Pentose Phosphate Pathway Inhibitor Research Articles

In vitro inhibition potency of malononitrile derivatives on the activity of two pentose phosphate pathway enzymes: accompanied by molecular docking evaluation.

Many disorders, including cancer and malaria, could be targeted via the pentose phosphate pathway (PPP),whose products are key in biosynthetic reactions incells. The goal of this study was to find new PPP inhibitors. The inhibition effects of malononitrile derivatives on Glucose 6-phosphate dehydrogenase (G6PD) and 6-phosphogluconate dehydrogenase (6PGD) were analyzed through invitro experiments. Besides, molecular docking studies were performed to predict the interactions having role in inhibition of compounds. K i constants of derivatives were found between 4.24±0.46-69.63±7.75 µM for G6PD and 1.91±0.12-95.07±11.08 µM for 6PGD. Derivatives indicated non-competitive inhibition on both enzymes except forcompound 4. The findings of the molecular docking studies revealed that free-binding energy estimations agreed with invitro data. The structure of these malononitrile derivatives may guide for drug discovery in targeting the PPP.

Abstract TMP83: Macrophage Metabolic Reprograming to Face Ferroptosis After ICH

Intracerebral Hemorrhage (ICH) is a destructive neurovascular disease with no efficient treatment. Trapped Red Blood Cells (RBCs) in the brain lyse releasing toxic heme from hemoglobin. Heme, a lipophilic molecule with iron, triggers severe oxidative stress, particularly to lipid-rich cell plasma membranes (PM), causing cell death via ferroptosis. Macrophages are specialized in hematoma phagocytosis; their survival determines hematoma detoxification in the brain. Using in vitro murine macrophage cultures treated with heme, we noted heme induced cell death (Fig 1A), along with heightened oxidized fatty acids (FAs), a marker of ferroptosis (Fig 1B). Heme-triggered cell death was countered by the ferroptosis inhibitors ferrostatin-1 (Fer-1), NAC, β-ME and Deferoxamine (DFO) (Fig 1C-F), which confirm that heme induces ferroptosis in macrophages. Macrophages may shift metabolism to the Pentose Phosphate Pathway (PPP) in the presence of heme to promotes NADPH synthesis, vital for heme degradation and oxidation defense. Dehydroisoandrosterone (DHEA), a potent PPP inhibitor, worsened heme-induced ferroptosis (Fig 1 G&H). This underscores the PPP's vital role against heme-driven ferroptosis. Ferroptosis involves PM oxidation and loss of integrity, causing cell death. Heme induced rapid extracellular ATP release (Fig I). Lysosomes contain abundant ATP, thus we hypothesized that ATP release triggers by heme is the result of PM repair by lysosomal exocytosis/endocytosis. Using metabolomics, we found that heme induced accumulation of membrane components like phospholipids, sphingolipids, and cholesterol (Fig J-L), all which require NADPH for synthesis. This suggests PPP aids NADPH production, aiding lysosomal synthesis for PM repair. Overall, the findings support a critical role for the PPP in macrophage resistance to ferroptosis in the presence of heme and suggest a possible new therapeutic target for macrophage survival after ICH.

Activation of the pentose phosphate pathway in macrophages is crucial for granuloma formation in sarcoidosis.

Sarcoidosis is a disease of unknown etiology in which granulomas form throughout the body and is typically treated with glucocorticoids, but there are no approved steroid-sparing alternatives. Here, we investigated the mechanism of granuloma formation using single-cell RNA-Seq in sarcoidosis patients. We observed that the percentages of triggering receptor expressed on myeloid cells 2-positive (TREM2-positive) macrophages expressing angiotensin-converting enzyme (ACE) and lysozyme, diagnostic makers of sarcoidosis, were increased in cutaneous sarcoidosis granulomas. Macrophages in the sarcoidosis lesion were hypermetabolic, especially in the pentose phosphate pathway (PPP). Expression of the PPP enzymes, such as fructose-1,6-bisphosphatase 1 (FBP1), was elevated in both systemic granuloma lesions and serum of sarcoidosis patients. Granuloma formation was attenuated by the PPP inhibitors in in vitro giant cell and in vivo murine granuloma models. These results suggest that the PPP may be a promising target for developing therapeutics for sarcoidosis.

Metabolic Alterations Associated with Loss of TET2 Potentiates Inflammation in Myeloid Cells Driving Breast Cancer Progression

Clonal hematopoiesis of indeterminate potential (CHIP) is characterized by the clonal expansion of hematopoietic stem cells possessing somatic mutations commonly associated with cancer, but with no detectable hematological cancer. Certain CHIP driver mutations, such as loss of function in Tet2 result in hyperinflammatory myeloid cells. The proinflammatory cytokine IL-1β promotes breast cancer progression and correlates with disease severity. Activation of the NLRP3 inflammasome in myeloid cells is the main source of IL-1β processing. Here, we demonstrate growth of the metastatic breast cancer cell line, E0771, in both germline and chimeric Tet2 +/- mice was 2-fold greater than that observed in Tet2 +/+ mice (Figure 1). The increased tumor burden in Tet2 +/- mice was shown to be NLRP3-dependent as Tet2 +/-mice receiving the NLRP3 inhibitor, OLT1177, exhibited significantly decreased tumor growth compared to Tet2 +/- mice with no treatment (Figure 1). Cellular composition of the tumor microenvironment was also assessed. Notably, we observed marked increases in tumor-infiltrating granulocytes in the Tet2 +/- mice. This observation was consistent with elevated serum cytokines associated with neutrophil recruitment and activation. Next, considering NLRP3 inflammasome activation is tightly linked to metabolic alterations, we sought to characterize the metabolic profile of Tet2 +/- myeloid cells. To start, global metabolic analysis revealed increased abundance of metabolites from glycolysis and pentose phosphate pathway in Tet2 +/- myeloid cells compared to Tet2 +/+ myeloid cells. Consistent with the metabolic demands of a hyperinflammatory phenotype, Seahorse Flux assays revealed significantly increased ECAR (glycolysis) and OCR (pentose phosphate pathway) as measured at baseline in Tet2 +/- myeloid cells compared to Tet2 +/+ myeloid cells. Lastly, to confirm whether metabolic alterations drive the hyperinflammatory phenotype in Tet2 +/- myeloid cells, we assessed cytokine production after LPS stimulation in Tet2 +/-and Tet2 +/+ myeloid cells treated with 2-DG (glycolysis inhibitor) or 6-AN (pentose phosphate pathway inhibitor). Expectedly, Tet2 +/- myeloid cells produced significantly more IL-1β, IL-6, TNFα and IL-18 compared to Tet2 +/+ myeloid cells after LPS stimulation. LPS induced IL-1β and IL-6 production was significantly decreased for both genotypes after 2-DG and 6-AN treatment. Importantly, LPS induced TNFα and IL-18 only decreased after 2-DG and 6-AN treatment in Tet2 +/- myeloid cells suggesting metabolic changes associated with loss of TET2 affect not only NLRP3 priming but TNFα secretion as well. Overall, these studies demonstrate how TET2-driven CHIP alters the tumor microenvironment in breast cancers through aberrant NLRP3 activation and characterizes the underlying metabolic alterations subsequent to loss of TET2, offering further mechanistic insight into the hyperinflammatory phenotype in myeloid cells associated with TET2-driven CHIP. Figure 1. Tet2+/- driven breast cancer progression is NLRP3 activation-dependent. Experimental designs; BM chimeras were generated by transplanting 75%WT:25%Tet2+/+ or Tet2+/- mixed BM, mice were orthotopically implanted with E0771 breast tumors and given standard or OLT1177 diet (OLT). Tumor growth curve from Tet2+/+(WT) or Tet2 +/- chimeric mice implanted with E0771 cells receiving standard or OLT1177 diet(OLT) (N=10, two independent experiments). ***p<0.001, **p<0.01, *p<0.05.

RADT-28. DISRUPTION OF BIOENERGETICS ENHANCES RADIO-SENSITIVITY OF PATIENT-DERIVED GLIOBLASTOMA TUMORSPHERES

Abstract INTRODUCTION Glioblastoma (GBM), the most common primary brain tumor, has been associated with poor prognosis despite various treatment modalities, the surgical resection accompanied by chemotherapy and radiotherapy. Although the importance of radiotherapy is well known, its combination with bioenergetics regulators has not been established in clinical practice. Here, we hypothesized that bioenergetics regulators improve the radio-sensitivity of GBM-tumorspheres (TS). METHODS Gene expression profiles of GBM patient-derived TSs were obtained from microarray. In vitro efficacies of the treatment were evaluated using clonogenic assay, 3D invasion assay, neurosphere-formation assay, and apoptosis-analysis. Protein expression was evaluated by western blot, and immunofluorescence was performed for detection of γH2AX foci. In vivo efficacies were confirmed using a mouse orthotopic-xenograft model. RESULTS GBM TSs with high-radioresistant or low-radioresistant were selected according to expression levels of radiation response associated genes. In order to select the bioenergetics regulators that significantly increased the radiotherapy effect of GBM-TSs, five bioenergetics regulators were combined which are oxidative-phosphorylation (OxPhos) inhibitor (IM156A), aldehyde-dehydrogenase inhibitor (gossypol) + OxPhos inhibitor (phenformin), fatty acid oxidation inhibitor (etomoxir), pentose-phosphate pathway inhibitor (dehydroepiandrosterone; DHEA) and glycolysis inhibitor (2-deoxy-D-glucose; 2DG). Amongst of these five bioenergetics regulators, DHEA and 2DG significantly augmented the therapeutic efficacies of ionizing radiation (IR) by reducing the survival fraction, stemness, and invasiveness in both high-radioresistant and low-radioresistant GBM TSs. IR combined with bioenergetics regulators (DHEA or 2DG) induced DNA damage response and apoptosis in low-radioresistant GBM TSs. In vivo experiments using a mouse orthotopic-xenograft model confirmed the tendency to prolong survival period and reduced the size in tumor after the combined treatment of 2DG with IR in low-radioresistant GBM TS. CONCLUSION These data proposes that the combination of bioenergetics regulators and radiotherapy is more effective than IR monotherapy itself. We expect that this combination regimen can strategically establish as a future treatment practice of GBM.

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.

TMET-26. PRIMARY OLIGODENDROGLIOMA CELL CULTURE VIABILITY: AN IN VITRO STUDY WITH METABOLIC MODULATORS

Abstract Oligodendrogliomas are tumors that develop from oligodendrocytes, the myelinating cells of the central nervous system. Oligodendrocytes are highly metabolically active cells that synthesize and transfer metabolites to neighboring cells. Given its intimate metabolic relation with neurons, we aim to investigate oligodendrocyte metabolism as an antitumoral target. A recurred oligodendroglioma WHO grade III was surgically removed from a 50-year patient after 10 years of progression-free disease. The tumor sample was mechanically digested and cultured at 37oC, 5% CO2, in DMEM: F12, 10 % FBS and antibiotics. Primary oligodendroglioma cells were trypsinized and seeded in a 96-well plate. After 24 hs, cells were treated with metabolic modulators: metformin (MET, mitochondrial complex II inhibitor, 5 mM), 2 deoxyglucose (2DG, hexokinase inhibitor, 1 mM), 6-aminonicotinamide (6AN, pentose phosphate pathway inhibitor, 50 µM) and/or 1400W and S-methylisothiourea (both iNOS inhibitors, 5 µM, and 50 µM respectively). Standard treatment with temozolomide (TMZ, 200 µM) was also performed. After 5 days of treatment, cells were stained with violet crystal. Two weeks after tumor sample digestion, a primary oligodendroglioma culture was successfully established. In vitro, proliferating cells appeared mostly undifferentiated with reduced branching complexity. Hexokinase inhibition by 2DG notoriously affected the viability of oligodendroglioma cells. Similar results were obtained with standard TMZ treatment. On the other hand, the inhibition of the pentose phosphate pathway by 6AN did not affect the cell monolayer. However, 6AN was able to increase the effect of MET as monotherapy. Both, MET and 2DG altered oligodendrocyte morphology inducing a more fusiform shape. Finally, iNOS inhibition modestly disrupted cell's monolayers and this effect did not seem to be improved by combinatory therapies. Glycolytic inhibitor 2DG resulted effective against oligodendroglioma cells. Whereas further studies are needed to validate these results, a better understanding of metabolic susceptibility could allow the development of better-targeted and more-effective therapeutic approaches.

Methyl benzoate derivatives as inhibitors of pentose phosphate pathway, which promotes cancer progression and drug resistance: An in silico study supported by in vitro results

The pentose phosphate pathway (PPP), whose products are vital in biosynthetic events, is targeted in the treatment of many diseases such as cancer and malaria. The objective of this study was to identify new PPP inhibitors. The inhibition effects of methyl 4-amino benzoates on glucose 6-phosphate dehydrogenase (G6PD) and 6-phosphogluconate dehydrogenase (6PGD) were analyzed through in vitro experiments and molecular docking studies were used to estimate inhibition mechanisms. IC50 values of compounds were found between 100.8 and 430.8μM for G6PD and 206 and 693.2μM for 6PGD. Molecular docking analysis showed that compound 1 was found the most effective inhibitor against hG6PD and compound 4 had the highest inhibitory potency against h6PGD with the estimated binding energy of -6.71 and -7.61kcal/mol, respectively. In conclusion, it was determined that in vitro and in silico outcomes of the study were highly correlated with each other. The structure of these benzoates may aid in the development of drugs that target the PPP.

Increasing nicotinamide adenine dinucleotide phosphate hydrogen levels via pentose phosphate pathway promotes phyenylpropanoid metabolism of fresh-cut white mushroom (Agaricus bisporus) under high O2/CO2 storage

Cutting usually shortens white mushroom shelf life due to wounding stress. The metabolic activities including phenylpropanoid metabolism can promote wound-healing ability in white mushroom, thus alleviating the negative effect of its cutting process. However, few reports have reported on the relationship between phenylpropanoid metabolism and nicotinamide adenine dinucleotide phosphate hydrogen (NADPH) content. In this study, the effects of high O2/CO2 treatment on the phenylpropanoid metabolism and NADPH level produced via the pentose phosphate pathway (PPP) in white mushroom slices were analyzed. White mushroom slices were first treated with 80% O2+20% CO2 controlled atmosphere for 3 h. The results showed that high O2/CO2 treatment enhanced phenylpropanoid metabolism, which is demonstrated by higher total phenol, flavonoid, and lignin content. High O2/CO2 treatment also reduced the total respiratory rate. However, it increased PPP and tricarboxylic acid cycle rates; inhibited decline in ATP content and improved NAD+ kinase activity. Moreover, NAD+ and NADH contents decreased, whereas NADP+ and NADPH contents increased. To further investigate the relationship between respiratory metabolism and phenylpropanoid metabolism, whole white mushroom were immersed in 0.1 mol L−1 Na3PO4 (a PPP inhibitor) and then the slices were treated with 80% O2+20% CO2 for 3 h. This led to lower key enzyme activity and metabolic product content in the phenylpropanoid metabolism compared with no Na3PO4 treatment. These results demonstrated that high oxygen treatment can effectively induce phenylpropanoid metabolism by increasing NADPH levels via stimulating the PPP. In conclusion, the PPP might play an important role of phenylpropanoid metabolism in fresh-cut white mushroom.

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.

The Pentose Phosphate Pathway and Its Involvement in Cisplatin Resistance.

Cisplatin is the first-line treatment for different types of solid tumors, such as ovarian, testicular, bladder, cervical, head and neck, lung, and esophageal cancers. The main problem related to its clinical use is the onset of drug resistance. In the last decades, among the studied molecular mechanisms of cisplatin resistance, metabolic reprogramming has emerged as a possible one. This review focuses on the pentose phosphate pathway (PPP) playing a pivotal role in maintaining the high cell proliferation rate and representing an advantage for cancer cells. In particular, the oxidative branch of PPP plays a role in oxidative stress and seems to be involved in cisplatin resistance. In light of these considerations, it has been demonstrated that overexpression and higher enzymatic activity of different enzymes of both oxidative and non-oxidative branches (such as glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, and transketolase) increase cisplatin resistance, and their silencing or combined treatment with cisplatin could restore cisplatin sensitivity. Moreover, drug delivery systems loaded with both PPP inhibitors and cisplatin give the possibility of reaching cancer cells selectively. In conclusion, targeting PPP is becoming a strategy to overcome cisplatin resistance; however, further studies are required to better understand the mechanisms.

A multiscale model to design therapeutic strategies that overcome drug resistance to tyrosine kinase inhibitors in multiple myeloma

Drug resistance (DR) is a phenomenon characterized by the tolerance of a disease to pharmaceutical treatment. In cancer patients, DR is one of the main challenges that limit the therapeutic potential of the existing treatments. Therefore, overcoming DR by restoring the sensitivity of cancer cells would be greatly beneficial. In this context, mathematical modeling can be used to provide novel therapeutic strategies that maximize the efficiency of anti-cancer agents and potentially overcome DR. In this paper, we present a new multiscale model devoted to the interaction of potential treatments with multiple myeloma (MM) development. In this model, MM cells are represented as individual objects that move, divide, and die by apoptosis. The fate of each cell depends on intracellular and extracellular regulation, as well as the administered treatment. The model is used to explore the combined effects of a tyrosine-kinase inhibitor (TKI) with a pentose phosphate pathway (PPP) inhibitor. We use numerical simulations to tailor effective and safe treatment regimens that may eradicate the MM tumors. The model suggests that an interval for the daily dose of the PPP inhibitor can maximize the responsiveness of MM cells to the treatment with TKIs. Then, it demonstrates that the combination of high-dose pulsatile TKI treatment with high-dose daily PPP inhibitor therapy can potentially eradicate the tumor.The predictions of numerical simulations using such a model can be considered as testable hypotheses in future pre-clinical experiments and clinical studies.

180 Glucose versus fatty acids: Different energy supplies for the bovine oocyte

Studies show that optimal concentrations of fatty acids (FA) and glucose are crucial for proper oocyte development, maturation, and further embryo quality support (Sutton McDowall et al. 2010 Reproduction 139, 685-95, https://doi.org/10.1530/REP-09-0345; Sutton McDowall et al. 2014 Theriogenology 82, 95-103, https://doi.org/10.1016/j.theriogenology.2014.03.011). A balance between metabolism of FA and glucose in oocytes needs to be maintained; however, it is not clear what the preferable pathway of energy production is and how it affects the oocyte. The aim of the experiment was to selectively block pathways of either glucose or FA metabolism during IVM of bovine oocytes to reveal changes within oocyte lipid droplets (LD) under crucial changes in energy metabolism. Cumulus-oocyte complexes were matured under standard conditions (Warzych et al. 2007Mol. Reprod. Dev. 74, 280-289; https://doi.org/10.1002/mrd.20610) without FA and glucose supplementation. The experimental groups were (1) control (IVM in basic medium), (2) group with inhibited glucose metabolism [supplementation with 1.5 µM iodoacetate (IO, inhibitor of glycolysis) and 150 µM dehydroepiandrosterone (DHEA, inhibitor of pentose phosphate pathway)], and (3) group with inhibited FA metabolism (150 µM etomoxir supplementation, ETO). Oocytes after 24h of IVM were stained with boron-dipyrromethene (BODIPY) 493/503 dye (lipid droplets) and 4′,6-diamidino-2-phenylindole (DAPI; chromatin) and analysed using a confocal microscope (LSM 880 AiryScan FAST; Zeiss). Obtained data were analysed using the Kruskal-Wallis test. The MII rate decreased to 60.5% for IO+DHEA (P<0.05) and to 78% for the ETO group compared with the control (83.8%). The average LD area (% of total oocyte area) significantly decreased in both experimental groups (IO+DHEA 6.72±2.5, ETO 6.28±3.2; P<0.01) compared with control (8.7±3.6). Total lipid content (intensity of the fluorescence), was significantly lower in experimental groups (IO+DHEA: 1.17×106±3×105, ETO: 7.1×104±2×104 vs. control: 1.68×106±4.6×105; P<0.01). With regard to the total number of LD, only in the IO+DHEA group were significantly fewer LD noted (987±343; P<0.05), whereas the ETO group did not differ significantly (1146±414) compared with control (1148±357). The ETO group had significantly lower total lipid content (P<0.01) and higher total LD number (P<0.05) compared with IO+DHEA. The area of lipid droplets did not differ between the experimental groups. These results show that blocking glucose metabolism strongly affects the nuclear maturation process of the oocyte, limiting the number of oocytes that reach the MII stage. A significant decrease in total lipid content in the ETO group may suggest strong utilisation of lipids during the maturation process, when de novo synthesis of lipids is blocked. Accumulation of lipids in LD before maturation allows this process to be passed even under the applied FA metabolism inhibitory conditions. In contrast, inhibition of glucose metabolism negatively affects oocyte development strictly after inhibition conditions, indicating the greater significance of glucose for proper oocyte maturation. Further studies are being conducted to investigate the effect of inhibitory systems on lipids in bovine cumulus cells and further embryos. Funding for this study was provided by National Science Centre, Poland (project no 2017/27/B/NZ9/00904).

CORM-401 induces calcium signalling, NO increase and activation of pentose phosphate pathway in endothelial cells.

Carbon monoxide-releasing molecules (CO-RMs) induce nitric oxide (NO) release (which requires NADPH), and Ca2+ -dependent signalling; however, their contribution in mediating endothelial responses to CO-RMs is not clear. Here, we studied the effects of CO liberated from CORM-401 on NO production, calcium signalling and pentose phosphate pathway (PPP) activity in human endothelial cell line (EA.hy926). CORM-401 induced NO production and two types of calcium signalling: a peak-like calcium signal and a gradual increase in cytosolic calcium. CORM-401-induced peak-like calcium signal, originating from endoplasmic reticulum, was reduced by thapsigargin, a SERCA inhibitor, and by dantrolene, a ryanodine receptors (RyR) inhibitor. In contrast, the phospholipase C inhibitor U73122 did not significantly affect peak-like calcium signalling, but a slow and progressive CORM-401-induced increase in cytosolic calcium was dependent on store-operated calcium entrance. CORM-401 augmented coupling of endoplasmic reticulum and plasmalemmal store-operated calcium channels. Interestingly, in the presence of NO synthase inhibitor (l-NAME) CORM-401-induced increases in NO and cytosolic calcium were both abrogated. CORM-401-induced calcium signalling was also inhibited by superoxide dismutase (poly(ethylene glycol)-SOD). Furthermore, CORM-401 accelerated PPP, increased NADPH concentration and decreased the ratio of reduced to oxidized glutathione (GSH/GSSG). Importantly, CORM-401-induced NO increase was inhibited by the PPP inhibitor 6-aminonicotinamide (6-AN), but neither by dantrolene nor by an inhibitor of large-conductance calcium-regulated potassium ion channel (paxilline). The results identify the primary role of CO-induced NO increase in the regulation of endothelial calcium signalling, that may have important consequences in controlling endothelial function.

Abstract 3548: Hypoxia-induced Cystathionine-β-synthase (CBS) expression: impact on colonic epithelial cells metabolism, proliferation and migration

Abstract Introduction: We have reported aberrant overexpression of CBS in human colorectal cancers, where its activity (H2S production) promotes tumor progression and metastasis. New data show that CBS expression increases in adenomatous polyps. The aims of this study were to identify possible mechanisms for CBS upregulation and determine the biological impact of its upregulation using a premalignant colonic epithelial cell line NCM356. Methods: Induction of CBS in response to LPS, TLR2&4 agonists, and hypoxia was assessed by immunoblotting. CBS overexpression was achieved using lentiviral expression vector (pReceiver-Lv103, GeneCopoeia™) in NCM356 cells. Global metabolic profiling (Metabolon, Durham) was performed on cellular extracts from NCM365 overexpressing cells (CBS-hi) and vector-transduced controls. High-resolution respirometry (Orobors Oxygraph-2k) assessed bioenergetics. H2S levels were measured using the fluorogenic probe 7-Azido-4-methylcoumarin. A Coulter counter was used to assess cell proliferation rates. Transwell assays were used to assess cell migration. CBS activity was inhibited with aminooxyacetic acid (AOAA). Results: CBS protein expression was increased by ~3-fold in pNCM after 2 and 24 hr of hypoxia (1% O2). Treatment with LPS but not the TLR agonists increased CBS expression 2-fold. Metabolic profiling of CBS-hi cells and controls identified 85 metabolites that were differentially expressed (65 increased and 20 decreased; p≤0.05 Welch’s 2-Sample t-Test). The metabolite lanthionine, which is produced by the CBS-dependent condensation of cysteine to produce H2S, showed the largest increase (12.3-fold increase, p=1.5E-06). A 4-fold increase in basal H2S production was measured in CBS-hi cells compared to control cultures. High-resolution respirometry showed that CBS-hi cells exhibited a significant increase in maximum respiration rate, reserve respiratory capacity and increased citrate synthase activity, suggesting increased cellular mitochondria mass. Treatment of CBS-hi cells either with pentose phosphate pathway inhibitor (oxythiamine) or lactate dehydrogenase inhibitor (XF-11) reducing growth by 90% and ~25% at day 5, respectively; the inhibitors had no effect on the vector control cells. Exposure of NCM356 cells to hypoxic conditions resulted in increased migration to conditioned media (p<0.001, NCM356 normoxia vs. hypoxia); inhibition of CBS using AOAA attenuated the hypoxia-induced migration. Conclusions: Hypoxia and LPS can induce CBS expression in a premalignant colonic epithelial cell line. Upregulation of CBS has a board impact on cellular metabolism including enhanced flux through the transsulfuration, glycolytic and pentose phosphate pathways, resulting in enhanced cellular bioenergetics, growth and migration. These data suggest that enhance CBS activity may be involved the adenoma to carcinoma sequence. Citation Format: Ches'Nique M. Phillips, John R. Zatarain, Michael E. Nicholls, Craig Porter, Steven Widen, Ketan Thanki, James W. Randall, Judith L. Hellmich, Manjit Maskey, Suimin Qiu, Thomas G. Wood, Nadiya Druzhyna, Bartosz Szczesny, Katalin Modis, Csaba Szabo, Celia Chao, Mark R. Hellmich. Hypoxia-induced Cystathionine-β-synthase (CBS) expression: impact on colonic epithelial cells metabolism, proliferation and migration [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3548. doi:10.1158/1538-7445.AM2017-3548

The Role of Glucose Metabolism on Porcine Oocyte Cytoplasmic Maturation and Its Possible Mechanisms.

In the present study, we investigated the potential role of glucose and pyruvate in the cytoplasmic maturation of porcine oocytes by investigating the effect of glucose and/or pyruvate supplementation, in the presence or absence of 10% porcine follicular fluid (PFF), on meiotic maturation and subsequent embryo development. In the absence of 10% PFF, without exogenous addition of glucose and pyruvate, the medium seemed unable to support maturation. In the presence of 10% PFF, the addition of 5.6 mM glucose and/or 2 mM pyruvate during in vitro maturation of cumulus enclosed oocytes increased MII oocyte and blastocyst rates. In contrast, oocytes denuded of cumulus cells were not able to take full advantage of the glucose in the medium, as only pyruvate was able to increase the MII rate and the subsequent early embryo developmental ability. Treatment of cumulus enclosed oocytes undergoing maturation with 200 μM dehydroepiandrosterone (DHEA), a pentose phosphate pathway inhibitor, or 2 μM iodoacetate (IA), a glycolysis inhibitor, significantly reduced GHS, intra-oocyte ATP, maternal gene expression, and MPF activity levels. DHEA was also able to increase ROS and reduce the levels of NADPH. Moreover, blastocysts of the DHEA- or IA-treated groups presented higher apoptosis rates and markedly lower cell proliferation cell rates than those of the non-treated group. In conclusion, our results suggest that oocytes maturing in the presence of 10% PFF can make full use of energy sources through glucose metabolism only when they are accompanied by cumulus cells, and that pentose phosphate pathway (PPP) and glycolysis promote porcine oocyte cytoplasmic maturation by supplying energy, regulating maternal gene expression, and controlling MPF activity.

Abstract 1058: Targeting pentose phosphate pathway (PPP) represents a novel therapeutic strategy for hepatocellular carcinoma (HCC) treatment

Abstract Background and objectives: Hepatocellular carcinoma (HCC) is the primary liver malignancy with an extremely low survival rate. HCC progression is frequently associated with accelerated glucose consumption that confers growth advantage to tumor cell through two essential metabolic pathways - glycolysis and the pentose phosphate pathway (PPP). Glycolysis utilizes glucose for ATP production, while the PPP converts glycolytic intermediates to generate NADPH, the cellular antioxidant, and ribose-5-phophate, the nucleotide precursor. Although glycolysis has been extensively studied in HCC, how PPP supports HCC growth remains largely unknown. Our study aims at delineating the clinical significance, regulation and functions of PPP in HCC development and explore the therapeutic potential of PPP inhibitors for HCC therapy. Methods: The expression and abundance of the the PPP genes were examined in 16 pairs of human HCC tumors and adjacent non-tumors by transcriptome sequencing. Level of reactive oxygen species (ROS) and glucose uptake were measured by CM-H2DCFDA and 2-NBDG stainings, respectively. Metabolomics study was performed with CE-TOF-MS analysis. Metabolic flux analysis was conducted using UPLC-MS/MS. Results: Transcriptome sequencing data showed that enzymes in the PPP were frequently upregulated in HCC. Transketolase (TKT), the gene encodes the reversible enzyme connecting PPP and glycolysis, is the most abundant and most overexpressed PPP gene in HCC. Overexpression of TKT was confirmed in an expanded sample cohort at the mRNA and protein level. Meanwhile, TKT overexpression was significantly correlated with venous invasion, microsatellite formation, tumor size and absence of tumor encapsulation. Notably, TKT expression was controlled by NRF2/KEAP1/BACH1 pathway, which is a major transcription regulator for redox homeostasis. CHIP assay revealed that NRF2 and BACH1 competitively bound to the same antioxidant responsive elements (ARE) in TKT. Functionally, knockdown of TKT in HCC cells retarded cell growth, attenuated glucose uptake and NADPH production, increased intracellular ROS, and induced oxidative stress-associated cell cycle delay. In line with these findings, knockdown of TKT greatly suppressed tumor growth in vivo. Metabolomics and metabolic flux analysis revealed that loss of TKT disrupted the PPP and subsequently reduced NADPH production. Intriguingly, genetic knockdown and pharmacological inhibition of TKT enhanced the efficacy of Sorafenib, the only FDA-approved drug for HCC treatment, both in vitro and in vivo. Conclusion: Our study suggested the clinical significance of TKT in HCC and illustrated the anti-oxidative role of TKT in HCC progression. We also proposed that disrupting the metabolic machinery by TKT inhibition might be a novel therapeutic strategy for HCC treatment. Citation Format: Ming Jing Xu, Kit Ho Lai, Shu Hai Lin, Pui Wah Tse, David Kung Chun Chiu, Hui Yu Koh, Cheuk Ting Law, Chun Ming Wong, Zong Wei Cai, Carmen Chak Lui Wong, Irene Oi Lin Ng. Targeting pentose phosphate pathway (PPP) represents a novel therapeutic strategy for hepatocellular carcinoma (HCC) treatment. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1058.

Implications of glycolytic and pentose phosphate pathways on the oxidative status and active mitochondria of the porcine oocyte during IVM

Glycolysis and the pentose phosphate pathway (PPP) were modulated in porcine cumulus-oocyte complexes during IVM by the addition of inhibitors and stimulators of key enzymes of the pathways to analyze their influence on the oxidative status, active mitochondria, and maturation of the oocyte. The influence of pharmacologic and physiological inhibitors of glycolysis (Sodium fluoride and ATP) and PPP (6-Aminonicotinamide and nicotinamide adenine dinucleotide phosphate) was validated by assessing glucose and lactate turnover and brilliant cresyl blue staining in oocytes. Inhibitors of glycolysis and PPP activity significantly perturbed nuclear maturation, oxidative metabolism (Redox Sensor Red CC-1), and active mitochondria (Mitotracker Green FM) within oocytes (P < 0.05). In comparison, physiological stimulators of glycolysis (adenosine monophosphate) and PPP (nicotinamide adenine dinucleotide phosphate) did not affect any of evaluated parameter. In the absence of modulators, fluctuations in the oocyte oxidative activity and active mitochondria were observed during porcine IVM. The inhibition of glycolysis and PPP modified the pattern of oxidation and mitochondrial fluctuation, resulting in impaired meiotic progression. We demonstrated the relationship between carbohydrate metabolism in COC and oocyte redox status necessary for porcine oocyte IVM.

Multiple myeloma acquires resistance to EGFR inhibitor via induction of pentose phosphate pathway.

Multiple myeloma (MM) was characterized by frequent mutations in KRAS/NRAS/BRAF within the EGFR pathway that could induce resistance to EGFR inhibitors. We here report that EGFR inhibition solely exhibited moderate inhibition in KRAS/NRAS/BRAF wildtype (triple-WT) MM cells, whilst had no effect in myeloma cells with any of the mutated genes. The moderate inhibitory effect was conferred by induction of pentose phosphate pathway (PPP) when cells were treated with Gefitinib, the EGFR inhibitor. Combination of Gefitinib with PPP inhibitor 6AN effected synergistically in triple-WT cells. The inhibition could be restored by addition of NADPH. Dual EGFR/ERBB2 inhibitor Afatinib also exhibited similar effects. Further genetic silencing of EGFR, ERBB2 and mTOR indicated that major effect conferred by ERBB2 was via convergence to EGFR pathway in MM. Our results contributed to the individualized targeted therapy with EGFR inhibitors in MM.

PO-0510 Inhibition Of Pentose Phosphate Pathway And Nadph Oxidase Impaired The Response Of Chicken Ductus Arteriosus To Oxygen

Introduction NADPH derived from the pentose phosphate pathway (PPP) is a key system involved in maintaining the function of several important redox and antioxidant defense mechanisms. NADPH oxidases contain a catalytic NOX subunit that transfers electrons from NADPH to oxygen, thereby forming reactive oxygen species (ROS). Normoxic contraction of the ductus arteriosus (DA), such as occurs at birth, appears to be dependent upon the increase of ROS in DA smooth muscle cells. We hypothesised a role for NOX-derived ROS in the signalling pathway of oxygen-induced contraction of the DA. Methods We investigated the effects of the inhibition of PPP and NOX in the ex vivo response of chicken DA to oxygen. Experiments were performed in myograph-mounted DA rings (pulmonary and aortic sides) isolated from chicken embryos incubated for 19 days (total incubation: 21-d). Results Exposure to oxygen (21%) induced a sustained contractile response in the pulmonary but relaxation in the aortic side of 19-d DA. Incubation with the PPP inhibitor epiandrosterone or with the NOX inhibitors GKT-136901, VAS2870 and VAS3947 elicited a partial or complete impairment of oxygen-induced contraction. Phenylephrine- and KCl-induced contraction of chicken DA were impaired by epiandrosterone and VAS3947 but not by the other NOX inhibitors. Moreover, VAS3947 evoked an irreversible impairment of the contractility of the vessel. Oxygen-induced relaxation in the aortic part of the DA was not affected by NOX inhibitors. Conclusions Our data indicate that PPP and NADPH oxidase activation are events involved in the signalling cascade ofnormoxic contraction of chicken DA.