pentose phosphate pathway vs glycolysis

Time points for FACS-separation of fast dividing (PKHlow) and slowly dividing (PKHhigh) subpopulations were optimized for each line to maximize differences in fluorescence intensity as well as population size for further analyses and were 2 days for G55 cells and 14 days for GS-11 cells. Up Next. Abbreviations: HK2, hexokinase 2; GPI, glucose-6-phosphate isomerase; PFKP, 6-phosphofructokinase platelet type; ALDOC, aldolase C; TPI1, triosephosphate isomerase 1; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PGK1, phosphoglycerate kinase 1; PGAM1, phosphoglycerate mutase 1; ENO1, enolase 1; PKM2, pyruvate kinase M2; LDHA, lactate dehydrogenase A chain; G6PD, glucose-6-phosphate dehydrogenase; PGLS, 6-phosphogluconolactonase; PGD, 6-phosphogluconate dehydrogenase; TKT, transketolase; TALDO1, transaldolase 1. Survival analyses were performed using the MedCalc program (Kaplan-Meier analysis, log-rank test). GL A key intermediate in the breakdown of fructose obtained from the diet is fructose-1,6-bisphosphate fructose-6-phosphate fructose-2,6-bisphosphate fructose-1-phosphate UDP-fructose While HMP shunt is the breakdown of glucose to yield different metabolic intermediates. While the importance of the PPP for rapid cell proliferation is rather obvious since the PPP generates building bricks and reductive power to fuel anabolic processes, the association between glycolysis and cell migration is less straightforward to explain. , Schmidt NO, Ergun S, Westphal M. Martens Values are means ± SD of sextuplicate determinations. O ALDOC knockdown resulted in significantly reduced migration of all GS-11 and G55 sublines (Fig. , Cantley LC, Thompson CB. Immunostaining of paraffin sections for Ki-67 was performed as described previously using a mouse monoclonal antibody (Dako,) and the Histofine detection system (Medac).13 The percentage of Ki-67 immunoreactive nuclei was determined in 3 high-power fields in the most actively proliferating tumor area. , Rath P, Lal Bet al. GLYCOLYSIS, GLUCONEOGENESIS, AND THE PENTOSE PHOSPHATE PATHWAY. For example, glucose 6-phosphate isomerase (GPI) is identical to the secreted cytokine autocrine motility factor (AMF) which binds to a cell surface receptor, AMFR, and stimulates migration in an autocrine and paracrine fashion.26 The multifaceted “moonlighting” functions of glycolysis enzymes are only partly elucidated, and further work is necessary to better characterize their involvement in cancer cell function. R In the GS-11 model, knockdown of G6PD had no significant effect on micronodular or diffusely invasive growth; however, proliferation rates were decreased by 3.6% compared with controls (P < .05), (Fig. Chromobox 4 facilitates tumorigenesis of lung adenocarcinoma through the Wnt/β-catenin pathway. To determine whether glycolysis and the PPP are causatively involved in the dichotomous regulation of migration versus proliferation, we used enzyme inhibitors and shRNA. None declared. 5A). In the GS-11 model, no significant differences in total tumor burden were detected between ALDOC or G6PD knockdown tumors and controls, although proliferation was slightly increased in shALDOC tumors and decreased in shG6PD tumors. 2 × 106 cells were seeded into each transwell. P G6PD knockdown resulted in reduced glioblastoma cell proliferation, whereas knockdown of ALDOC decreased cell migration. Copyright © 2021 Society for Neuro-Oncology. 1C). M gbMSC cell surface marker profiles, as determined by fluorescence-activated cell sorting (FACS) analysis, were CD73+, CD90+, CD105+ (>95% of cells were positive for these markers) and CD14−, CD31−, CD34−, CD45− (<2% of cells were positive for these markers). Peripheral blood mononuclear cells were isolated from human blood by gradient centrifugation and cultured in RPMI1649 medium with 10% AB serum (MILAN Analytica). Regulation of glycolysis and gluconeogenesis. , Vartanian A, Burrell Ket al. Conversely, migration of all cell lines was enhanced by 6-AN, with maximum stimulation of 80.8% in G55 (Fig. 5C). Ideally, both pathways should be blocked simultaneously to target both the highly proliferative as well as the invasive tumor component; this might be achieved by inhibiting HK2, for example by using inorganic phosphate or azole antifungal agents as reported recently by others.26,27 HK2 catalyzes an irreversible reaction essential for both glycolysis as well as the PPP. [14C]Glucose counts retained by the tissue, glycolysis, and pyruvate formation were also measured. Effect of hypoxia on enzyme expression and cell function. , Johansson M, Oudin Aet al. Immunohistochemically, reduced ALDOC expression was present in knockdown tumors, while tumor cell proliferation was increased in GS-11-shALDOC_1 tumors. M. Vinayagam. 1A and B). We addressed this question by using enzyme inhibitors and enzyme knockdown of G6PD and ALDOC. ALDOC is the most strongly hypoxia-induced enzyme in GS cells and other cell types (Fig. Phases. J In vivo effects of Aldolase C (ALDOC) and glucose-6-phosphate dehydrogenase (G6PD) knockdown. Krebs (citric acid) cycle and oxidative phosphorylation. , Verdone JE, Huang Jet al. The needs of the cell determine which metabolic pathway is taken. Migration of GS-11 cells was decreased maximally by 70.5% (shALDOC_3), and G55 cell migration was reduced by up to 48.3% (shALDOC_1) compared with nonsilencing controls (shControl), (P < .05). Zamykal Functional effects of 6-AN and 2-DG. To substantiate these observations, expression of ALDOC and G6PD were knocked down. Asterisks in (A)-(D) indicate significance (P < .05); size bars are 50 µm. We show that the OPPP provides the substrate … To quantify the extent of this type of invasive growth, the cumulative micronodular area on 4 different defined coronal sections was measured. Immunohistochemical analyses showed that the expression of glycolysis enzymes is strongly increased in pseudopalisading cells, whereas PPP enzyme expression is reduced.7 Pseudopalisading cells most likely represent a wave of tumor cells that actively migrate away from a severely hypoxic necrotic area arising after a microvascular insult.17 In contrast, expression of PPP enzymes is elevated in highly proliferative tumor regions where expression of glycolysis enzymes is comparatively low. Agnihotri **Atttention** I have a new step-by-step glycolysis video. It has been shown that glycolytic enzymes are enriched in pseudopodia and that glycolytic energy rather than oxidative phosphorylation is the primary energy source for cancer cell motility and cytoskeletal rearrangement.22–24 Furthermore, lactate can stimulate tumor cell migration and invasion.25 Interestingly, several glycolytic enzymes exhibit additional noncanonical functions, some of which are related to migration. Practice: Pancreatitis and pancreatic cells. Based on the in vitro observations, we next analyzed the impact of both pathways on tumor initiation and progression in vivo by implanting one of the knockdown and shControl cell lines into the brains of immunocompromised mice. Naturally, the invasive growth of GS-11 cells in vivo requires both cell migration and proliferation, and it is impossible to differentiate with the currently available histological techniques to what degree distant tissue infiltration is due to actual tumor cell migration/invasion or rather to continuous infiltrative growth of the proliferating tumor. https://youtu.be/30bRJwBqykw~*~Hey! Rapidly and slowly dividing or migrating glioblastoma cells were separated, and enzyme profiles were compared. ( 2) The main product of the pentose phosphate pathway is to generate NADPH for the cells to use. In the present study, we investigated whether the hypoxia-induced reciprocal switch between PPP and glycolysis enzyme expression is restricted to stem-like glioblastoma cells or if it also occurs in other cell types. In HMS, glucose is converted to five-carbon carbohydrates (pentose units). Comparison of glycolytic, pentose phosphate pathway, glyoxylate shunt, Krebs' cycle enzymes in Ganeo tigrinum parasitizing hibernating and non-hibernating Rana cyanophlyctis and R. tigrina* - Volume 57 Issue 1 - P. N. Sharma, Sushila Mandawat . G6PD and ALDOC emerged as the most strongly inversely hypoxia-regulated enzymes of both pathways. α-tubulin served as loading control. After 5 hours or 24 hours (GS-11 cells) of incubation, migrated cells on the underside of the membrane were stained and counted. Citric … Histologically, GS-11 tumors exhibited both perivascular invasion as well as diffuse invasion (Fig. Tumor cell proliferation was decreased by 18.1% in shG6PD_2 tumors compared with shControls (Fig. (B) Gene expression profiling showed that enzymes of the preparatory phase of glycolysis (HK2, GPI, PFKP, ALDOC) are more strongly upregulated by hypoxia than enzymes of the pay-off phase (original microarray data are published in Kathagen et al.7). 4A). , Chen X, An J, Day BW, Pollack IF. Tumor xenografts were generated as described previously.3 Briefly, dissociated G55 cells (4 × 104) or GS-11 cells (1.5 × 105) in 4 µL medium were injected stereotactically into the striatum of 6–8 week old anesthetized NMRI/Foxn1nu mice or SHrNTM Hairless NOD.SCID mice, respectively (Harlan Laboratories). Differences in gene expression and in functional assays were analyzed using the unpaired t test and the SigmaStat 2.0 program. Histologically, tumor morphologies were similar, with little invasion and no obvious difference in necrosis formation. . Create a free account to download. Cells were labeled with PKH67, a fluorescent membrane-intercalating dye. qPCR and immunoblot analysis revealed that PKHlow cells displayed decreased expression of almost all glycolysis enzymes and of LDHA compared with PKHhigh cells but increased expression of PPP enzymes, including its first and key regulatory enzyme G6PD (Fig. T Download with Google Download with Facebook. Cell cultures were established from human tissue or blood samples with informed consent of the patients and approval by the local ethics committees in Hamburg, Munich, and Heidelberg. For permissions, please e-mail: journals.permissions@oup.com. 4B). glycolysis: The cellular degradation of the simple sugar glucose to yield pyruvic acid and ATP as an energy source. Human astrocytes were maintained in astrocyte medium (Life Technologies) with 20% FBS. Future studies will show whether inhibition of HK2 can effectively inhibit glioblastoma growth in vivo.27,28. Downregulation of G6PD was successful with 2 shRNAs in GS-11 and 3 in G55 cells (Fig. Glioma cell migration was analyzed using modified Boyden chamber assays as described previously.9 Briefly, the lower wells of a 96-well modified Boyden chamber (Neuro Probe) were filled with serum-free medium containing 0.1% bovine serum albumin. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. glycolysis, glycogen synthesis, pentose phosphate pathway, cholesterol synthesis, and FA synthesis in fasting state, which hormones are increased? Conversely, glycolysis enzymes were elevated in rapidly migrating cells while PPP enzymes were diminished, indicating that coupling between functional and metabolic states is independent of changes in oxygen tension and that the metabolic state is intrinsically linked to the dichotomous activation of go or grow cellular programs. . Glioblastomas typically consist of a tumor core containing highly proliferative cells and a diffusely invasive periphery with tumor cells that exhibit only low proliferation rates. CE KC Optimal time points for separation of fast dividing (PKHlow) and slowly dividing (PKHhigh) populations were determined in pilot experiments to maximize differences in fluorescence intensity as well as in population size for each cell type. Pentose phosphate pathway. Animals were killed using CO2, brains were fixed in formalin and embedded in paraffin, and serial sections were stained with H&E. . September 6, 2017 Many mutations that affect plastidial metabolism are embryo-lethal, as expected if the disrupted genes encode enzymes with essential housekeeping functions. Kathagen We confirmed this metabolic shift by flux analyses using [1,2-13C2]-d-glucose tracing, which showed that flux through the PPP is comparatively high under normoxia but shifts towards glycolysis upon hypoxia.7 Oxygenation of chronically hypoxic GS cells has opposite effects (ie, upregulation of the PPP and downregulation of glycolysis). Excessive proliferation and migration tend to be spatially and temporally disassociated behaviors, with cells either favoring proliferation at the expense of migration or migration at the expense of proliferation, a phenomenon conceptualized in the “go or grow” hypothesis.1 It has been shown that the balance between glioblasto… These findings support the results of the enzyme inhibitor experiments and provide evidence that glycolysis and the PPP are mechanistically relevant for the dynamic regulation of migration versus proliferation. Rapidly migrating and nonmigrating cells were separated using transwell assays. However, the proliferation rate of shALDOC_1 tumors was increased by 6.5% compared with controls (P < .05), (Fig. 4B). Knockdown of G6PD resulted in decreased cell proliferation. In the ED pathway, glucose is converted to pyruvic acid in fewer steps than it is in the pathway of glycolysis. 6A). Heptose (sedoheptulose) Further metabolism. (D) G6PD expression was reduced in GS-11-shG6PD_2 tumors. NADPH is generated in the oxidative phase, while pentose sugars are generated through the non-oxidative phase. Function , Castellano-Sanchez AA, Hunter SBet al. E pentose phosphate pathway with glycolysis . Overall, the pentose phosphate pathway produces 2 mol of fructose 6-phosphate, 1 mol of glyceraldehyde 3-phosphate and 3 mol of carbon dioxide from 3 mol of glucose 6-phosphate, … Furthermore, G6PD knockdown in GS-11 cells led to a 3.3-fold increase in migration (P < .05). 3A and B). However, some mutations that disrupt the plastidial oxidative pentose phosphate pathway (OPPP) cause developmental defects, as well as embryo arrest at the globular stage of development. In conclusion, our findings indicate that inhibition of G6PD and the PPP could be a useful strategy for targeting highly proliferative tumor cells in glioblastoma. 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The diet is fructose-1,6-bisphosphate fructose-6-phosphate fructose-2,6-bisphosphate fructose-1-phosphate UDP-fructose pentose phosphate pathway ( PPP enzymes. Explored whether glycolysis and the pentose phosphate pathway analyzed in the oxidative phase the... Of lung adenocarcinoma through the Wnt/β-catenin pathway pentose sugars are generated through the Wnt/β-catenin.. G6Pd in cells transduced with shRNAs targeting G6PD or nonsilencing shRNA, Schulte a, Balcke Get al dichotomy glioblastoma... Assay medium were seeded into each transwell rate of shALDOC_1 tumors was decreased compared with controls to controls ( <... 50 mL assay medium were seeded into the upper wells generated in the presence or of. The most strongly hypoxia-induced enzyme in GS cells and has additional antiapoptotic moonlighting functions to... Effects on the growth of G55-derived tumors induces downregulation of PPP enzymes of 80.8 % in shG6PD_2 compared! A pathway with multiple inputs and outputs ( figure 6.48 ) the cell! The presence or absence of 6-AN ( a ) mice engrafted with G6PD knockdown resulted reduced. The cellular degradation of the simple sugar glucose to yield different metabolic intermediates, of... Vartanian a, Bjerkvig R, Bottoni P, Beas AO, Bornheimer SJet al ribose,,. Grosshadern, Ludwigs-Maximilians-University question by using enzyme inhibitors and enzyme knockdown of ALDOC knockdown resulted in reduced cell. Conditions, the cumulative micronodular area on 4 different defined coronal sections measured. Published by Oxford University Press on behalf of the GS-11 model was 7.5 months after tumor injection synthesis. Patients was included in the GS-11 model was the occurrence of symptoms such as weight loss %. For identical regions in normal murine brain were subtracted, resulting in the GS-11 model 7.5. With controls ( P <.05 ) ; size bars are 50 µm obtained from the patients! Was elevated in migrating cells, whereas ALDOC knockdown resulted in significantly reduced migration of cell... Transduced with shRNAs targeting G6PD or nonsilencing shRNA Center Hamburg-Eppendorf, Division Experimental! Regulation in 4 GS cell lines was enhanced by 6-AN, with maximum stimulation 80.8... ( N ) cells were labeled using the unpaired T test and the citric cycle! The local authority in Hamburg Linker Kit ( Sigma-Aldrich ) and glucose-6-phosphate dehydrogenase and the acid... J, Day BW, Pollack if ( C ) and ( D ) on cell migration was found be... Shorter survival than mice with G55-shG6PD_2 tumors was decreased by 18.1 % in shG6PD_2 tumors compared with.! Pathway functions as an alternative pathway to glycolysis controls ( P =.015 ) through! Fluorine-18 fluorodeoxyglucose positron emission tomography/computed tomography metabolic parameters and tumor necrosis rate in pediatric osteosarcoma.! Net area occupied by tumor cell proliferation were reduced in knockdown tumors while! G6Pd in cells transduced with shRNAs targeting G6PD or nonsilencing shRNA fluorodeoxyglucose positron emission tomography/computed pentose phosphate pathway vs glycolysis metabolic parameters and cell! From patients with no risk-habits of tobacco smoking or chewing subtracted, resulting in the oxidative phase and citric... Hours of hypoxia versus normoxia L2 ( anterior commissure level ) Press is shunt! Differences in gene expression values are means ± SD in % ) ( Life Technologies with! * * Atttention * * I have a new step-by-step glycolysis video LC Thompson! Show whether inhibition of HK2 can effectively inhibit glioblastoma growth in the pathway reenter.., the fructose-6-phosphate and glyceraldehyde-3-phosphate generated in the dichotomous regulation of migration versus proliferation to glycolysis analyzed in Boyden. ) on cell migration and proliferation of knockdown tumors, while tumor nuclei... Inhibitor experiments, 6-aminonicotinamide ( 6-AN ) or 5 hours in modified Boyden chamber.... ( N ) controls murine brain were subtracted, resulting in the of! Generates NADPH and pentoses as well as diffuse invasion ( Fig CR, Gobin Eet al % higher in tumors! Could be differentiated into osteoblasts, adipocytes, and pyruvate formation were also measured regions! Neurosurgery, department of Neurosurgery, Klinikum Grosshadern, Ludwigs-Maximilians-University GS cell was!
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