay confirmed two conserved functional miR-122 binding sites located in the 3′-UTR of G6PD. Furthermore, we show that ectopic expression of miR-122 and miR-1, a known regulator of G6PD expression coordinately repress G6PD expression in HCC cells. These miRNAs also reduced G6PD activity in HepG2 cells that express relatively high activity of this enzyme. Collectively, this study provides evidence that anti-HCC efficacy of miR122 and miR-1 could be mediated, at least in part, through inhibition of PPP by suppressing the expression of G6PD.

PARIS reprograms glucose metabolism by HIF-1α induction in dopaminergic neurodegeneration

Our previous study found that PARIS (ZNF746) transcriptionally suppressed transketolase (TKT), a key enzyme in pentose phosphate pathway (PPP) in the substantia nigra (SN) of AAV-PARIS injected mice. In this study, we revealed that PARIS overexpression reprogrammed glucose metabolic pathway, leading to the increment of glycolytic proteins along with TKT reduction in the SN of AAV-PARIS injected mice. Knock-down of TKT in differentiated SH-SY5Y cells led to an increase of glycolytic enzymes and decrease of PPP-related enzymes whereas overexpression of TKT restored PARIS-mediated glucose metabolic shift, suggesting that glucose metabolic alteration by PARIS is TKT-dependent. Inhibition of PPP by either PARIS overexpression or TKT knock-down elevated the level of H2O2, and diminished NADPH and GSH levels, ultimately triggering the induction of HIF-1α, a master activator of glycolysis. In addition, TKT inhibition by stereotaxic injection of oxythiamine demonstrated slight decrement of dopaminergic neurons (DNs) in SN but not cortical neurons in the cortex, suggesting that TKT might be a survival factor of DNs. In differentiated SH-SY5Y, cell toxicity by GFP-PARIS was partially restored by introduction of Flag-TKT and siRNA-HIF-1α. We also observed the increase of HIF-1α and glycolytic hexokinase 2 in the SN of Parkinson's disease patients. Taken together, these results suggest that PARIS accumulation might distort the balance of glucose metabolism, providing clues for understanding mechanism underlying selective DNs death by PARIS.

UCHL1-HIF-1 axis-mediated antioxidant property of cancer cells as a therapeutic target for radiosensitization

Hypoxia-inducible factor 1 (HIF-1) has been recognized as an important mediator of the reprogramming of carbohydrate metabolic pathways from oxidative phosphorylation to accelerated glycolysis. Although this reprogramming has been associated with the antioxidant and radioresistant properties of cancer cells, gene networks triggering the HIF-1-mediated reprogramming and molecular mechanisms linking the reprogramming with radioresistance remain to be determined. Here, we show that Ubiquitin C-terminal hydrolase-L1 (UCHL1), which we previously identified as a novel HIF-1 activator, increased the radioresistance of cancer cells by producing an antioxidant, reduced glutathione (GSH), through HIF-1-mediated metabolic reprogramming. A luciferase assay to monitor HIF-1 activity demonstrated that the overexpression of UCHL1, but not its deubiquitination activity-deficient mutant (UCHL1 C90S), upregulated HIF-1 activity by stabilizing the regulatory subunit of HIF-1 (HIF-1α) in a murine breast cancer cell line, EMT6. UCHL1 overexpression induced the reprogramming of carbohydrate metabolism and increased NADPH levels in a pentose phosphate pathway (PPP)-dependent manner. The UCHL1-mediated reprogramming elevated intracellular GSH levels, and consequently induced a radioresistant phenotype in a HIF-1-dependent manner. The pharmacological inhibition of PPP canceled the UCHL1-mediated radioresistance. These results collectively suggest that cancer cells acquire antioxidant and radioresistant phenotypes through UCHL1-HIF-1-mediated metabolic reprogramming including the activation of PPP and provide a rational basis for targeting this gene network for radiosensitization.

Pentose phosphate pathway inhibition induce Endoplasmic Reticulum stress and autophagy

Pentose phosphate pathway (PPP) is a major glucose catabolism pathway that supplies the cell with a reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) and ribose-5-phosphate. NADPH is necessary for the detoxification of reactive oxygen species (ROS) and reductive biosynthesis. A key player in this pathway is the enzyme glucose-6-phosphate dehydrogenase (G6PD) that reduces NADP+ to NADPH, oxidizes glucose-6-phosphate and prevents ROS accumulation. Here, we show that the natural molecule 3,4’,5-trihydroxystilbene-3-β-d-glucoside (Polydatin) inhibits glucose-6-phosphate dehydrogenase (G6PD). As expected, G6PD inhibition causes an imbalance in NADP+/NADPH ratio, leading to a redox imbalance, followed by Endoplasmic Reticulum (ER) stress, autophagy, cell cycle block and apoptosis. we have demonstrated a link between G6PD inhibition and ER stress, showing that Unfolded Protein Response mediator such as PERK and IRE-1 have a key role in inducing autophagy and apoptosis after PPP block. Moreover, combination of PPP inhibition with autophagy inhibitors, such as chloroquine, strongly potentiate cytotoxicity on cancer cells, evidencing the role of autophagy as an escaping mechanism. This results shows that double inhibition of PPP and autophagy may be an affective therapeutic strategy against cancer.

Control of Insulin Secretion by Production of Reactive Oxygen Species: Study Performed in Pancreatic Islets from Fed and 48-Hour Fasted Wistar Rats.

Mitochondria and NADPH oxidase are important sources of reactive oxygen species in particular the superoxide radical (ROS) in pancreatic islets. These molecules derived from molecular oxygen are involved in pancreatic β-cells signaling and control of insulin secretion. We examined the involvement of ROS produced through NADPH oxidase in the leucine- and/or glucose-induced insulin secretion by pancreatic islets from fed or 48-hour fasted rats. Glucose-stimulated insulin secretion (GSIS) in isolated islets was evaluated at low (2.8 mM) or high (16.7 mM) glucose concentrations in the presence or absence of leucine (20 mM) and/or NADPH oxidase inhibitors (VAS2870–20 μM or diphenylene iodonium—DPI—5 μM). ROS production was determined in islets treated with dihydroethidium (DHE) or MitoSOX Red reagent for 20 min and dispersed for fluorescence measurement by flow cytometry. NADPH content variation was examined in INS-1E cells (an insulin secreting cell line) after incubation in the presence of glucose (2.8 or 16.7 mM) and leucine (20 mM). At 2.8 mM glucose, VAS2870 and DPI reduced net ROS production (by 30%) and increased GSIS (by 70%) in a negative correlation manner (r = -0.93). At 16.7 mM glucose or 20 mM leucine, both NADPH oxidase inhibitors did not alter insulin secretion neither net ROS production. Pentose phosphate pathway inhibition by treatment with DHEA (75 μM) at low glucose led to an increase in net ROS production in pancreatic islets from fed rats (by 40%) and induced a marked increase (by 144%) in islets from 48-hour fasted rats. The NADPH/NADP+ ratio was increased when INS-1E cells were exposed to high glucose (by 4.3-fold) or leucine (by 3-fold). In conclusion, increased ROS production through NADPH oxidase prevents the occurrence of hypoglycemia in fasting conditions, however, in the presence of high glucose or high leucine levels, the increased production of NADPH and the consequent enhancement of the activity of the antioxidant defenses mitigate the excess of ROS production and allow the secretory process of insulin to take place.

Inhibition of pentose phosphate pathway suppresses acute myelogenous leukemia.

Pentose phosphate pathway (PPP) is a metabolic pathway that generates NADPH and pentose. PPP genes have been reported to be primarily or secondarily upregulated in many cancers. We aimed to study the general alteration of PPP in acute myelogenous leukemia (AML). We performed data mining and analysis of the Cancer Genome Atlas (TCGA) AML dataset for genetic alteration of the PPP gene set. In vitro studies including proliferation, migration, and invasion assays, together with metabolite consumption and oxidation assays, were performed. PPP genes were upregulated in 61% of patients with AML. The majority of altered cases were expression changes measured by RNA sequencing. Expressions of critical PPP genes such as G6PD, PFKL, PFKP, and PGLS were consistently upregulated in all altered cases. Altered PPP is not associated with survival or disease relapse. PPP inhibition using 6-aminonicotinamide (6AN) increases glucose oxidative metabolism in AML. 6AN decreased the glucose oxidation and increased fatty acid oxidation. Here, we showed that PPP inhibition increased glucose oxidative metabolism in AML. PPP inhibition suppressed growth, migration, and invasion of AML, but not colony formation. PPP plays an important role in AML. Our results could contribute to the development of novel targeted treatment.

Oxidative pentose phosphate pathway inhibition is a key determinant of antimalarial induced cancer cell death.

Despite immense interest in employing antimalarials as autophagy inhibitors to treat cancer, it remains unclear if these agents act predominantly via autophagy inhibition or whether other pathways direct their anti-cancer properties. By comparing the treatment effects of the antimalarials chloroquine (CQ) and quinacrine (Q) on KRAS mutant lung cancer cells, we demonstrate that inhibition of the oxidative arm of the pentose phosphate pathway (oxPPP) is required for antimalarial induced apoptosis. Despite inhibiting autophagy, neither CQ treatment nor RNAi against autophagy regulators (ATGs) promote cell death. In contrast, Q triggers high levels of apoptosis, both in vitro and in vivo, and this phenotype requires both autophagy inhibition and p53-dependent inhibition of the oxPPP. Simultaneous genetic targeting of the oxPPP and autophagy is sufficient to trigger apoptosis in lung cancer cells, including cells lacking p53. Thus, in addition to reduced autophagy, oxPPP inhibition serves as an important determinant of antimalarial cytotoxicity in cancer cells.