Enzymes Of Glycolytic Pathway Research Articles

PKM1 Regulates the Expression of Autophagy and Neuroendocrine Markers in Small Cell Lung Cancer

Small cell lung cancer (SCLC) is known as recalcitrant cancer with high malignancy and heterogeneity. Immunotherapy has changed the treatment pattern of extensive-disease SCLC (ED-SCLC), but the beneficiary population is limited. Therefore, exploring new therapeutic strategies is an urgent clinical problem to be solved for SCLC. SCLC is characterized by highly active glycolytic metabolism and pyruvate kinase M1 (PKM1) is one of the isozymes of PK, an important rate-limiting enzyme in glycolysis pathway. Previous studies have shown that PKM1 is related to autophagy and drug sensitivity, however, how PKM1 regulates drug sensitivity in SCLC and its mechanism remain unclear. The aim of this study was to investigate the biological functions of PKM1 in SCLC, including its effects on proliferation, migration, autophagy, drug sensitivity, and expression of neuroendocrine (NE)-related markers in SCLC. Western blot was used to detect the expression level of PKM1 in SCLC cells. PKM1 gene-overexpressed SCLC cell lines were constructed by stable lentivirus transfection. Proliferation of cells and drug sensitivity were detected by MTT, and migration ability of cells was determined by Transwell. The level of autophagy was detected by flow cytometry. Western blot was used to determine the expression levels of NE-related proteins. PKM1 was differentially expressed among various SCLC cell lines, and was lower in H1092 cells (P<0.01). Compared with the control group, there was no significant difference in proliferation level of PKM1 overexpressing H1092 cell, but the migration ability was significantly increased (P<0.001), the drug sensitivity was reduced, and the level of autophagy was inhibited (P<0.001). Additionally, overexpression of PKM1 could upregulate the expression of non-neuroendocrine (non-NE)-related proteins (P<0.01) and decrease the expression of NE-related proteins (P<0.01). PKM1 was differentially expressed in SCLC cell lines, and high expression of PKM1 did not affect the proliferation, but affected the migration of SCLC cells. PKM1 might affect drug sensitivity by inhibiting autophagy and regulating the expression of NE markers. These results provide a theoretical basis for exploring the role of PKM1 in SCLC.

Design and synthesis of pyrano[2,3-c]pyrazole-4-aminoquinoline hybrids as effective antimalarial compounds

In this work, a series of nineteen novel pyrano[2,3-c]pyrazole-4-aminoquinoline hybrids were synthesized as potent antimalarial agents by covalently linking the scaffolds of 4-aminoquinoline and pyrano[2,3-c]pyrazoles via an ethyl linker and characterized using Fourier transform infrared spectroscopy (FTIR) and nuclear magnetic resonance spectroscopy (NMR). Molecular docking was used to test each hybrid's and standard chloroquine's ability to bind to Plasmodium falciparum lactate dehydrogenase enzyme (PfLDH), an important enzyme in the parasite's glycolytic pathway. The hybrid compounds had a stronger binding affinity than the standard chloroquine (CQ). The schizontical antimalarial test of pyrano[2,3-c]pyrazole-4-aminoquinoline hybrid compound shows that all nineteen hybrid compounds were potent with the IC50 values ranging from 0.0151 to 0.301 μM against the CQ-sensitive 3D7 P. falciparum strain, and were active against the CQ-resistant K1 P. falciparum strain with the IC50 values ranging from 0.01895 to 2.746 μM. All the tested hybrid compounds were less potent than the standard drug chloroquine dipaspate (CQDP) against the CQ-sensitive 3D7 strain. In contrast, nine of the nineteen hybrids (16d, 16g, 16h, 16i, 16l, 16n, 16o, 16r, and 16s) displayed superior antimalarial activity than the CQDP against the CQ-resistant K1 P. falciparum strain. Among all the tested hybrids, 16c against the 3D7 strain and 16h against the K1 strain were the most promising antimalarial agents with 0.0151 and 0.01895 μM of IC50 values, respectively. In addition, the compounds were selective, showing moderate to low cytotoxic activity against a human normal liver WRL68 cell line. The synthesis of pyrano[2,3-c]pyrazole-4-aminoquinoline hybrids introduces new chemical entities that have the potential to exhibit potent antimalarial activity. It could address the ongoing challenge of drug resistance in malaria treatment.

CRISPR/Cas9-mediated gene editing of L. bulgaricus d-lactate dehydrogenase (ldh) gene to monitor yogurt characteristics

Yogurt is a coagulated dairy product that results from the fermentation of milk and milk products by lactic acid bacteria, specifically Lactobacillus bulgaricus and Streptococcus thermophilus. Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 is an emerging tool in molecular biology research which can be used extensively in prokaryotes and eukaryotes. Lactate dehydrogenase (LDH) is an important enzyme of glycolytic pathway. In this research, we have edited L. bulgaricus d-lactate dehydrogenase (ldh) gene using CRISPR/Cas9 system. Genome editing was accomplished through synthesizing gDNA on the basis of PAM region of ldh gene and ligation of synthesized gDNA with pCRISPR-SacB plasmid which was later co-transformed with mutagenic donor DNA into competent cells containing pCasRed. This method combined CRISPR/Cas9 with γ Red machinery to achieve editing of ldh gene. The PCR screening and sequencing results confirmed that, base deletion has been occurred in the target region of ldh gene. Differences in several characteristics including texture, pH were observed on the yogurt samples from newly developed L. bulgaricus strains compared to normally produced yogurt. Our results suggest that, other genes of lactobacillus can be edited through CRISPR/Cas9 technology which can be extensively used for metabolic engineering.

Vibration Emissions Reduce Boar Sperm Quality via Disrupting Its Metabolism.

Artificial insemination (AI) with liquid-preserved semen has recently become common in pig breeding. The semen doses are produced in a centralized manner at the boar stud and then subsequently distributed and transported to sow farms. However, vibration emissions during transportation by logistic vehicles may adversely affect the quality of boar sperm. Therefore, this study aimed to explore the impact of vibration-induced emissions on sperm quality and function under simulated transportation conditions. Each time, ejaculates from all 15 boars were collected and then pooled together to minimize individual variations, and the sample was split using an extender for dilution. Different rotational speeds (0 rpm, 80 rpm, 140 rpm, 200 rpm) were utilized to simulate varying intensities of vibration exposure using an orbital shaker, considering different transportation times (0 h, 3 h, and 6 h). Subsequently, evaluations were conducted regarding sperm motility, plasma membrane integrity, acrosome integrity, mitochondrial function, adenosine triphosphate (ATP) levels, mitochondrial reactive oxygen species (ROS) levels, pH, glycolytic pathway enzyme activities, and capacitation following exposure to vibration emissions. Both vibration time and intensity impact sperm motility, plasma membrane integrity, and acrosomal integrity. Vibration exposure significantly reduced sperm ATP levels, mitochondrial membrane potential, and the levels of mitochondria-encoded proteins (MT-ND1, MT-ND6) (p < 0.05). After vibration emission treatment, the pH value and mitochondrial ROS levels significantly increased (p < 0.05). Inhibition of sperm glycolysis was observed, with reduced activities of hexokinase (HK), pyruvate kinase (PK), and lactate dehydrogenase (LDH), along with decreased lactate levels (p < 0.05). Additionally, sperm tyrosine phosphorylation levels were significantly reduced by vibration emissions compared to the control group (p < 0.05). After the vibration emission treatment, the number of sperm bound to each square millimeter of oviduct explants decreased significantly compared to the control group (p < 0.05). Similarly, compared to the control group, using semen subjected to vibration stress for AI results in significantly reduced pregnancy rates, total born litter size, live-born litter size, and healthy born litter size (p < 0.05).

Abstract 448: Alterations in glycolytic pathway enzymes and metabolic plasticity in prostate cancer progression

Abstract Background: Although oxidative phosphorylation (OXPHOS) is the main ATP source in primary prostate cancer, as the tumor progresses to castration resistance and/or metastatic disease, glycolysis becomes increasingly prevalent. Dependence on glycolysis can further increase under hypoxic conditions found in advanced cancers. We previously demonstrated that a novel small molecule, BKIDC-1553, inhibits proliferation and tumor growth of prostate cancer cell lines and xenografts through a hexokinase-2 (HK2) dependent inhibition of glycolysis. HK2 is a glucose 6-phosphorylating enzyme that works in conjunction with glucose transporter-1 (GLUT1) to drive glycolysis in cells. While our previous work suggests that BKIDC-1553 works primarily through a HK2 dependent mechanism, the objective of this current project is to further examine how expression and function of HK2 and GLUT1 are altered in response to BKIDC-1553 treatment. Methods: Hypoxic conditions are common in late-stage prostate cancer. Effects of hypoxia on cell proliferation and expression levels of HK1, HK2, and GLUT1 were examined with and without BKIDC-1553 treatment. To examine role of HK1, HK2, and GLUT1 in response to BKIDC-1553 treatment, we created CRISPR knockouts and shRNA knockdowns of HK1 and HK2 in the LNCaP prostate cancer cell line. GLUT1 was inhibited using a commercially available inhibitor, BAY-876. PCR and Western blots were used to confirm expression levels of HK1, HK2, and GLUT1. MTS proliferation assays were used to examine effect of KO and KD on response to BKIDC-1553 and BAY-876. Results: GLUT1 levels, but not HK1 or 2, increased at 72 hrs under hypoxic conditions. HK1, HK2, and GLUT1 levels all increased in cells in response to 144 hrs of BKIDC-1553 compared with 72 hrs treatment in both normoxic and hypoxic conditions. The KO and KD studies were all conducted in normoxic conditions. Westerns blots demonstrated that HK2 KO, but not HK1 KO, results in increased levels of GLUT1. HK1 or HK2 KD, however, did not alter GLUT1 levels. HK2 KD cells, with GLUT1 levels remaining the same, displayed less growth inhibition than control cells in response to BKIDC-1553 treatment, while HK2 KO cells, with increased levels of GLUT1, remained sensitive to BKIDC-1553. Because GLUT1 levels increase in response to HK2 KO, we wanted to see if inhibiting GLUT1 in these cells could decrease their proliferation. We found that KO cells were indeed more sensitive to growth inhibition of BAY-876. Conclusion/Summary: These studies indicate that KO of HK2 increases levels of GLUT1. These cells remain sensitive to BKIDC-1553 treatment, which could in part be due to the cells' continued reliance on glycolysis through increased GLUT1. A continued reliance on glycolysis allows BKIDC-1553 to continue to inhibit cell growth. Future studies examining how combination treatment with BKIDC-1553 and BAY-876 affects growth of advanced prostate cancer cells is underway. Citation Format: Cynthia Sprenger, Mika Munari, Shihua Sun, Kathryn Soriano Epilepsia, Stephen Plymate. Alterations in glycolytic pathway enzymes and metabolic plasticity in prostate cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 448.

Hypoxia-inducible factor-1α promotes macrophage functional activities in protecting hypoxia-tolerant large yellow croaker (Larimichthys crocea) against Aeromonas hydrophila infection.

The immune system requires a high energy expenditure to resist pathogen invasion. Macrophages undergo metabolic reprogramming to meet these energy requirements and immunologic activity and polarize to M1-type macrophages. Understanding the metabolic pathway switching in large yellow croaker (Larimichthys crocea) macrophages in response to lipopolysaccharide (LPS) stimulation and whether this switching affects immunity is helpful in explaining the stronger immunity of hypoxia-tolerant L. crocea. In this study, transcript levels of glycolytic pathway genes (Glut1 and Pdk1), mRNA levels or enzyme activities of glycolytic enzymes [hexokinase (HK), phosphofructokinase (PFK), pyruvate kinase (PK), and lactate dehydrogenase A (LDHA)], aerobic respiratory enzymes [pyruvate dehydrogenase (PDH), isocitrate dehydrogenase (IDH), and succinate dehydrogenase (SDH)], metabolites [lactic acid (LA) and adenosine triphosphate (ATP)], levels of bactericidal products [reactive oxygen species (ROS) and nitric oxide (NO)], and transcripts and level changes of inflammatory factors [IL1β, TNFα, and interferon (IFN) γ] were detected in LPS-stimulated L. crocea head kidney macrophages. We showed that glycolysis was significantly induced, the tricarboxylic acid (TCA) cycle was inhibited, and metabolic reprogramming occurred, showing the Warburg effect when immune cells were activated. To determine the potential regulatory mechanism behind these changes, LcHIF-1α was detected and found to be significantly induced and transferred to the nucleus after LPS stimulation. LcHif-1α interference led to a significant reduction in glycolytic pathway gene transcript expression, enzyme activity, metabolites, bactericidal substances, and inflammatory factor levels; a significant increase in the aerobic respiration enzymes; and decreased migration, invasion, and phagocytosis. Further ultrastructural observation by electron microscopy showed that fewer microspheres contained phagocytes and that more cells were damaged after LcHif-1α interference. LcHif-1α overexpression L. crocea head kidney macrophages showed the opposite trend, and promoter activities of Ldha and Il1β were significantly enhanced after LcHif-1α overexpression in HEK293T cells. Our data showed that LcHIF-1α acted as a metabolic switch in L. crocea macrophages and was important in polarization. Hypoxia-tolerant L. crocea head kidney showed a stronger Warburg effect and inhibited the TCA cycle, higher metabolites, and bactericidal substance levels. These results collectively revealed that LcHif-1α may promote the functional activities of head kidney macrophages in protecting hypoxia-tolerant L. crocea from Aeromonas hydrophila infection.

Deficits in mitochondrial function and glucose metabolism seen in sporadic and familial Alzheimer’s disease derived Astrocytes are ameliorated by increasing hexokinase 1

AbstractBackgroundAstrocytes have multiple roles including providing neurons with metabolic substrates and maintaining neurotransmitters at neuronal synapses. Astrocyte glucose metabolism plays a key role in learning and memory with astrocytic glycogen a key substrate supporting memory encoding. The homeostatic role the astrocyte provides for neurons leads to metabolic demands, meaning that abnormalities in the function of astrocyte mitochondria and glycolysis could affect this relationship. Changes to cellular metabolism are seen early in Alzheimer’s disease (AD). Understanding cellular metabolism changes in AD astrocytes could be exploited as a new biomarker or synergistic therapeutic agent when combined with anti‐amyloid treatments in AD.MethodsIn this project, we characterised mitochondrial and glycolytic dysfunction in astrocytes derived from patients with sporadic (n = 6) and familial (PSEN1, n = 3) AD, and associated controls (n = 9). Astrocytes were derived using direct reprogramming technology. Astrocyte metabolic outputs; ATP, and extracellular lactate levels were measured using luminescent and fluorescent protocols. Mitochondrial respiration and glycolytic function were measured using a Seahorse XF Analyzer. Hexokinase deficits identified where corrected by transfecting astrocytes with an adenovirus viral vector containing the hexokinase 1 gene.ResultsIn sAD astrocytes a 20% reduction (p = 0.05) and in fAD a 48% (p&lt;0.01) reduction in total cellular ATP was seen. A 44% reduction (p&lt;0.05), and 80% reduction in Mitochondrial spare capacity was seen in sAD and fAD respectively. Reactive oxygen species (ROS) where increased in both AD astrocyte types (p = 0.05). Mitochondrial complex I and II was significantly increased in sAD (p&lt;0.05) but not in fAD. Astrocyte glycolytic reserve and extracellular lactate was significantly reduced when compared to controls in both sAD and fAD (p&lt;0.05). We identified that astrocytes had a deficit in the glycolytic pathway enzyme hexokinase, and that correcting this deficit restored total cellular ATP levels, extracellular lactate and reduced ROS in sAD but not fAD astrocytes.ConclusionAD astrocytes have abnormalities in functional capacity of mitochondria and the process of glycolysis. These deficits are likely to impede metabolic support for neurons. These functional deficits can be improved by correcting hexokinase deficits. This suggests that hexokinase 1 deficiency could potentially be exploited as a new therapeutic target for AD.

PGK1 Represents a Therapeutic Target for Pediatric Acute Myeloid Leukemia Via Regulating c-Myc/SLC7A5/mTOR Pathway

Acute myeloid leukemia (AML) is a clonal disorder of hematopoietic stem and progenitor cells characterized by abnormal growth, differentiation blockade, and accumulation of immature myeloblasts, which accounts for 15-20% of pediatric leukemia. Emerging literature has shown that metabolism disorder and their related pathways, play pivotal roles in leukemogenesis and disease progression. Phosphoglycerate kinase 1 (PGK1), an essential enzyme in glycolysis pathway, has been demonstrated to be up-regulated in several malignancies, and was related with tumor stage and poor prognosis. This study aimed to investigate the correlation between PGK1 expression and tumorigenesis of AML, explore the possible underlying mechanism as well as provide a potential therapeutic target for pediatric AML. The research included 64 AML patients and 10 healthy donors recruited from July, 2012 to June, 2020 in Children's Hospital of Soochow University. Expression of PGK1 mRNA in bone marrow of newly-diagnosed AML patients was higher than normal group (p&amp;lt;0.001). Based on median expression of PGK1, 64 patients were divided into PGK1high group (n=32) and PGK1low group (n=32). Patients in PGK1high group had lower complete remission (CR) rate after first course of chemotherapy (65.6% vs 75.0%, p=0.027), overall survival (OS) (53.1%±8.8% vs 84.4%±6.4%, p=0.004) and relapse-free survival (RFS)(63.5%±10.4% vs 90.1%±5.4%, p=0.018). PGK1-knockdown cell lines were constructed to figure out the correlation between PGK1 expression and AML development in vitro. Knockdown of PGK1 inhibited the proliferation, suppressed colony formation and promoted apoptosis of AML cells. In addition, PGK1KD AML cells became more sensitive to Cytarabine (Ara-C) and daunorubicin (DNR). AML model was established by injecting PGK1KD P388-D1 cells of BALB/c mice via tail veins. The results indicated that growth of PGK1KD cells in vivo was significantly suppressed compared to negative control group. Moreover, the median survival time of PGK1 KD group was shorter (10 days vs 13 days, p=0.035). NG52, a newly reported small molecule kinase inhibitor of PGK1, could block glycolysis pathway by suppressing the kinase activity of PGK1. NG52 promoted MV4-11 and Kasumi-1 apoptosis in a dose-dependant manner, and showed synergism with Ara-C and DNR in MV4-11 and Kasumi-1. To identify downstream targets of PGK1, RNA-sequencing analysis was performed in PGK1 KD cell lines. SLC7A5 was found to be the most significant down-regulated gene of differentially expressed genes (DEGs). Meanwhile, c-Myc, p-S6 and p-4EBP1 expression were reduced in PGK1 KD cell lines, indicating that c-Myc/SLC7A5/mTOR pathway was suppressed in PGK1 KD AML cells. We subsequently established SLC7A5 KD cells. Inhibition of proliferation and increased apoptosis was found in SLC7A5 KD cells, which was consistent with the results in PGK1 KD cells. Expression of c-Myc and activation of mTOR pathway were observably decreased in SLC7A5 KD cells, which supported the hypothesis above. We found that SLC7A5 expression level was also higher in AML patients than in healthy donors. In conclusion, our study indicated that PGK1 is a molecular biomarker of poor prognosis and a potential therapeutic target of pediatric AML. Inhibition of PGK1 suppressed progression of AML by regulating c-Myc/SLC7A5/mTOR pathway, providing a promising intervention for AML precision medicine.

Abstract 9328: Sirt5 Deficiency Promotes Metabolic Reprogramming to Aggravate Cardiac Fibrosis

Introduction: Metabolic remodeling is critically involved in the pathogenesis of cardiac fibrosis. Glycolytic switch provides rapid energy production and biosynthetic intermediates to support proliferation and collagen synthesis of fibroblast. Sirtuin 5 (Sirt5) is a protein lysine de-succinylase that impacts diverse metabolic pathways. Objective: This study aimed to determine the function and mechanism of Sirt5 in cardiac fibroblast activation and cardiac fibrosis. Methods and Results: SIRT5 expression was examined in human and mouse myocardia, and was found to be downregulated in fibrotic myocardial tissues compared to non-fibrotic tissues. In a mouse model of myocardial infarction, Sirt5-knockout (KO) mice showed more severe cardiac fibrosis compared with wild-type mice. The aggravating cardiac fibrosis of Sirt5-KO mice were also found after transverse aortic constriction or angiotensin II plus phenylephrine-infusion. Proliferation and collagen synthesis of cardiac fibroblasts (CFBs) were both significantly enhanced by SIRT5 deficiency after AngII/PE-infusion in vivo and transform growth factor (TGF)-β1-treatment in vitro , but were attenuated by overexpression of Sirt5 in vitro . Moreover, Sirt5 deficiency enhanced a metabolic switch from oxidative phosphorylation to glycolysis in the isolated CFBs, and inhibition of glycolysis diminished the effect of Sirt5 deficiency on CFBs activation. Mechanistically, lysine succinylome analysis identified phosphoenolpyruvate carboxykinase 2 (Pck2), a rate-limiting enzyme in glycolysis pathway, as the protein mostly succinylated after Sirt5 deficiency. Activity of Pck2 was significantly inhibited by Sirt5 deficiency-mediated hypersuccinylation both in vivo and in vitro . K489R mutation of Pck2 diminished its succinylation and the reduction of its enzymatic activity mediated by Sirt5 deficiency, which also blunted the effect of Sirt5 deficiency on CFBs activation. Conclusions: Sirt5 deficiency enhances a metabolic reprogramming from oxidative phosphorylation to glycolysis in CFBs by succinylating Pck2, which promotes CFBs activation and aggravates cardiac fibrosis.

Identification of Natural Compounds as Inhibitors of Pyruvate Kinase M2 for Cancer Treatment

The reliance of tumor cells on aerobic glycolysis is one of the emerging hallmarks of cancer. Pyruvate kinase M2 (PKM2), an important enzyme of glycolytic pathway, is highly expressed in a number of cancer cells. Tumor cells heavily depend on PKM2 to fulfill their divergent energetic and biosynthetic requirements, suggesting it as novel drug target for cancer therapies. Based on this context, we performed enzymatic-assay-based screening of the in-house phenolic compounds library for the identification of PKM2 inhibitors. This screening identified silibinin, curcumin, resveratrol, and ellagic acid as potential inhibitors of PKM2 with IC50 values of 0.91 µM, 1.12 µM, 3.07 µM, and 4.20 µM respectively. For the determination of Ki constants and the inhibition type of hit compounds, Lineweaver–Burk graphs were plotted. Silibinin and ellagic acid performed the competitive inhibition of PKM2 with Ki constants of 0.61 µM and 5.06 µM, while curcumin and resveratrol were identified as non-competitive inhibitors of PKM2 with Ki constants of 1.20 µM and 7.34 µM. The in silico screening of phenolic compounds against three binding sites of PKM2 provided insight into the binding pattern and functionally important amino residues of PKM2. Further, the evaluation of cytotoxicity via MTT assay demonstrated ellagic acid as potent inhibitor of cancer cell growth (IC50 = 20 µM). These results present ellagic acid, silibinin, curcumin, and resveratrol as inhibitors of PKM2 to interrogate metabolic reprogramming in cancer cells. This study has also provided the foundation for further research to validate the potential of identified bioactive entities for PKM2 targeted-cancer therapies.

Modulations in human neutrophil metabolome and S-glutathionylation of glycolytic pathway enzymes during the course of extracellular trap formation

Neutrophil extracellular trap formation (NETosis) has been irrefutably referred to as a distinct and unique form of active cell death with the purpose to counteract invading pathogens or augmenting the inflammatory cascade. Since the discovery, consistent efforts have been made to understand the various aspects of the initiation and sustenance of NETosis. In this study, using a global metabolomics approach during the phorbol 12-myristate 13-acetate (PMA) induced NETosis in human neutrophils, various metabolic pathways were found to be altered which includes intermediates related to, carbohydrate metabolism, and redox related metabolites, nucleic acid metabolism, and amino acids metabolism. Enrichment analysis of the metabolite sets highlighted the importance of the pentose phosphate pathway (PPP) and glutathione metabolism PMA-induced NETotic neutrophils. Further, analysis of the glutathyniolation status of neutrophil proteins by Matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) indicated six different glutathionylated proteins: among them, two metabolically important proteins were α-enolase and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) with MALDI score 166 and 70 respectively. Other proteins were lactoferrin, β-actin, c-myc promoter-binding protein, and uracil DNA glycosylase with MALDI scores of 96, 167, 104, and 68 respectively. Besides, activation of signalling proteins involved in metabolic regulation is also correlated with NETosis. Altogether, a balance between reactive oxygen species-glutathione metabolism seems to regulate the activity of glycolytic enzymes such as GAPDH and α-enolase during PMA-induced NETosis in a time-dependent manner.

Abstract 5243: 3-Bromopyruvate in combination with radiation inhibits pancreatic cancer growth by dismantling mitochondria and ATP generation in a preclinical mouse model

Abstract 3-Bromopyruvate (3 BP) is a promising anticancer drug against pancreatic cancer. It exerts potent anticancer effects by inhibiting hexokinase II enzyme of glycolysis pathway and ATP generation in cancer cells. In present investigation we have examined the effect of 3-BromoPyruvate employing panc-2 cancer cells, and syngeneic pancreatic cancer mouse model. The clonogenic survival assay demonstrated dose dependent inhibition of cell growth, and we observed 95% cell death at 15 uM conc. Further, ATP production was severely inhibited in cancer cells treated with same concentration of 3-BP. We also observed that 3-BP in combination with low doses of irradiation was more effective in killing cancer cells than 3-BP alone. Electron microscopy revealed that 3-BromoPyruvate severely damaged mitochondrial integrity which might have severely affected ATP generation in cancer cells. We further tested 3-BromoPyruvate (2.5-10.0 mg/kg) in syngeneic panc-2 tumor mouse model and observed that 3-BP at10.0 mg/kg dose significantly reduced tumor growth (volume) about 35-40%. Immunohistochemistry (IHC) data showed that 3-BP inhibits hexokinase II (HK2) and TGFbeta1 and enhanced active caspase-3 expression in tumor tissues as compared to untreated control. The disruption of the interaction between VDAC1 and HK2 due to 3BP treatment was confirmed by CO-IP. Interestingly, 3 BP at higher dose induces caspase-3 activation in tumor derived organoid without showing any effect on normal pancreatic organoid. Altogether, the results in this study demonstrated that 3 BP could be a promising anti-tumor drug against pancreatic cancer and its efficacy could be improved in combination with radiation therapy. Citation Format: Sanjit K. Roy, Tijana Dukic, Binny Bhandary, Aneesh Acharya, Kevin J. Tu, Young H. Ko, Hem D. Shukla. 3-Bromopyruvate in combination with radiation inhibits pancreatic cancer growth by dismantling mitochondria and ATP generation in a preclinical mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5243.

The WWOX/HIF1A Axis Downregulation Alters Glucose Metabolism and Predispose to Metabolic Disorders.

Recent reports indicate that the hypoxia-induced factor (HIF1α) and the Warburg effect play an initiating role in glucotoxicity, which underlies disorders in metabolic diseases. WWOX has been identified as a HIF1α regulator. WWOX downregulation leads to an increased expression of HIF1α target genes encoding glucose transporters and glycolysis’ enzymes. It has been proven in the normoglycemic mice cells and in gestational diabetes patients. The aim of the study was to determine WWOX’s role in glucose metabolism regulation in hyperglycemia and hypoxia to confirm its importance in the development of metabolic disorders. For this purpose, the WWOX gene was silenced in human normal fibroblasts, and then cells were cultured under different sugar and oxygen levels. Thereafter, it was investigated how WWOX silencing alters the genes and proteins expression profile of glucose transporters and glycolysis pathway enzymes, and their activity. In normoxia normoglycemia, higher glycolysis genes expression, their activity, and the lactate concentration were observed in WWOX KO fibroblasts in comparison to control cells. In normoxia hyperglycemia, it was observed a decrease of insulin-dependent glucose uptake and a further increase of lactate. It likely intensifies hyperglycemia condition, which deepen the glucose toxic effect. Then, in hypoxia hyperglycemia, WWOX KO caused weaker glucose uptake and elevated lactate production. In conclusion, the WWOX/HIF1A axis downregulation alters glucose metabolism and probably predispose to metabolic disorders.

Pyruvate Kinase M2 Regulates Hif-1alpha Activity in Myeloid Dendritic Cells from Patients with Severe Aplastic Anemia

Hypoxia-inducible factor 1 (HIF-1) is a nuclear protein with transcriptional activity. HIF-1 can be activated by immune cells exposed to hypoxia and regulate glycolysis. In this regulation, pyruvate kinase M2 (PKM2), a key enzyme in the cell glycolytic pathway, is an important regulatory site. Our previous studies suggest that PKM expression is increased in myeloid dendritic cells (mDCs) in SAA patients. Therefore, we investigated the expression level of HIF-1α in mDCs and its interaction with PKM2 in SAA patients. 1.The HIF-1α expression of mRNA and protein on mDCs in SAA untreated patients was significantly higher than that in SAA remission patients and normal controls.2.In the SAA patients, the HIF-1α expression on mDCs was positively correlated with the numbers of mDCs (p < 0.01), CD80+mDC/mDC (r = 0.689, p < 0.01), and CD86+mDC/mDC in PB (p < 0.05).3.In SAA patients, the HIF-1α expression on mDCs was negatively correlated with the CD4+T/CD8+T ratio in peripheral blood (PB) (p < 0.05), the percentage of granulocytoid and erythroid cells in bone marrow (p < 0.05), the WBC count in PB (p < 0.05), ANC in PB (p < 0.05), and the percentage of Rets in the PB (p < 0.05); was positively correlated with the percentage of lymphoid cells in bone marrow(p < 0.05); and was not statistically correlated with the megakaryocyte number in bone marrow, absolute PBL count, HGB in PB, absolute Ret count in PB, or PLT count in PB.4.In the correlation analysis we observed that there was a positively correlation between HIF-1α and PKM2 expression on mDCs in SAA patients (p < 0.001). To evaluate whether there is a mutual adjustment relationship between HIF-1α and PKM2, we successfully reduced PKM2 gene expression in this cell population via siRNA transfection. This process resulted in significantly lower levels of PKM2 protein expression relative to cells transfected with siControl which were evaluated by western blotting. We observed that the relative expression levels of HIF-1α mRNA in mDCs transfected with PKM2 siRNA was lower than siControl group（P< 0.01）.Conclusions 1.In this study, we found that untreated patients with SAA had higher HIF-1α expression on mDCs compared with recovering SAA patients and normal controls. The expression of HIF-1α was correlated with the number and function in mDCs and the severity of pancytopenia of SAA.2.The results indicated that the mRNA expression level of HIF-1α in mDC of SAA patients at the onset was significantly higher than that of the remission group and the control group, and was significantly positively correlated with the expression of PKM2.3.The mRNA expression level of HIF-1α in mDC of SAA patients at the onset was significantly higher than that of the remission group and the control group, and was significantly positively correlated with the expression of PKM2. The above experimental results confirmed that HIF-1α plays an important role in the abnormal immune response in patients with severe aplastic anemia, mainly by regulating the activity of PKM2 and thereby affecting the energy metabolism in immune cells. DisclosuresNo relevant conflicts of interest to declare.

PTCSC3-mediated glycolysis suppresses thyroid cancer progression via interfering with PGK1 degradation.

The Warburg effect (aerobic glycolysis), a hallmark of cancer, serves as a promising target for diagnosis and therapy. Growing evidence indicates that long non‐coding RNAs (lncRNAs) play an important role in aerobic glycolysis of various tumours. However, the correlation between lncRNAs and glycolysis in thyroid cancer cells is still poorly understood. In this study, we showed that lncRNA papillary thyroid cancer susceptibility candidate 3 (PTCSC3) was significantly downregulated in papillary thyroid carcinoma (PTC). Overexpression of PTCSC3 significantly inhibited the aerobic glycolysis and tumour growth of PTC cells. Consistently, PTCSC3 overexpression suppressed tumour progress in vivo. Mechanistically, PTCSC3 inhibits aerobic glycolysis and proliferation of PTC by directly interacting with PGK1, a key enzyme in glycolytic pathway. As a result, PTCSC3 performs its role in PTC development via PGK1 and may be a potential therapeutic target for PTC treatment.

Base editing-mediated perturbation of endogenous PKM1/2 splicing facilitates isoform-specific functional analysis in vitro and in vivo.

Objectives PKM1 and PKM2, which are generated from the alternative splicing of PKM gene, play important roles in tumourigenesis and embryonic development as rate‐limiting enzymes in glycolytic pathway. However, because of the lack of appropriate techniques, the specific functions of the 2 PKM splicing isoforms have not been clarified endogenously yet.Materials and methodsIn this study, we used CRISPR‐based base editors to perturbate the endogenous alternative splicing of PKM by introducing mutations into the splicing junction sites in HCT116 cells and zebrafish embryos. Sanger sequencing, agarose gel electrophoresis and targeted deep sequencing assays were utilized for identifying mutation efficiencies and detecting PKM1/2 splicing isoforms. Cell proliferation assays and RNA‐seq analysis were performed to describe the effects of perturbation of PKM1/2 splicing in tumour cell growth and zebrafish embryo development.ResultsThe splicing sites of PKM, a 5’ donor site of GT and a 3’ acceptor site of AG, were efficiently mutated by cytosine base editor (CBE; BE4max) and adenine base editor (ABE; ABEmax‐NG) with guide RNAs (gRNAs) targeting the splicing sites flanking exons 9 and 10 in HCT116 cells and/or zebrafish embryos. The mutations of the 5’ donor sites of GT flanking exons 9 or 10 into GC resulted in specific loss of PKM1 or PKM2 expression as well as the increase in PKM2 or PKM1 respectively. Specific loss of PKM1 promoted cell proliferation of HCT116 cells and upregulated the expression of cell cycle regulators related to DNA replication and cell cycle phase transition. In contrast, specific loss of PKM2 suppressed cell growth of HCT116 cells and resulted in growth retardation of zebrafish. Meanwhile, we found that mutation of PKM1/2 splicing sites also perturbated the expression of non‐canonical PKM isoforms and produced some novel splicing isoforms.ConclusionsThis work proved that CRISPR‐based base editing strategy can be used to disrupt the endogenous alternative splicing of genes of interest to study the function of specific splicing isoforms in vitro and in vivo. It also reminded us to notice some novel or undesirable splicing isoforms by targeting the splicing junction sites using base editors. In sum, we establish a platform to perturbate endogenous RNA splicing for functional investigation or genetic correction of abnormal splicing events in human diseases.

Distinct glycolytic pathway regulation in liver, tumour and skeletal muscle of mice with cancer cachexia.

Energetically inefficient inter-organ substrate shuttles are proposed contributors to cachexia-related weight loss. Here, we examined glycolytic pathway metabolites, enzyme activity and transport proteins in skeletal muscle, liver and tumours of mice with cachexia-related weight loss induced by colon-26 cancer cells. Skeletal muscle of cachexic mice had increased [L-lactate]/[pyruvate], LDH activity and lactate transporter MCT1. Cachexic livers also showed increased MCT1. This is consistent with the proposal that the rate of muscle-derived lactate shuttling to liver for use in gluconeogenesis is increased, that is, an increased Cori cycle flux in weight-losing cachexic mice. A second shuttle between liver and tumour may also contribute to disrupted energy balance and weight loss. We found increased high-affinity glucose transporter GLUT1 in tumours, suggesting active glucose uptake, tumour MCT1 detection and decreased intratumour [L-lactate]/[pyruvate], implying increased lactate efflux and/or intratumour lactate oxidation. Last, high [L-lactate]/[pyruvate] and MCT1 in cachexic muscle provides a potential muscle-derived lactate supply for the tumour (a 'reverse Warburg effect'), supporting tumour growth and consequent cachexia. Our findings suggest several substrate shuttles among liver, skeletal muscle and tumour contribute to metabolic disruption and weight loss. Therapies that aim to normalize dysregulated substrate shuttling among energy-regulating tissues may alleviate unintended weight loss in cancer cachexia. SIGNIFICANCE OF THE STUDY: Cachexia is a serious complication of cancer characterized by severe weight loss, muscle atrophy and frailty. Cachexia occurs in roughly half of all cancer patients, and in up to 80% of patients with advanced disease. Cachexia independently worsens patient prognosis, lowers treatment efficacy, increases hospitalization cost and length of stay, and accounts for 20-30% of cancer-related deaths. There are no effective treatments. Our findings suggest several substrate shuttles among liver, skeletal muscle and tumour contribute to metabolic disruption and weight loss in cancer cachexia. Identifying therapies that normalize dysregulated substrate shuttling among energy-regulating tissues may protect against cachexia-related weight loss.

Expression and clinical significance of pyruvate kinase M2 in breast cancer: A protocol for meta-analysis and bioinformatics validation analysis.

Background:Breast cancer is a common malignant tumor in women. In recent years, its incidence is increasing year by year, and its morbidity and mortality rank the first place among female malignant tumors. Some key enzymes and intermediates in glycolysis are closely related to tumor development. Pyruvate kinase M2 (PKM2) is an important rate-limiting enzyme in glycolysis pathway. Meanwhile, it is highly expressed in proliferative cells, especially in tumor cells, and plays an important role in the formation of Warburg effect and tumorigenesis. Previous studies have explored the effects of PKM2 expression on the prognosis and clinical significance of breast cancer patients, while the results are contradictory and uncertain. This study uses controversial data for meta-analysis to accurately evaluate the problem. We collected relevant Oncomine and The Cancer Genome Atlas (TCGA) data to further verify the results. Through bioinformatics analysis, the mechanism and related pathways of PKM2 in breast cancer are explored.Methods:We searched Wanfang, Chinese Biomedical Literature Database, Chinese National Knowledge Infrastructure, the Chongqing VIP Chinese Science and Technology Periodical Database, PubMed, Embase, and Web of Science databases from inception to March 2021. The language restrictions are Chinese and English. The published literatures on PKM2 expression and prognosis or clinicopathological characteristics of breast cancer patients were statistically analyzed. Combined hazard ratios (HRs), odds ratios (ORs), and 95% confidence intervals (95% CIs) were used to evaluate the effects of PKM2 on the prognosis and clinicopathological features of breast cancer. Stata 14.0 software was applied for meta-analysis. Oncomine and TCGA databases were used to meta-analyze the differences of PKM2 mRNA expression between breast cancer and normal breast tissues. The expression of PKM2 protein was verified by Human Protein Atlas (HPA) database. The relationship between the gene and the survival of breast cancer patients was analyzed by Gene Expression Profiling Interactive Analysis (GEPIA). The relationship between PKM2 gene and clinicopathological characteristics was analyzed by using LinkedOmics, and the Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment pathway analysis was performed by using Metascape. Protein–protein interaction (PPI) network was constructed by String website.Results:The results of this meta-analysis will be submitted to a peer-reviewed journal for publication.Conclusion:This study provides high-quality medical evidence for the correlation between the expression of PKM2 and the prognosis and clinicopathological features of breast cancer. Through bioinformatics analysis, this study further deepens the understanding of the mechanism and related pathways of PKM2 in breast cancer.Ethics and dissemination:The private information from individuals will not be published. This systematic review also should not damage participants’ rights. Ethical approval is not available. The results may be published in a peer-reviewed journal or disseminated in relevant conferences.OSF REGISTRATION NUMBER:DOI 10.17605/OSF.IO/W52HB.

CircATP2B1 Promotes Aerobic Glycolysis in Gastric Cancer Cells Through Regulation of the miR-326 Gene Cluster.

The discovery of circular RNA (circRNA) enormously complimented the repertoire of traditional gene expression theory. As a type of endogenous noncoding RNA, circRNA participates in the occurrence of many kinds of tumors in addition to regulating their development. The Warburg effect (aerobic glycolysis is taken with priority for cancer cells instead of oxidative phosphorylation) is one of the most important factors involved in the excessive proliferation of gastric cancer cells. Our data showed that circRNA circATP2B1 (also called hsa_circ_000826) was overexpressed in gastric cancer tissues instead of linear ATP2B1 mRNA, and it promoted aerobic glycolysis in gastric cancer cells. Bioinformatic Gene Ontology analysis showed that the potential downstream targets of circATP2B1 include the microRNA miR-326 gene cluster (miR-326-3p/miR-330-5p), which is functionally focused on cell growth and metabolic processes. The expressions of miR-326-3p/miR-330-5p were downregulated in gastric cancer, and circATP2B1 functionally targeted miR-326-3p/miR-330-5p in an RNA-induced silencing complex (RISC) dependent manner. Dual-luciferase reporter assays demonstrated that pyruvate kinase M2 (PKM2) was one of the targets of miR-326-3p/miR-330-5p. As a rate-limiting enzyme in the aerobic glycolytic pathway, PKM2 accelerated gastric cancer cells’ glucose uptake and increased cell viability. Taken together, circATP2B1 captured miR-326-3p/miR-330-5p and decreased the suppression of PKM2 by miR-326-3p/miR-330-5p, thus aiding the aerobic glycolysis and proliferation of gastric cancer cells. This study identified a novel molecular pathway in gastric cancer that may provide more targets for reversing cancer metabolic reprogramming, as well as a potential strategy for targeted therapy of gastric cancer.

Proteomic analyses of the oleaginous and carotenogenic yeast Rhodotorula diobovata across growth phases under nitrogen- and oxygen-limited conditions

Carotenoids and triacylglycerols from yeasts are important bioproducts that can be utilized for the nutraceutical and biodiesel industries respectively. Rhodotorula diobovata is capable of producing these bioproducts under varied culture conditions. These productions have been linked to the early stationary growth phase and their levels only start to decline at the late stationary phase when carbon becomes limiting. While nitrogen-limitation influences the onset of lipogenesis, continuous synthesis and accumulation of neutral lipids (triacylglycerides) may be dependent on other culture conditions such as aeration. Proteomic analyses were conducted to enhance our understanding of changes in gene product expression under culture conditions with nitrogen-limitation, coupled with insufficient aeration, and revealed a correlation between the upregulation of proteins in the lipolysis pathways and the reduced synthesis of fatty acids at the early stationary phase. Upregulation of glycolytic pathway enzymes suggested that glucose was quickly converted into pyruvate and then acetyl-CoA. However, acetyl-CoA flux favoured carotenoids biosynthesis over fatty acid synthesis, as cells transitioned into the stationary phase. This work provides insights into how culture conditions influence gene product expression levels, pathway utilization, and end-product synthesis patterns.