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1
Li, Lin, Siyuan Cheng, and Xiuping Yu. "The expression of PKM1 and PKM2 in benign and cancerous prostatic tissues." Journal of Clinical Oncology 41, no. 16_suppl (June 1, 2023): e17058-e17058. http://dx.doi.org/10.1200/jco.2023.41.16_suppl.e17058.
Анотація:
e17058 Background: Prostate cancer (PCa) is the most diagnosed cancers in American men. After androgen deprivation therapy (ADT) fails, there is an increase in the aggressive neuroendocrine (NE) phenotype. Currently, there is no effective treatment for NE prostate cancer (NEPC). The metabolic reprogramming, one of the cancer hallmarks, regulates PCa progression and therapy resistance. However, the energy metabolism in NEPC has not been well studied yet. Pyruvate kinase (PK), catalyzing the final step of glycolysis, has PKM1, PKM2, PKL and PKR four isoforms. PKL and PKR are expressed in the liver and erythrocytes, respectively. Alternative splicing of PKM results in two isoforms, PKM1 and PKM2, which are expressed in most tissues. In prostate adenocarcinoma, loss of PKM1 promotes PCa progression whereas loss of PKM2 suppresses tumor growth. However, the expression pattern of PKM1 and PKM2 in NEPCa remains largely unknown. Methods: Immunofluorescence (IF), immunohistochemistry (IHC), Western blot and RT-qPCR were conducted to examine the expression of PKM1 and PKM2 in both murine and human prostatic tissues. The bioinformatics analysis was done using the publicly available RNA-Seq data obtained from the cBioportal, the Cancer Genome Atlas (TCGA), and the Cancer Cell Line Encyclopedia websites (CCLE). Results: TRAMP is a widely used PCa mouse model. TRAMP mice develop prostatic intraepithelial neoplasia (PIN) and the tumors progress into NEPC following castration. We found that PKM1 expression was detected in normal prostate but not in the PIN lesions. In the TRAMP NEPC tumors, PKM1 expression was detected in the NE areas but not in the adjacent PIN lesions. Compared with the adjacent PIN, NEPC cells displayed lower PKM2 expression. Further, we examined the expression of PKM1 and PKM2 in human prostatic tissues including benign prostatic hyperplasia (BPH), low-grade adenocarcinomas (AdPCa), high-grade AdPCa, and NEPC. We found that in BPH, basal epithelial cells express both PKM1 and PKM2. In PCa, PKM1 was robustly expressed in the stromal cells but its expression was absent in the cancer cells in majority of the specimens examined except a small number of samples where low level of PKM1 was detected in the cancer cells. However, PKM1 expression was detected in 9 out of 12 NEPC samples and colocalized with NE marker chromogranin A. This co-expression was also detected in the NE cells scattered on the prostate adenocarcinoma tissues. As for PKM2, its expression was detected in all the samples examined. However, different from previous report, PKM2 expression levels did not correlate with cancer grade in this cohort. Conclusions: PKM1 is expressed in the basal epithelial cells of benign prostates, a small subset of prostate adenocarcinomas, and 75% NEPC tumors. PKM2 is expressed throughout prostate development, in both benign and cancerous prostate. However, PKM2 expression does not correlate with tumor grade.
2
Kim, Seong Ho, Ji Hun Wi, HyeRan Gwak, Eun Gyeong Yang, and So Yeon Kim. "Single-Cell FISH Analysis Reveals Distinct Shifts in PKM Isoform Populations during Drug Resistance Acquisition." Biomolecules 12, no. 8 (August 6, 2022): 1082. http://dx.doi.org/10.3390/biom12081082.
Анотація:
The Warburg effect, i.e., the utilization of glycolysis under aerobic conditions, is recognized as a survival advantage of cancer cells. However, how the glycolytic activity is affected during drug resistance acquisition has not been explored at single-cell resolution. Because the relative ratio of the splicing isoform of pyruvate kinase M (PKM), PKM2/PKM1, can be used to estimate glycolytic activity, we utilized a single-molecule fluorescence in situ hybridization (SM-FISH) method to simultaneously quantify the mRNA levels of PKM1 and PKM2. Treatment of HCT116 cells with gefitinib (GE) resulted in two distinct populations of cells. However, as cells developed GE resistance, the GE-sensitive population with reduced PKM2 expression disappeared, and GE-resistant cells (Res) demonstrated enhanced PKM1 expression and a tightly regulated PKM2/PKM1 ratio. Our data suggest that maintaining an appropriate PKM2 level is important for cell survival upon GE treatment, whereas increased PKM1 expression becomes crucial in GE Res. This approach demonstrates the importance of single-cell-based analysis for our understanding of cancer cell metabolic responses to drugs, which could aid in the design of treatment strategies for drug-resistant cancers.
3
Verbrugge, Sander A. J., Sebastian Gehlert, Lian E. M. Stadhouders, Daniel Jacko, Thorben Aussieker, Gerard M. J. de Wit, Ilse S. P. Vogel, et al. "PKM2 Determines Myofiber Hypertrophy In Vitro and Increases in Response to Resistance Exercise in Human Skeletal Muscle." International Journal of Molecular Sciences 21, no. 19 (September 25, 2020): 7062. http://dx.doi.org/10.3390/ijms21197062.
Анотація:
Nearly 100 years ago, Otto Warburg investigated the metabolism of growing tissues and discovered that tumors reprogram their metabolism. It is poorly understood whether and how hypertrophying muscle, another growing tissue, reprograms its metabolism too. Here, we studied pyruvate kinase muscle (PKM), which can be spliced into two isoforms (PKM1, PKM2). This is of interest, because PKM2 redirects glycolytic flux towards biosynthetic pathways, which might contribute to muscle hypertrophy too. We first investigated whether resistance exercise changes PKM isoform expression in growing human skeletal muscle and found that PKM2 abundance increases after six weeks of resistance training, whereas PKM1 decreases. Second, we determined that Pkm2 expression is higher in fast compared to slow fiber types in rat skeletal muscle. Third, by inducing hypertrophy in differentiated C2C12 cells and by selectively silencing Pkm1 and/or Pkm2 with siRNA, we found that PKM2 limits myotube growth. We conclude that PKM2 contributes to hypertrophy in C2C12 myotubes and indicates a changed metabolic environment within hypertrophying human skeletal muscle fibers. PKM2 is preferentially expressed in fast muscle fibers and may partly contribute to the increased potential for hypertrophy in fast fibers.
4
Buneeva, Olga, Arthur Kopylov, Oksana Gnedenko, Marina Medvedeva, Alexander Veselovsky, Alexis Ivanov, Victor Zgoda, and Alexei Medvedev. "Proteomic Profiling of Mouse Brain Pyruvate Kinase Binding Proteins: A Hint for Moonlighting Functions of PKM1?" International Journal of Molecular Sciences 24, no. 8 (April 21, 2023): 7634. http://dx.doi.org/10.3390/ijms24087634.
Анотація:
Affinity-based proteomic profiling is widely used for the identification of proteins involved in the formation of various interactomes. Since protein–protein interactions (PPIs) reflect the role of particular proteins in the cell, identification of interaction partners for a protein of interest can reveal its function. The latter is especially important for the characterization of multifunctional proteins, which can play different roles in the cell. Pyruvate kinase (PK), a classical glycolytic enzyme catalyzing the last step of glycolysis, exists in four isoforms: PKM1, PKM2, PKL, and PKR. The enzyme isoform expressed in actively dividing cells, PKM2, exhibits many moonlighting (noncanonical) functions. In contrast to PKM2, PKM1, predominantly expressed in adult differentiated tissues, lacks well-documented moonlighting functions. However, certain evidence exists that it can also perform some functions unrelated to glycolysis. In order to evaluate protein partners, bound to PKM1, in this study we have combined affinity-based separation of mouse brain proteins with mass spectrometry identification. The highly purified PKM1 and a 32-mer synthetic peptide (PK peptide), sharing high sequence homology with the interface contact region of all PK isoforms, were used as the affinity ligands. This proteomic profiling resulted in the identification of specific and common proteins bound to both affinity ligands. Quantitative affinity binding to the affinity ligands of selected identified proteins was validated using a surface plasmon resonance (SPR) biosensor. Bioinformatic analysis has shown that the identified proteins, bound to both full-length PKM1 and the PK peptide, form a protein network (interactome). Some of these interactions are relevant for the moonlighting functions of PKM1. The proteomic dataset is available via ProteomeXchange with the identifier PXD041321.
5
Kurihara-Shimomura, Miyako, Tomonori Sasahira, Chie Nakashima, Hiroki Kuniyasu, Hiroyuki Shimomura, and Tadaaki Kirita. "The Multifarious Functions of Pyruvate Kinase M2 in Oral Cancer Cells." International Journal of Molecular Sciences 19, no. 10 (September 25, 2018): 2907. http://dx.doi.org/10.3390/ijms19102907.
Анотація:
Head and neck cancers, including oral squamous cell carcinoma (OSCC), are the sixth most common malignancies worldwide. OSCC frequently leads to oral dysfunction, which worsens a patient’s quality of life. Moreover, its prognosis remains poor. Unlike normal cells, tumor cells preferentially metabolize glucose by aerobic glycolysis. Pyruvate kinase (PK) catalyzes the final step in glycolysis, and the transition from PKM1 to PKM2 is observed in many cancer cells. However, little is known about PKM expression and function in OSCC. In this study, we investigated the expression of PKM in OSCC specimens and performed a functional analysis of human OSCC cells. We found that the PKM2/PKM1 ratio was higher in OSCC cells than in adjacent normal mucosal cells and in samples obtained from dysplasia patients. Furthermore, PKM2 expression was strongly correlated with OSCC tumor progression on immunohistochemistry. PKM2 expression was higher during cell growth, invasion, and apoptosis in HSC3 cells, which show a high energy flow and whose metabolism depends on aerobic glycolysis and oxidative phosphorylation. PKM2 expression was also associated with the production of reactive oxygen species (ROS) and integration of glutamine into lactate. Our results suggested that PKM2 has a variety of tumor progressive functions in OSCC cells.
6
Williams, Allison Lesher, Vedbar Khadka, Mingxin Tang, Abigail Avelar, Kathryn J. Schunke, Mark Menor, and Ralph V. Shohet. "HIF1 mediates a switch in pyruvate kinase isoforms after myocardial infarction." Physiological Genomics 50, no. 7 (July 1, 2018): 479–94. http://dx.doi.org/10.1152/physiolgenomics.00130.2017.
Анотація:
Alternative splicing of RNA is an underexplored area of transcriptional response. We expect that early changes in alternatively spliced genes may be important for responses to cardiac injury. Hypoxia inducible factor 1 (HIF1) is a key transcription factor that rapidly responds to loss of oxygen through alteration of metabolism and angiogenesis. The goal of this study was to investigate the transcriptional response after myocardial infarction (MI) and to identify novel, hypoxia-driven changes, including alternative splicing. After ligation of the left anterior descending artery in mice, we observed an abrupt loss of cardiac contractility and upregulation of hypoxic signaling. We then performed RNA sequencing on ischemic heart tissue 1 and 3 days after infarct to assess early transcriptional changes and identified 89 transcripts with altered splicing. Of particular interest was the switch in Pkm isoform expression (pyruvate kinase, muscle). The usually predominant Pkm1 isoform was less abundant in ischemic hearts, while Pkm2 and associated splicing factors (hnRNPA1, hnRNPA2B1, Ptbp1) rapidly increased. Despite increased Pkm2 expression, total pyruvate kinase activity remained reduced in ischemic myocardial tissue. We also demonstrated HIF1 binding to PKM by chromatin immunoprecipitation, indicating a direct role for HIF1 in mediating this isoform switch. Our study provides a new, detailed characterization of the early transcriptome after MI. From this analysis, we identified an HIF1-mediated alternative splicing event in the PKM gene. Pkm1 and Pkm2 play distinct roles in glycolytic metabolism and the upregulation of Pkm2 is likely to have important consequences for ATP synthesis in infarcted cardiac muscle.
7
Lee, Yuumi, Yuko Ito, Kohei Taniguchi, Takashi Nuri, SangWoong Lee, and Koichi Ueda. "Experimental Study of Warburg Effect in Keloid Nodules: Implication for Downregulation of miR-133b." Plastic and Reconstructive Surgery - Global Open 11, no. 8 (August 2023): e5202. http://dx.doi.org/10.1097/gox.0000000000005202.
Анотація:
Background: A keloid is composed of several nodules, which are divided into two zones: the central zone (CZ; a hypoxic region) and the marginal zone (MZ; a normoxic region). Keloid nodules play a key role in energy metabolic activity for continuous growth by increasing in number and total area. In this study, we aimed to investigate the roles of the zones in the execution of the Warburg effect and identify which microRNAs regulate this phenomenon in keloid tissue. Methods: Eleven keloids from patients were used. Using immunohistochemical analysis, 179 nodules were randomly chosen from these keloids to identify glycolytic enzymes, autophagic markers, pyruvate kinase M (PKM) 1/2, and polypyrimidine tract binding protein 1 (PTBP1). Western blot and qRT-PCR tests were also performed for PKM, PTBP1, and microRNAs (miR-133b and miR-200b, c). Results: Immunohistochemical analysis showed that the expression of the autophagic (LC3, p62) and glycolytic (GLUT1, HK2) were significantly higher in the CZ than in the MZ. PKM2 expression was significantly higher than PKM1 expression in keloid nodules. Furthermore, PKM2 expression was higher in the CZ than in the MZ. However, PKM1 and PTBP1 expression levels were higher in the MZ than in the CZ. The qRT-PCR analysis showed that miR-133b-3p was moderately downregulated in the keloids compared with its expression in the normal skin tissue. Conclusions: The Warburg effect occurred individually in nodules. The MZ presented PKM2-positive fibroblasts produced by activated PTBP1. In the CZ, PKM2-positive fibroblasts produced lactate. MiR-133b-3p was predicted to control the Warburg effect in keloids.
8
Kuranaga, Yuki, Nobuhiko Sugito, Haruka Shinohara, Takuya Tsujino, Kohei Taniguchi, Kazumasa Komura, Yuko Ito, Tomoyoshi Soga, and Yukihiro Akao. "SRSF3, a Splicer of the PKM Gene, Regulates Cell Growth and Maintenance of Cancer-Specific Energy Metabolism in Colon Cancer Cells." International Journal of Molecular Sciences 19, no. 10 (October 2, 2018): 3012. http://dx.doi.org/10.3390/ijms19103012.
Анотація:
Serine and arginine rich splicing factor 3 (SRSF3), an SR-rich family protein, has an oncogenic function in various kinds of cancer. However, the detailed mechanism of the function had not been previously clarified. Here, we showed that the SRSF3 splicer regulated the expression profile of the pyruvate kinase, which is one of the rate-limiting enzymes in glycolysis. Most cancer cells express pyruvate kinase muscle 2 (PKM2) dominantly to maintain a glycolysis-dominant energy metabolism. Overexpression of SRSF3, as well as that of another splicer, polypyrimidine tract binding protein 1 (PTBP1) and heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1), in clinical cancer samples supported the notion that these proteins decreased the Pyruvate kinase muscle 1 (PKM1)/PKM2 ratio, which positively contributed to a glycolysis-dominant metabolism. The silencing of SRSF3 in human colon cancer cells induced a marked growth inhibition in both in vitro and in vivo experiments and caused an increase in the PKM1/PKM2 ratio, thus resulting in a metabolic shift from glycolysis to oxidative phosphorylation. At the same time, the silenced cells were induced to undergo autophagy. SRSF3 contributed to PKM mRNA splicing by co-operating with PTBP1 and hnRNPA1, which was validated by the results of RNP immunoprecipitation (RIP) and immunoprecipitation (IP) experiments. These findings altogether indicated that SRSF3 as a PKM splicer played a positive role in cancer-specific energy metabolism.
9
Xia, Yong, Xing Wang, Yan Liu, Ellen Shapiro, Herbert Lepor, Moon-Shong Tang, Tung-Tien Sun, and Xue-Ru Wu. "PKM2 Is Essential for Bladder Cancer Growth and Maintenance." Cancer Research 82, no. 4 (December 13, 2021): 571–85. http://dx.doi.org/10.1158/0008-5472.can-21-0403.
Анотація:
Abstract Pyruvate kinase M2 (PKM2) has been shown to promote tumorigenesis by facilitating the Warburg effect and enhancing the activities of oncoproteins. However, this paradigm has recently been challenged by studies in which the absence of PKM2 failed to inhibit and instead accelerated tumorigenesis in mouse models. These results seem inconsistent with the fact that most human tumors overexpress PKM2. To further elucidate the role of PKM2 in tumorigenesis, we investigated the effect of PKM2 knockout in oncogenic HRAS-driven urothelial carcinoma. While PKM2 ablation in mouse urothelial cells did not affect tumor initiation, it impaired the growth and maintenance of HRAS-driven tumors. Chemical inhibition of PKM2 recapitulated these effects. Both conditions substantially reduced complex formation of PKM2 with STAT3, their nuclear translocation, and HIF1α- and VEGF-related angiogenesis. The reduction in nuclear STAT3 in the absence of PKM2 also correlated with decreased autophagy and increased apoptosis. Time-controlled, inducible PKM2 overexpression in simple urothelial hyperplasia did not trigger tumorigenesis, while overexpression of PKM2, but not PKM1, in nodular urothelial hyperplasia with angiogenesis strongly accelerated tumorigenesis. Finally, in human patients, PKM2 was overexpressed in low-grade nonmuscle-invasive and high-grade muscle-invasive bladder cancer. Based on these data, PKM2 is not required for tumor initiation but is essential for tumor growth and maintenance by enhancing angiogenesis and metabolic addiction. The PKM2–STAT3–HIF1α/VEGF signaling axis may play a critical role in bladder cancer and may serve as an actionable therapeutic target. Significance: Genetic manipulation and pharmacologic inhibition of PKM2 in mouse urothelial lesions highlight its essential role in promoting angiogenesis and metabolic addiction, events indispensable for tumor growth and maintenance.
10
Grant, Melissa M. "Pyruvate Kinase, Inflammation and Periodontal Disease." Pathogens 10, no. 7 (June 22, 2021): 784. http://dx.doi.org/10.3390/pathogens10070784.
Анотація:
Pyruvate kinase (PK) is the final and rate-limiting enzyme in glycolysis. It has four isoforms PKM1, PKM2, PKL and PKR. PK can form homo tetramers, dimers or monomers. The tetrameric form has the most catalytic activity; however, the dimeric form has non-canonical functions that contribute to the inflammatory response, wound healing and cellular crosstalk. This brief review explores these functions and speculates on their role in periodontal disease.
11
Lemos, Fernanda O., Ian de Ridder, Martin D. Bootman, Geert Bultynck, and Jan B. Parys. "The Complex Effects of PKM2 and PKM2:IP3R Disruption on Intracellular Ca2+ Handling and Cellular Functions." Cells 12, no. 21 (October 26, 2023): 2527. http://dx.doi.org/10.3390/cells12212527.
Анотація:
Pyruvate kinase M (PKM) 2 was described to interact with the inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) and suppress its activity. To further investigate the physiological importance of the PKM2:IP3R interaction, we developed and characterized HeLa PKM2 knockout (KO) cells. In the HeLa PKM2 KO cells, the release of Ca2+ to the cytosol appears to be more sensitive to low agonist concentrations than in HeLa wild-type (WT) cells. However, upon an identical IP3-induced Ca2+ release, Ca2+ uptake in the mitochondria is decreased in HeLa PKM2 KO cells, which may be explained by the smaller number of contact sites between the ER and the mitochondria. Furthermore, in HeLa PKM2 KO cells, mitochondria are more numerous, though they are smaller and less branched and have a hyperpolarized membrane potential. TAT-D5SD, a cell-permeable peptide representing a sequence derived from IP3R1 that can disrupt the PKM2:IP3R interaction, induces Ca2+ release into the cytosol and Ca2+ uptake into mitochondria in both HeLa WT and PKM2 KO cells. Moreover, TAT-D5SD induced apoptosis in HeLa WT and PKM2 KO cells but not in HeLa cells completely devoid of IP3Rs. These results indicate that PKM2 separately regulates cytosolic and mitochondrial Ca2+ handling and that the cytotoxic effect of TAT-D5SD depends on IP3R activity but not on PKM2. However, the tyrosine kinase Lck, which also interacts with the D5SD sequence, is expressed neither in HeLa WT nor PKM2 KO cells, and we can also exclude a role for PKM1, which is upregulated in HeLa PKM2 KO cells, indicating that the TAT-D5SD peptide has a more complex mode of action than anticipated.
12
Mojzeš, Anamarija, Marko Tomljanović, Lidija Milković, Renata Novak Kujundžić, Ana Čipak Gašparović, and Koraljka Gall Trošelj. "Cell-Type Specific Metabolic Response of Cancer Cells to Curcumin." International Journal of Molecular Sciences 21, no. 5 (February 28, 2020): 1661. http://dx.doi.org/10.3390/ijms21051661.
Анотація:
In order to support uncontrolled proliferation, cancer cells need to adapt to increased energetic and biosynthetic requirements. One such adjustment is aerobic glycolysis or the Warburg effect. It is characterized by increased glucose uptake and lactate production. Curcumin, a natural compound, has been shown to interact with multiple molecules and signaling pathways in cancer cells, including those relevant for cell metabolism. The effect of curcumin and its solvent, ethanol, was explored on four different cancer cell lines, in which the Warburg effect varied. Vital cellular parameters (proliferation, viability) were measured along with the glucose consumption and lactate production. The transcripts of pyruvate kinase 1 and 2 (PKM1, PKM2), serine hydroxymethyltransferase 2 (SHMT2) and phosphoglycerate dehydrogenase (PHGDH) were quantified with RT-qPCR. The amount and intracellular localization of PKM1, PKM2 and signal transducer and activator of transcription 3 (STAT3) proteins were analyzed by Western blot. The response to ethanol and curcumin seemed to be cell-type specific, with respect to all parameters analyzed. High sensitivity to curcumin was present in the cell lines originating from head and neck squamous cell carcinomas: FaDu, Detroit 562 and, especially, Cal27. Very low sensitivity was observed in the colon adenocarcinoma-originating HT-29 cell line, which retained, after exposure to curcumin, a higher levels of lactate production despite decreased glucose consumption. The effects of ethanol were significant.
13
Huang, Rong, Rong Han, Yucheng Yan, Jifan Yang, Guoxuan Dong, Miao Wang, Zhiguo Su, Hu Jiao, and Jincai Fan. "PTB Regulates the Metabolic Pathways and Cell Function of Keloid Fibroblasts through Alternative Splicing of PKM." International Journal of Molecular Sciences 24, no. 6 (March 8, 2023): 5162. http://dx.doi.org/10.3390/ijms24065162.
Анотація:
Keloids, benign fibroproliferative cutaneous lesions, are characterized by abnormal growth and reprogramming of the metabolism of keloid fibroblasts (KFb). However, the underlying mechanisms of this kind of metabolic abnormality have not been identified. Our study aimed to investigate the molecules involved in aerobic glycolysis and its exact regulatory mechanisms in KFb. We discovered that polypyrimidine tract binding (PTB) was significantly upregulated in keloid tissues. siRNA silencing of PTB decreased the mRNA levels and protein expression levels of key glycolytic enzymes and corrected the dysregulation of glucose uptake and lactate production. In addition, mechanistic studies demonstrated that PTB promoted a change from pyruvate kinase muscle 1 (PKM1) to PKM2, and silencing PKM2 substantially reduced the PTB-induced increase in the flow of glycolysis. Moreover, PTB and PKM2 could also regulate the key enzymes in the tricarboxylic acid (TCA) cycle. Assays of cell function demonstrated that PTB promoted the proliferation and migration of KFb in vitro, and this phenomenon could be interrupted by PKM2 silencing. In conclusion, our findings indicate that PTB regulates aerobic glycolysis and the cell functions of KFb via alternative splicing of PKM.
14
Nayak, Manasa, Nirav Dhanesha, Manish Jain, and Anil Chauhan. "Manipulating Metabolic Plasticity By Targeting Pyruvate Kinase M2 in Platelets Inhibits Arterial Thrombosis." Blood 132, Supplement 1 (November 29, 2018): 868. http://dx.doi.org/10.1182/blood-2018-99-112704.
Анотація:
Abstract Background: Most of the cellular responses initiated upon platelet activation are energy consuming. Like normal cells, resting platelets rely primarily on oxidative phosphorylation (OXPHOS) to generate ATP, whereas activated platelets exhibit a high level of aerobic glycolysis (conversion of glucose to lactate in the presence of oxygen, a phenomenon referred to as the Warburg effect in tumor cells) suggesting that metabolic plasticity exists in activated platelets. Although aerobic glycolysis yields less total ATP when compared to OXPHOS, the rate of ATP generation is faster in aerobic glycolysis compared to OXPHOS, which is well suited for high-energy demands during platelet activation. Pyruvate kinases (PKs) catalyzes the final step of glycolysis, the formation of pyruvate and ATP from phosphoenolpyruvate and ADP. Four PK isoforms exist in mammals: L and R isoforms are expressed in the liver and red blood cells; the M1 isoform is expressed in most adult tissues that have high catabolic demands including muscle and brain; M2 is expressed in cells including activated platelets and leukocytes. While PKM1 and tetrameric PKM2 favor ATP production from OXPHOS through the TCA cycle, dimeric PKM2 drives aerobic glycolysis. Objective: We tested an innovative concept that by manipulating the energy demand of activated platelets (metabolic plasticity), by targeting PKM2, will inhibit platelet function and thrombosis. Methods: Using a specific inhibitor of PKM2 (inhibits PKM2 dimerization and stabilizes tetramers) and standardized platelet in vitro assays, we determined the mechanistic role of PKM2 in modulating platelet function in human and mice. To provide definitive evidence, we generated a megakaryocyte or platelet-specific PKM2-/- mouse (PKM2fl/flPF4Cre). Lactate assay was performed in WT and PKM2 null platelets. Susceptibility to thrombosis was evaluated in vitro (microfluidics flow chamber) and in vivo (FeCl3-induced carotid artery thrombosis and laser injury models) by utilizing intravital microscopy. Results: We found that PKM2 is relatively highly expressed compared to PKM1 in human and murine platelets. Transmission electron microscopy (immunogold staining) revealed that PKM2 is found in the cytoplasm and a- granule in resting platelets, whereas most of PKM2 translocated to cytoplasm upon activation. Human and mouse platelets pretreated with PKM2 inhibitor exhibited decreased platelet aggregation to sub-optimal doses of collagen and convulxin but not to thrombin. In microfluidics flow chamber assay, human and whole mouse blood pretreated with PKM2 inhibitor formed small thrombi when perfused over collagen for 5 min at an arterial shear rate of 1500s-1 (P<0.05 vs. vehicle control). Platelets from PKM2fl/flPF4Cre mice exhibited decreased platelet aggregation to sub-optimal doses of collagen and convulxin, but not to thrombin, compared to PKM2fl/fl mice concomitant with decrease lactate production. In microfluidics flow chamber assay, whole blood from PKM2fl/flPF4Cre mice formed smaller thrombi when perfused over collagen for 5 min at an arterial shear rate of 1500s-1, compared to PKM2fl/fl mice. PKM2fl/flPF4Cre mice were less susceptible to thrombosis in the FeCl3-induced carotid and laser injury-induced mesenteric artery thrombosis models (P<0.05 vs. vehicle control, N=10 mice/group), without altering hemostasis. PKM2 regulates the phosphorylation signal transducer and activator of transcription 3 (STAT3) and p-STAT3 act as a protein scaffold that facilitates the catalytic process of activating PLCg by kinase Syk in response to low-doses of collagen and CRP, but not TRAP or ADP in human and murine platelets. Interestingly, we found that PKM2 and STAT3 colocalized in the convulixn- stimulated control platelets and less phosphorylation of STAT-3 was observed in activated PKM2 null platelets (P<0.05 vs. WT), suggesting a non-glycolytic role of the PKM2 in regulating collagen signaling. Conclusions: Our results suggest that dimeric PKM2 regulates platelet function and arterial thrombosis most likely via GPVI signaling pathway. We suggest that manipulating metabolic plasticity by targeting dimeric PKM2 may be explored as a novel strategy to inhibit platelet function and arterial thrombosis. Disclosures No relevant conflicts of interest to declare.
15
Goldberg, Michael S., and Phillip A. Sharp. "Pyruvate kinase M2-specific siRNA induces apoptosis and tumor regression." Journal of Experimental Medicine 209, no. 2 (January 23, 2012): 217–24. http://dx.doi.org/10.1084/jem.20111487.
Анотація:
The development of cancer-specific therapeutics has been limited because most healthy cells and cancer cells depend on common pathways. Pyruvate kinase (PK) exists in M1 (PKM1) and M2 (PKM2) isoforms. PKM2, whose expression in cancer cells results in aerobic glycolysis and is suggested to bestow a selective growth advantage, is a promising target. Because many oncogenes impart a common alteration in cell metabolism, inhibition of the M2 isoform might be of broad applicability. We show that several small interfering (si) RNAs designed to target mismatches between the M2 and M1 isoforms confer specific knockdown of the former, resulting in decreased viability and increased apoptosis in multiple cancer cell lines but less so in normal fibroblasts or endothelial cells. In vivo delivery of siPKM2 additionally causes substantial tumor regression of established xenografts. Our results suggest that the inherent nucleotide-level specificity of siRNA can be harnessed to develop therapeutics that target isoform-specific exons in genes exhibiting differential splicing patterns in various cell types.
16
Ensink, Elliot, and Sophia Y. Lunt. "Abstract 3031: Impact of pyruvate kinase isoforms on cysteine metabolism in pancreatic cancer." Cancer Research 82, no. 12_Supplement (June 15, 2022): 3031. http://dx.doi.org/10.1158/1538-7445.am2022-3031.
Анотація:
Abstract Pancreatic cancer cells undergo metabolic alterations to survive within a nutrient-depleted tumor microenvironment. Cysteine metabolism in particular is critical for supporting pancreatic cancer cell survival through its role in redox homeostasis. Targeting cysteine metabolism presents an underexplored opportunity for therapeutic exploitation, but much remains unknown about how cysteine metabolism is influenced by microenvironmental conditions and the overall metabolic state of the cell. One critical metabolic shift in pancreatic cancer cells occurs through altered isoform expression of the glycolytic enzyme, pyruvate kinase (PKM). Pancreatic cancer cells preferentially switch from the constitutively active M1 isoform to the allosterically regulated M2 isoform of PKM. Overexpression of PKM2 in pancreatic cancer cells produces a profound reprogramming of many metabolic pathways; however, the precise relationship between PKM isoform expression and cysteine metabolism is not well understood. We hypothesize that PKM isoform selection enables pancreatic cancer cell survival within a low cystine tumor microenvironment by reprogramming metabolic activity to increase cystine import and glutathione production and modulate glucose and glutamine metabolism to resist oxidative stress. We used lentiviral CRISPR-Cas9 vectors to genetically modify two human pancreatic ductal adenocarcinoma cell lines, AsPC-1 and Panc-1, to exclusively express either PKM1 or PKM2. We evaluated the effects of PKM isoform selection on tolerance of low cystine environments by assessing viability under a range of cystine concentrations (0 – 50 μM) reflective of physiologic and cystine starvation conditions. We observe altered viability and proliferation in PKM1 vs. PKM2 expressing cells under normoxic (21%) and hypoxic (1% O2) conditions. Targeted metabolomics using stable isotope labeling tracers further reveal impacts of PKM isoform expression in central carbon metabolism when cystine is limited. Additional experiments to elucidate the precise mechanism by which PKM isoform expression impacts pancreatic cancer cells are currently under investigation. This study provides valuable insight into the complexities of metabolic reprogramming within the tumor microenvironment and enhances our understanding of how cysteine metabolism could be targeted for the treatment of pancreatic cancer. Citation Format: Elliot Ensink, Sophia Y. Lunt. Impact of pyruvate kinase isoforms on cysteine metabolism in pancreatic cancer [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 3031.
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Warenius, Hilmar M. "Selectively depleting the energy of cancer cells: A new therapeutic paradigm." Journal of Cancer Biology 5, no. 1 (January 29, 2024): 1–10. http://dx.doi.org/10.46439/cancerbiology.5.057.
Анотація:
Over the past fifty years the somatic mutation theory of cancer has emerged as the most successful explanation of the molecular phenotype of human cancer cells. Normal non-mutated genes may, however, also play a role in carcinogenesis. In particular these may contribute to aerobic glycolysis and the potential interaction of PKM2 and Cdk4 in helping the nascent cancer cell avoid apoptosis by the interaction of their respective amino acid sequences: anionic SDPTEA and cationic PRGPRP. It is proposed that cancer first occurs in normal tissues cells which, as part of the premalignant phenotype have switched from PKM1 to PKM2 expression, and this phenotype persists as cancers age and further molecular biological mechanisms to avoid apoptosis and encourage aerobic glycolysis emerge. PRGPRP and several congeners have been shown to kill a wide range of in vitro cancers by necrosis by producing a fall in ATP without harming normal diploid cells. The ATP depletion is suggested to result from inhibition of aerobic glycolysis and has resulted in the first therapeutic agent that globally selectively kills cancer cells by depriving them of energy.
18
Bluemlein, Katharina, Nana-Maria Grüning, René G. Feichtinger, Hans Lehrach, Barbara Kofler, and Markus Ralser. "No evidence for a shift in pyruvate kinase PKM1 to PKM2 expression during tumorigenesis." Oncotarget 2, no. 5 (May 22, 2011): 393–400. http://dx.doi.org/10.18632/oncotarget.278.
19
Wang, Ying-Hua, William J. Israelsen, Dongjun Lee, Vionnie W. C. Yu, Nathaniel T. Jeanson, Clary B. Clish, Lewis C. Cantley, Matthew G. Vander Heiden, and David T. Scadden. "Differential Dependence On Aerobic Glycolysis In Normal and Malignant Hematopoietic Stem and Progenitor Cells To Sustain Daughter Cell Production." Blood 122, no. 21 (November 15, 2013): 793. http://dx.doi.org/10.1182/blood.v122.21.793.793.
Анотація:
Abstract How glucose is metabolized can influence cell function, but whether differences in glucose metabolism reflect, or dictate, cell state is not clear and is of particular interest given the association of cancer with aerobic glycolysis. Studies on cancer cell lines have indicated that increased glucose uptake with lactate production regardless of oxygen concentration, a phenomenon also known as the Warburg effect, is promoted in part by expression of the M2 isoform of pyruvate kinase (PKM2) and the muscle form of lactate dehydrogenase A (LDHA). Normal somatic cells thought to also preferentially use glycolytic metabolism are tissue stem cells, particularly the self-renewing hematopoietic stem cells (HSC) resident in the hypoxic microenvironment of the bone marrow. It remains to be defined, however whether proliferating hematopoietic progenitor cells rely on aerobic glycolysis and whether malignant and normal hematopoietic cells are dependent on the same metabolic regulation. We observed that PKM2 and LDHA are the predominant isoforms expressed by all BM hematopoietic cells. To further understand the role of glycolytic metabolism in hematopoiesis and hematological malignancy, we utilized a mouse strain that allows conditional deletion of the PKM2 specific exon 10. Deletion of PKM2 in hematopoetic cells leads to expression of PKM1, accompanied with partial inhibition of lactate production and decreased glycolytic intermediates in the hematopoietic stem/progenitor cell (HSPC) population. Loss of PKM2 compromises the long-term repopulation capacity of HSPCs as revealed by serial transplantation assay. Interestingly, the repopulating defects resulting from PKM2 depletion appear to involve progenitors, perhaps due to inadequate biomass generation necessary for robust cell proliferation. To confirm that the effect of PKM2 deletion on HSPC function is due to metabolic changes rather than other putative PKM2 functions, we engineered a mouse strain that allowed conditional knockout of LDHA to more potently impair aerobic glycolysis. LDHA deletion completely inhibited lactate production, enhanced ROS levels in hematopoietic cells and impaired long-term BM repopulating activity. In contrast to PKM2 deletion that affects progenitor but not stem cells, LDHA depletion impacts both stem cell maintenance and progenitor cell proliferation. Deletion of either PKM2 or LDHA markedly suppressed leukemia initiation by either putative stem cell (BCR-ABL) or progenitor (MLL-AF9) transforming alleles. Therefore, modulating aeroblic glycolysis has effects on normal hematopoietic cells that depend upon cell state and negatively impacts leukemic growth regardless of cell state. The differential sensitivity of normal and malignant cells to modulation of aerobic glycolysis suggests a potential therapeutic opportunity for leukemia intervention. Disclosures: No relevant conflicts of interest to declare.
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Nayak, Manasa K., Madankumar Ghatge, Nirav Dhanesha, Gagan D. Flora, Manish Jain, Omar Rodriguez, Kathleen Markan, Mathhhew Potthoff, Steven R. Lentz, and Anil K. Chauhan. "Targeting Metabolic Enzyme Pyruvate Kinase M2: A Novel Strategy to Inhibit Platelet Function and Arterial Thrombosis." Blood 134, Supplement_1 (November 13, 2019): 1056. http://dx.doi.org/10.1182/blood-2019-129027.
Анотація:
Background: The cellular responses initiated upon platelet activation are energy consuming. Activated platelets, in comparison to their resting state, exhibit a high level of aerobic glycolysis (conversion of glucose to lactate in the presence of oxygen) relative to oxidative phosphorylation (OXPHOS), suggesting that metabolic plasticity exists in platelets. Although aerobic glycolysis yields less total ATP when compared to OXPHOS, the rate of ATP generation is faster in aerobic glycolysis compared to OXPHOS, which we hypothesize is well suited for high-energy requirement during platelet activation. The glycolytic enzyme pyruvate kinases (PKs) catalyzes the final step of glycolysis and contributes to net ATP production. Four PK isoforms (L, R, M1 and M2) exist in mammals: L and R isoforms are expressed in the liver and red blood cells; the M1 isoform is expressed in most adult tissues that have high catabolic demands including muscle and brain; M2 is expressed in cells including activated platelets and leukocytes. Unlike other isoforms of PK that function only as tetramers, PKM2 can exist in either a tetrameric state or a dimeric state. PKM2 is allosterically regulated by the upstream metabolite fructose-1, 6 biphosphate. While PKM1 and tetrameric PKM2 favor ATP production from OXPHOS through the TCA cycle, dimeric PKM2 drives aerobic glycolysis. The glycolytic and non-glycolytic functions of PKM2 in platelets have not investigated yet. Objective: We tested an innovative concept that whether targeting metabolic enzyme PKM2 will inhibit platelet function and arterial thrombosis. Methods: Using a specific inhibitor of PKM2 (that prevents PKM2 dimerization and stabilizes tetramers) and a range of standardized platelet in vitro assays, we determined the mechanistic role of PKM2 in modulating platelet function in human and mice. To provide definitive evidence, we generated a megakaryocyte or platelet-specific PKM2-/- mouse (PKM2fl/flPF4Cre). Susceptibility to thrombosis was evaluated in vitro (microfluidics flow chamber) and in vivo (FeCl3-induced carotid and laser-injury induced mesenteric artery thrombosis models) by utilizing intravital microscopy. Susceptibility to hemostasis was evaluated in tail bleeding assay. Results: Human and mouse platelets pretreated with PKM2 inhibitor significantly decreased platelet aggregation to sub-optimal doses of collagen, convulxin, thrombin, and ADP. Consistent with this, inhibiting PKM2 dimerization reduced αIIbβ3 activation, alpha and dense granule secretion, clot retraction that was concomitant with decreased glucose uptake. Furthermore, treatment with PKM2 inhibitor reduced Akt and GSK3β phosphorylation, that are predominantly involved in PI3K/Akt signaling, suggesting a non-glycolytic role of the PKM2 in regulating platelet function. In microfluidics flow chamber assay, human and whole mouse blood pretreated with PKM2 inhibitor formed small thrombi when perfused over collagen for 5 minutes at an arterial shear rate of 1500s-1 (P<0.05 vs. vehicle). In agreement with PKM2 inhibitor studies, platelets from PKM2fl/flPF4Cre mice exhibited decreased agonist-induced platelet aggregation, which was in agreement with decreased alpha and dense granule secretion, αIIbβ3 activation, clot retraction, lactate production, and Akt and GSK3β phosphorylation (P<0.05 vs. PKM2fl/fl littermate controls). Wild-type mice-treated with PKM2 inhibitor and/or PKM2fl/flPF4Cre were less susceptible to thrombosis in the FeCl3-induced carotid and laser injury-induced mesenteric artery thrombosis models. Lack of effect on tail bleeding time suggested normal hemostasis in PKM2fl/flPF4Cre mice and PKM2 inhibitor-treated wild-type mice. No sex-based differences were observed. Currently, we are performing platelet metabolomics to determine the effect of targeting PKM2 on metabolic pathways. Conclusions: Our results suggest that manipulating metabolic plasticity by targeting dimeric PKM2 may be explored as a novel strategy to inhibit platelet function and arterial thrombosis. Disclosures Lentz: Novo Nordisk Inc.: Consultancy, Honoraria, Research Funding.
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Shi, Weiye, Xu Yao, Xueyu Cao, Yu Fu, and Yingze Wang. "Serine/Arginine-Rich Splicing Factor 7 Knockdown Inhibits Aerobic Glycolysis and Growth in HepG2 Cells by Regulating PKM2 Expression." Current Issues in Molecular Biology 46, no. 5 (May 20, 2024): 5023–36. http://dx.doi.org/10.3390/cimb46050301.
Анотація:
Serine/arginine-rich splicing factors (SRSFs), part of the serine/arginine-rich (SR) protein family, play a crucial role in precursor RNA splicing. Abnormal expression of SRSFs in tumors can disrupt normal RNA splicing, contributing to tumor progression. Notably, SRSF7 has been found to be upregulated in hepatocellular carcinoma (HCC), yet its specific role and molecular mechanisms in HCC pathogenesis are not fully understood. We investigated the expression and prognostic significance of SRSF7 in HCC using bioinformatics database analysis. In HepG2 cells, the expressions of SRSF7 and glycolytic enzymes were analyzed using qRT-PCR, and Western blot. Glucose uptake and lactate production were quantified using relevant reagent kits. Additionally, cell proliferation, clonogenicity, invasion, and apoptosis were evaluated using MTS assay, clonal formation assay, Transwell assay, and mitochondrial membrane potential assay, respectively. This study demonstrated significant overexpression of SRSF7 in HCC tissue, correlating with poor prognosis. Knockdown of SRSF7 in HepG2 cells resulted in inhibited proliferation, clonogenicity, and invasion, while apoptosis was enhanced. This knockdown also decreased glucose uptake and lactate production, along with a reduction in the expression of glucose transporter 1 (GLUT1) and lactate dehydrogenase A (LDHA). Furthermore, SRSF7 downregulation increased the pyruvate kinase muscle 1 (PKM1)/PKM2 ratio. The glycolytic boost due to PKM2 overexpression partially counteracted the effects of SRSF7 silencing on HepG2 cell growth. The knockdown of SRSF7 impairs aerobic glycolysis and growth in HepG2 cells by downregulating PKM2 expression.
22
Taniguchi, Kohei, Nobuhiko Sugito, Minami Kumazaki, Haruka Shinohara, Nami Yamada, Yoshihito Nakagawa, Yuko Ito, et al. "MicroRNA-124 inhibits cancer cell growth through PTB1/PKM1/PKM2 feedback cascade in colorectal cancer." Cancer Letters 363, no. 1 (July 2015): 17–27. http://dx.doi.org/10.1016/j.canlet.2015.03.026.
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Yan, Qiuxia, Peng Zeng, Xiuqin Zhou, Xiaoying Zhao, Runqiang Chen, Jing Qiao, Ling Feng, Zhenjie Zhu, Guozhi Zhang, and Cairong Chen. "RBMX suppresses tumorigenicity and progression of bladder cancer by interacting with the hnRNP A1 protein to regulate PKM alternative splicing." Oncogene 40, no. 15 (February 9, 2021): 2635–50. http://dx.doi.org/10.1038/s41388-021-01666-z.
Анотація:
AbstractThe prognosis for patients with metastatic bladder cancer (BCa) is poor, and it is not improved by current treatments. RNA-binding motif protein X-linked (RBMX) are involved in the regulation of the malignant progression of various tumors. However, the role of RBMX in BCa tumorigenicity and progression remains unclear. In this study, we found that RBMX was significantly downregulated in BCa tissues, especially in muscle-invasive BCa tissues. RBMX expression was negatively correlated with tumor stage, histological grade and poor patient prognosis. Functional assays demonstrated that RBMX inhibited BCa cell proliferation, colony formation, migration, and invasion in vitro and suppressed tumor growth and metastasis in vivo. Mechanistic investigations revealed that hnRNP A1 was an RBMX-binding protein. RBMX competitively inhibited the combination of the RGG motif in hnRNP A1 and the sequences flanking PKM exon 9, leading to the formation of lower PKM2 and higher PKM1 levels, which attenuated the tumorigenicity and progression of BCa. Moreover, RBMX inhibited aerobic glycolysis through hnRNP A1-dependent PKM alternative splicing and counteracted the PKM2 overexpression-induced aggressive phenotype of the BCa cells. In conclusion, our findings indicate that RBMX suppresses BCa tumorigenicity and progression via an hnRNP A1-mediated PKM alternative splicing mechanism. RBMX may serve as a novel prognostic biomarker for clinical intervention in BCa.
24
Sturgill, Eric M., and Monica L. Guzman. "Cytokine Induced Nuclear Localization Of Pyruvate Kinase M2 In Acute Myeloid Leukemia." Blood 122, no. 21 (November 15, 2013): 5406. http://dx.doi.org/10.1182/blood.v122.21.5406.5406.
Анотація:
Abstract A common characteristic among nearly all cancers, including leukemia, is the cell’s metabolic proclivity for glycolysis over the more energy efficient process of oxidative phosphorylation (OXPHOS) in the presence of oxygen. This altered state of aerobic glycolysis was observed in tumor cells by Otto Warburg over fifty years ago (Warburg, 1956) and continues to be intensely investigated in hopes of ultimately exploiting this “Warburg effect” in the treatment of cancer (Vander Heiden et al. 2009). Recent studies have revealed that the M2 isoform of the enzyme pyruvate kinase (PKM2) plays a critical role in the maintenance of aerobic glycolysis in tumor cells and is important for their growth and development (Christofk et al. 2008). Pyruvate kinase produces pyruvate and one molecule of adenosine 5’-triphosphate (ATP) in the rate-limiting step of glycolysis. Pyruvate kinase coded for by PKM has two splice isoforms, the constitutively active PKM1 that exists only as a tetramer and PKM2 that can shift between a more active tetramer and less active dimers or monomers. The dynamic enzymatic activity of PKM2 is key to its preferential expression in tumor cells. By utilizing the less active form of PKM2, tumor cells can limit the levels of pyruvate available for OXPHOS and instead shunt glycolytic carbons towards anabolic processes. However, recent studies have revealed novel activities of PKM2 outside the realm of energy metabolism that also contribute to tumor formation, maintenance, and growth. The less active PKM2 dimer, whose structure is favored upon phosphorylation at Tyr105 (Hitosugi et al. 2009), can also translocate to the nucleus and act as a transcription factor for cell cycle associated genes like MYC and CCND1 upon stimulation with epidermal growth factor in certain cancer cell lines (Gao et al. 2012). Acute myeloid leukemia (AML) is a malignancy of hematopoietic progenitor cells characterized by the extraordinarily rapid growth of abnormal myeloid cells, making the proliferative influences of PKM2 an intriguing target for therapy. We have found that PKM2 is abundantly expressed in AML cell lines and primary AML patient samples and that low basal levels of PKM2 can be detected in their nuclei. Interestingly, stimulation with various cytokines such as IL-6 or GM-CSF can induce the nuclear translocation of PKM2 and association with histone H3 in these cells and concomitant treatment with PKM2 activating compounds that have been shown to promote its tetrameric structure and suppress tumor growth (Anastasiou et al. 2012) can inhibit this effect. These data show that the role of PKM2 in regulating transcription in addition to its metabolic activity may be important for the proliferation and maintenance of hematopoietic malignancies. Using fluorescence-activated cell sorting to isolate specific sub-populations of primary AML patient cells and elucidating PKM2’s interaction with protein kinases involved in known signaling pathways like JAK/STAT, ERK1/2, and FLT3, we show that the proliferative influences of PKM2 function and activity differ between AML cell phenotypes. For example, cells from AML patient samples sorted based on high or low levels of reactive oxygen species (ROS) differ in relative phosphorylation of PKM2 at Tyr105. These data, along with reports that the PKM2 dimer specifically plays a role in tumor cell antioxidant response (Anastasiou, et al. 2011) suggests that PKM2 may contribute to the maintenance of phenotypically ROS-low leukemia stem cells that are thought to contribute to patient relapse after achieving remission (Hope et al. 2004). Our data suggests that the broad cellular functions of PKM2 employed by AML cells and its direct influence on tumor growth and survival make it a promising potential target for therapy. Disclosures: No relevant conflicts of interest to declare.
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Sun, Ye, Yushi Chen, Ming Xu, Chunying Liu, Hai Shang, and Chun Wang. "Shenmai Injection Supresses Glycolysis and Enhances Cisplatin Cytotoxicity in Cisplatin-Resistant A549/DDP Cells via the AKT-mTOR-c-Myc Signaling Pathway." BioMed Research International 2020 (June 22, 2020): 1–10. http://dx.doi.org/10.1155/2020/9243681.
Анотація:
Tumor cells, especially drug-resistant cells, predominately support growth by glycolysis even under the condition of adequate oxygen, which is known as the Warburg effect. Glucose metabolism reprogramming is one of the main factors causing tumor resistance. Previous studies on Shenmai injection (SMI), a Chinese herbal medicine, have shown enhanced efficacy in the treatment of tumors in combination with chemotherapy drugs, but the mechanism is not clear. In this study, we investigated the effect of SMI combined with cisplatin on cisplatin-resistant lung adenocarcinoma A549/DDP cells. Our results showed that cisplatin-resistant A549/DDP cells exhibited increased glucose consumption, lactate production, and expression levels of key glycolytic enzymes, including hexokinase 2 (HK2), pyruvate kinase M1/2 (PKM1/2), pyruvate kinase M2 (PKM2), glucose transporter 1 (GLUT1), and lactate dehydrogenase A (LDHA), compared with cisplatin-sensitive A549 cells. SMI combined with cisplatin in A549/DDP cells, led to significantly lower expression levels of key glycolytic enzymes, such as HK2, PKM1/2, GLUT1, and pyruvate dehydrogenase (PDH). In addition, we found that the combination of SMI and cisplatin could inhibit cell proliferation and promote apoptosis by reducing the expression levels of p-Akt, p-mTOR, and c-Myc, and then, it reduced the glycolysis level. These results suggest that SMI enhances the antitumor effect of cisplatin via glucose metabolism reprogramming. Therefore, the combination of SMI and cisplatin may be a potential therapeutic strategy to treat cisplatin-resistant nonsmall cell lung cancer.
26
Chiavarina, Barbara, Diana Whitaker-Menezes, Ubaldo E. Martinez-Outschoorn, Agnieszka K. Witkiewicz, Ruth Birbe, Anthony Howell, Richard G. Pestell, et al. "Pyruvate kinase expression (PKM1 and PKM2) in cancer-associated fibroblasts drives stromal nutrient production and tumor growth." Cancer Biology & Therapy 12, no. 12 (December 15, 2011): 1101–13. http://dx.doi.org/10.4161/cbt.12.12.18703.
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Akbar Napitupulu, Alief, Ni Nyoman Puspawati, and I. Desak Putu Kartika Pratiwi. "CEMARAN ASPERGILLUS FLAVUS PENGHASIL AFLATOKSIN B1 PADA JAGUNG MANIS (ZEA MAYS SACCARATA) YANG DIJUAL DI PASAR TRADISIONAL DAN PASAR MODERN DI KECAMATAN DENPASAR BARAT, KOTA MADYA DENPASAR – BALI." Jurnal Ilmu dan Teknologi Pangan (ITEPA) 7, no. 2 (July 29, 2018): 11. http://dx.doi.org/10.24843/itepa.2018.v07.i02.p02.
Анотація:
This study aims to determine contamination, Aspergillus flavus in sweet corn sold in traditional and modern markets in West Denpasar district and to know whether A. flavus contaminating sweet corn can produce aflatoxin B1. The design of this study applied survey methods with sampling techniques of purposive sampling. Sampling determination was conducted on a number of markets, which were Traditional Markets and Modern Markets in West Denpasar District. West Denpasar District has 7 traditional markets and 3 modern markets. The variables observed were total mold of yeast, total mold, total of A. flavus, and aflatoxin content of B1. The results showed that the lowest A, flavus population was found on sweet corn sold in the traditional market sample which was <1 x 106 CFU/g. and the highest population was in the PKP sample of 7.0 x 106 CFU/g. The population of A. flavus in sweet corn sold in the modern market was <1 x 106 CFU/g. The result of aflatoxin test showed that the total samples taken in sweet corn merchants in traditional markets and modern markets in West Denpasar, 11 samples (34,37%) did not contain aflatoxin while 16 samples < 20 ppb (50%) contained aflatoxin B1 which was on samples of PBP1, PBP2, PPK, PKM1, PKM2, PBM1, PBM2, PBM3, PAS1, PAS2, PAS3, TD, GSR, SE, PKP, PBM2 and 5 samples (15,63%) were declared in accordance with BPOM and FDA regulations which was on samples PKM1, PAS1, PKP, GSR, and SE.
28
Bartyś, Natalia, Anna Pasternak, and Jolanta Lisowiec-Wąchnicka. "Optimization of Bifunctional Antisense Oligonucleotides for Regulation of Mutually Exclusive Alternative Splicing of PKM Gene." Molecules 27, no. 17 (September 3, 2022): 5682. http://dx.doi.org/10.3390/molecules27175682.
Анотація:
Oligonucleotide tools, as modulators of alternative splicing, have been extensively studied, giving a rise to new therapeutic approaches. In this article, we report detailed research on the optimization of bifunctional antisense oligonucleotides (BASOs), which are targeted towards interactions with hnRNP A1 protein. We performed a binding screening assay, Kd determination, and UV melting experiments to select sequences that can be used as a high potency binding platform for hnRNP A1. Newly designed BASOs were applied to regulate the mutually exclusive alternative splicing of the PKM gene. Our studies demonstrate that at least three repetitions of regulatory sequence are necessary to increase expression of the PKM1 isoform. On the other hand, PKM2 expression can be inhibited by a lower number of regulatory sequences. Importantly, a novel branched type of BASOs was developed, which significantly increased the efficiency of splicing modulation. Herein, we provide new insights into BASOs design and show, for the first time, the possibility to regulate mutually exclusive alternative splicing via BASOs.
29
Balakumbahan, R., C. Ravindran, and V. Sivakumar. "Standardization of Propagation Techniques in Annual Moringa (Moringa oleifera Lam.) for Enhancing Crop Uniformity." Journal of Advances in Biology & Biotechnology 27, no. 7 (July 2, 2024): 1315–20. http://dx.doi.org/10.9734/jabb/2024/v27i71093.
Анотація:
An investigation on annual Moringa, Moringa oleifera Lam., var., (PKM 1 and PKM2) with the objective of standardizing the method of vegetative propagation was carried out at Horticultural College and Research Institute, Periyakulam. the treatment consisted of three factors viz., cultivars (Factor 1: Moringa variety PKM 1 and PKM2), propagation methods (Factor 2: hardwood cuttings, semi hardwood cuttings and air layering) and application of different concentration of growth regulator IBA (Factor 3:0, 1500, 2500 and 4000 ppm) and the trial was laid out with twenty four treatments using Factorial Randomized Block Design with three replications. The overall result revealed that the treatment combinations of PKM 1 air layering treated with IBA 4000 ppm registered the highest dry matter content of root (70.00%) and PKM 1 hardwood cuttings treated with IBA 4000 ppm recorded the highest dry shoot to root ratio (9.19). In PKM 2 semi hardwood cuttings treated with IBA 1500 ppm registered the highest root length (16.30cm) and dry matter content of shoot (61.99%). PKM 2 air layers treated with IBA 4000 ppm took minimum number of days to root (17.99 days), days to sprout (9.85 days) had the highest root fresh weight (2.70g), root dry weight (1.80g), shoot fresh weight (12.52g) and shoot dry weight (6.90g). This study concluded that air layering with 2500 to 4000 ppm IBA treatment is best pro pragation method for PKM1 and PKM 2 annual moringa.
30
Singh, Smriti, Sathiya Pandi Narayanan, Kajal Biswas, Amit Gupta, Neha Ahuja, Sandhya Yadav, Rajendra Kumar Panday, Atul Samaiya, Shyam K. Sharan, and Sanjeev Shukla. "Intragenic DNA methylation and BORIS-mediated cancer-specific splicing contribute to the Warburg effect." Proceedings of the National Academy of Sciences 114, no. 43 (October 9, 2017): 11440–45. http://dx.doi.org/10.1073/pnas.1708447114.
Анотація:
Aberrant alternative splicing and epigenetic changes are both associated with various cancers, but epigenetic regulation of alternative splicing in cancer is largely unknown. Here we report that the intragenic DNA methylation-mediated binding of Brother of Regulator of Imprinted Sites (BORIS) at the alternative exon of Pyruvate Kinase (PKM) is associated with cancer-specific splicing that promotes the Warburg effect and breast cancer progression. Interestingly, the inhibition of DNA methylation, BORIS depletion, or CRISPR/Cas9-mediated deletion of the BORIS binding site leads to a splicing switch from cancer-specific PKM2 to normal PKM1 isoform. This results in the reversal of the Warburg effect and the inhibition of breast cancer cell growth, which may serve as a useful approach to inhibit the growth of breast cancer cells. Importantly, our results show that in addition to PKM splicing, BORIS also regulates the alternative splicing of several genes in a DNA methylation-dependent manner. Our findings highlight the role of intragenic DNA methylation and DNA binding protein BORIS in cancer-specific splicing and its role in tumorigenesis.
31
Li, Yuchao, Shuwei Zhang, Yuexian Li, Junchao Liu, Qian Li, Wenli Zang, and Yaping Pan. "The Regulatory Network of hnRNPs Underlying Regulating PKM Alternative Splicing in Tumor Progression." Biomolecules 14, no. 5 (May 9, 2024): 566. http://dx.doi.org/10.3390/biom14050566.
Анотація:
One of the hallmarks of cancer is metabolic reprogramming in tumor cells, and aerobic glycolysis is the primary mechanism by which glucose is quickly transformed into lactate. As one of the primary rate-limiting enzymes, pyruvate kinase (PK) M is engaged in the last phase of aerobic glycolysis. Alternative splicing is a crucial mechanism for protein diversity, and it promotes PKM precursor mRNA splicing to produce PKM2 dominance, resulting in low PKM1 expression. Specific splicing isoforms are produced in various tissues or illness situations, and the post-translational modifications are linked to numerous disorders, including cancers. hnRNPs are one of the main components of the splicing factor families. However, there have been no comprehensive studies on hnRNPs regulating PKM alternative splicing. Therefore, this review focuses on the regulatory network of hnRNPs on PKM pre-mRNA alternative splicing in tumors and clinical drug research. We elucidate the role of alternative splicing in tumor progression, prognosis, and the potential mechanism of abnormal RNA splicing. We also summarize the drug targets retarding tumorous splicing events, which may be critical to improving the specificity and effectiveness of current therapeutic interventions.
32
FOSTER, HANNAH R., THUONG HO, EVGENIY POTAPENKO, SOPHIE L. LEWANDOWSKI, SOPHIA SDAO, HALENA R. VANDEUSEN, REBECCA L. CARDONE, RICHARD KIBBEY, and MATTHEW J. MERRINS. "127-OR: In Vivo Genetic Evidence That the Pyruvate Kinase Isoforms PKM1 and PKM2 Differentially Control Beta-Cell Fuel Sensing." Diabetes 70, Supplement 1 (June 2021): 127—OR. http://dx.doi.org/10.2337/db21-127-or.
33
Méndez-Lucas, Andrés, Xiaolei Li, Junjie Hu, Li Che, Xinhua Song, Jiaoyuan Jia, Jingxiao Wang, et al. "Glucose Catabolism in Liver Tumors Induced by c-MYC Can Be Sustained by Various PKM1/PKM2 Ratios and Pyruvate Kinase Activities." Cancer Research 77, no. 16 (June 19, 2017): 4355–64. http://dx.doi.org/10.1158/0008-5472.can-17-0498.
34
Dogsom, Oyungerel, Amir Hamza, Shohel Mahmud, Jung-Ki Min, Yoon-Beom Lee, and Jae-Bong Park. "The Complex of p-Tyr42 RhoA and p-p65/RelA in Response to LPS Regulates the Expression of Phosphoglycerate Kinase 1." Antioxidants 12, no. 12 (December 8, 2023): 2090. http://dx.doi.org/10.3390/antiox12122090.
Анотація:
Inflammation plays a crucial role in tumorigenesis, primarily mediated by NF-κB. RhoA GTPases are instrumental in regulating the activation of NF-κB. Specifically, the phosphorylation of Tyrosine 42 on RhoA ensures the activation of NF-κB by directly activating the IKKβ associated with IKKγ (NEMO). This study aimed to uncover the molecular mechanism through which p-Tyrosine 42 RhoA, in conjunction with NF-κB, promotes tumorigenesis. Notably, we observed that p-Tyrosine 42 RhoA co-immunoprecipitated with the p-Ser 536 p65/RelA subunit in NF-κB in response to LPS. Moreover, both p-Tyrosine 42 RhoA and p-p65/RelA translocated to the nucleus, where they formed a protein complex associated with the promoter of phosphoglycerate kinase 1 (PGK1) and regulated the expression of PGK1. In addition, p-p65/RelA and p-Tyr42 RhoA co-immunoprecipitated with p300 histone acetyltransferase. Intriguingly, PGK1 exhibited an interaction with β-catenin, PKM1 and PKM2. Of particular interest, si-PGK1 led to a reduction in the levels of β-catenin and phosphorylated pyruvate dehydrogenase A1 (p-PDHA1). We also found that PGK1 phosphorylated β-catenin at the Thr551 and Ser552 residues. These findings discovered that PGK1 may play a role in transcriptional regulation, alongside other transcription factors.
35
Li, Dongliang, Yan Xie, Jisan Sun, Li Zhang, and Wentao Jiang. "LncRNA ZNF674-AS1 Hinders Proliferation and Invasion of Hepatic Carcinoma Cells through the Glycolysis Pathway." Journal of Oncology 2022 (July 13, 2022): 1–9. http://dx.doi.org/10.1155/2022/8063382.
Анотація:
Purpose. Long noncoding RNAs (lncRNAs) play important roles in regulating various functions of cells at the levels of transcription and posttranscription. Extensive investigations have illustrated that lncRNAs are critical in the glucose metabolism of tumor cells, but their mechanisms of action need to be further explored. This study evaluates the role of lncRNA ZNF674-AS1 on the apoptosis and proliferation of human hepatic carcinoma cells in vitro through the glucose metabolism and its related mechanisms. Methods. Real-time quantitative PCR was employed for detecting the level of expressions for lncRNA ZNF674-AS1 in liver cancer tissues (25 cases), paracancerous tissues, and liver cancer cell lines. The lncRNA ZNF674-AS1 high expression cell strain was constructed by the lentiviral overexpression vector. CCK-8, plate colony formation, transwell assay, lactate production, glucose consumption, and ATP levels were used to detect the change of cell proliferation, colony formation, migration, and invasion, as well as glycolytic capability. Western blot was carried out to detect the expression of HK2, PFKL, PKM2, GLUT1, and PKM1, which are the key proteins of glycolysis in cells. Result. The lncRNA ZNF674-AS1 was undesirably expressed in liver cancer cell lines and tissues. Cell function assessments showed that compared with the blank control group (vector), overexpression of lncRNA ZNF674-AS1 could substantially hinder the proliferation, colony formation, migration, and invasion capability of liver cancer cells. Furthermore, overexpression of lncRNA ZNF674-AS1 could inhibit cell glycolysis (inhibit glucose consumption and reduce intracellular lactate and ATP levels) by inhibiting the expression of key proteins (such as PFKL, HK2, PKM2, and GLUT1) in the process of glycolysis. Conclusion. As a tumor repressor gene, lncRNA ZNF674-AS1 inhibits the expression of key proteins in glycolysis to inhibit glycolysis level, thereby inhibiting cell migration and proliferation. Therefore, lncRNA ZNF674-AS1 could be a potent therapeutic target or a novel diagnostic molecule for patients suffering from liver cancer.
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Li, Qi, Xin Qi, and Wenjun Jia. "3,3′,5-triiodothyroxine inhibits apoptosis and oxidative stress by the PKM2/PKM1 ratio during oxygen-glucose deprivation/reperfusion AC16 and HCM-a cells." Biochemical and Biophysical Research Communications 475, no. 1 (June 2016): 51–56. http://dx.doi.org/10.1016/j.bbrc.2016.05.030.
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Clear, Andrew James, Annalisa D'Avola, Samir G. Agrawal, Laura Z. Rassenti, Thomas J. Kipps, John G. Gribben, and John Riches. "B-Cell Receptor Signaling Drives Glycolysis in Chronic Lymphocytic Leukemia Cells." Blood 132, Supplement 1 (November 29, 2018): 3121. http://dx.doi.org/10.1182/blood-2018-99-112860.
Анотація:
Abstract A key feature of tumor cell energetics is preferential metabolism of glucose to lactate even in the presence of oxygen (aerobic glycolysis). While this is an inefficient way of producing energy, it enables cancer cells to use glucose to generate biomass to support cellular proliferation. It is unclear whether this process is playing a role in the pathogenesis of chronic lymphocytic leukemia (CLL). Previous studies have noted that CLL cells have increased numbers of mitochondria when compared with healthy B cells (Carew Leukaemia 2004, Jitschin Blood 2014). As CLL cells have increased expression of lipoprotein lipase it has been hypothesized that CLL cells adopt a predominantly mitochondrial metabolism. In this study, we investigated the role of metabolism in the pathogenesis of CLL. While initial analysis of mitochondrial mass by flow cytometry showed that CLL cells do have increased mitochondrial content compared to healthy B cells, these and previous observations were comparing a monoclonal population of CLL cells with a CD27+ activated/memory phenotype with polyclonal healthy B cells predominantly composed of naïve CD27- cells. When we investigated this further we found that healthy CD27+ memory B cells have a higher mitochondrial mass when compared with healthy CD27- naïve B cells. CLL cells actually had reduced mitochondrial mass when compared healthy CD27+ memory B cells, which fell further with disease progression. B-cell receptor (BCR) signaling plays a vital role in the pathogenesis of CLL, as shown by the clinical efficacy of inhibitors of this pathway. Despite this little is known regarding the impact of BCR-signaling on CLL-cell metabolism. Primary human CLL cells were stimulated with either anti-IgM, anti-IgD or anti-IgG (isotype control) for up to 72 hours before measuring the residual glucose concentration of the media to assess glucose uptake. Anti-IgM treated CLL cells showed an increase in glucose uptake compared to control. The impact of anti-IgM on the expression of enzymes involved in glycolysis including glucose transporters (GLUTs), hexokinase, phosphofructokinase, enolase, and pyruvate kinase was assessed by immunoblotting. As myc is induced by BCR-signaling in CLL cells we focused on known myc targets hexokinase 2 (HK2), enolase 1 (ENOL-1), lactate dehydrogenase A (LDHA) and the heterogenous nuclear ribonuclearproteins (hnRNPs) A1, A2/B1 and PTBP1. GLUT3, HK2 and the hnRNPs were all expressed at low levels in resting CLL cells but increased significantly (at 24 hours) after anti-IgM stimulation. The other myc targets ENOL-1 and LDHA were constitutively expressed and did not increase further after stimulation. There was significant heterogeneity in response to anti-IgM stimulation with IGHV unmutated cases showing a trend toward greater glucose uptake and enzyme induction, while anti-IgD stimulation had a similar but weaker effect. Immunohistochemistry on lymph node biopsies from CLL patients showed a significant increase in expression of GLUT3 and HK2 within CLL proliferation centres. hnRNP induction has been shown to promote a switch to use of the M2 isoform of pyruvate kinase (PKM2): a key feature of many cancers. Interestingly, both circulating and lymph node CLL cells were already switched to using PKM2 and so anti-IgM stimulation had little further effect. However, when the relative levels of PKM1 and PKM2 were compared between early- and advanced-stage patients there was a significant shift to use of PKM2 with disease progression. Treatment of CLL cells in vitro by ibrutinib, idelalisib and the MEK inhibitor U0126 all blocked the anti-IgM induced increase in glucose uptake and GLUT3, HK2 and hnRNP expression. Investigation of the expression of GLUT3 and HK2 in CLL cells obtained from ibrutinib-treated patients also showed a reduction in the expression of these proteins demonstrating that ibrutinib is metabolically reprogramming CLL cells in vivo. We conclude that previous observations regarding an increase in mitochondrial mass in CLL cells compared to healthy B cells reflects their differentiation states. In contrast, we show that BCR-signaling increases glucose uptake and glycolytic enzyme expression in a myc-dependent manner as part of a switch to aerobic glycolysis. Treatment with BCR inhibitors block this effect. Therefore, we anticipate that many of the novel anti-glycolytic therapies currently in development will prove useful in the treatment of CLL. Disclosures Kipps: F. Hoffmann-La Roche Ltd: Consultancy, Research Funding; Verastem: Membership on an entity's Board of Directors or advisory committees; AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy; Gilead: Consultancy, Honoraria, Research Funding; Genentech Inc: Consultancy, Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Verastem: Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees. Gribben:Acerta Pharma: Honoraria, Research Funding; Novartis: Honoraria; Wellcome Trust: Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Abbvie: Honoraria; Kite: Honoraria; TG Therapeutics: Honoraria; Cancer Research UK: Research Funding; Unum: Equity Ownership; Medical Research Council: Research Funding; Janssen: Honoraria, Research Funding; NIH: Research Funding; Roche: Honoraria; Pharmacyclics: Honoraria.
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Kabir, Mohammad Faujul, Adam Karami, Anbin Mu, Don-Gerard Conde, and Kelly A. Whelan. "Abstract 5808: Diclofenac inhibits esophageal cancer cell growth by depleting mitochondrial functions." Cancer Research 82, no. 12_Supplement (June 15, 2022): 5808. http://dx.doi.org/10.1158/1538-7445.am2022-5808.
Анотація:
Abstract Esophageal cancer is one of the most aggressive forms of human malignancy with a 5-yearsurvival rate of less than 20%. Mitochondria are dynamic organelles that play essential roles in various cellular processes, including energy metabolism, redox homeostasis, and apoptotic cell death. Alterations in mitochondrial biology are associated with esophageal carcinogenesis and esophageal cancer cell response to therapy. As the anti-inflammatory drug diclofenac (DCF)induces mitochondrial dysfunction, we hypothesized that DCF may inhibit esophageal carcinogenesis by affecting mitochondrial processes. In the human esophageal squamous cell carcinoma (ESCC) cell line TE11, we performed MTT assay and Annexin-V/Propidium Iodide (PI) to evaluate the impact of DCF on proliferation and cell death, respectively. RNA sequencing and Ingenuity Pathway Analysis (IPA) identified differentially expressed genes (DEGs) and altered canonical pathways in DCF-treated TE11 cells. To assess metabolic function, we measured the levels of pyruvate, lactate, and ATP. Mitochondrial membrane potential and ROS production were assessed by flow cytometry for MitoTracker red/green and MitoSox red, respectively. Mice bearing subcutaneous syngeneic ESCC tumors were treated with DCF.DCF inhibited proliferation of TE11 in dose dependent manner with an IC50 concentration of 76.7µM. By contrast, the IC50 concentration of DCF in normal esophageal keratinocytes was 876.2µM, supporting selectivity of DCF for esophageal cancer cells. In TE11, 200 µM DCF induced apoptosis in 60% of cells. RNA-sequencing identified that DCF significantly altered expression of3287 genes (FC ≥ 1.50) in TE11, including the top cancer-associated genes: PLK1, MCM2, MCM3,MCM7, MCM10 and SKP2. IPA analysis revealed that DCF activated p53 signaling while inhibited Gluconeogenesis I, Glycolysis I and Oxidative Phosphorylation. DCF significantly inhibited glycolysis as demonstrated by reduction in concentrations of pyruvate (1.76-fold) and lactate (7.30-fold) and downregulation of the glycolysis- associated genes PKM2, PFKM, LDHA, and PKM1. A decreased production of ATP (5-fold) was also noted in DCF-treated TE11 cells. In addition, DCF depleted mitochondrial membrane potential (2.5-fold) and increased mitochondrial ROS production (5-fold). A reduction in ROS by mitochondrial antioxidant MitoTempo increased viability of the DCF-treated TE11 cells, indicating that ROS contributes to the anticancer activity of DCF. Consistent with antitumor activity of DCF in TE11, the drug significantly decreased the tumor volume in syngeneic ESCC tumors in vivo. Our preclinical findings indicate that DCF may limit esophageal cancer cell growth through the inhibition of mitochondrial functions, identifying a novel experimental therapeutic for ESCC. Future studies will define the precise molecular mechanisms through which DCF promotes cell death in vitro and in vivo. Citation Format: Mohammad Faujul Kabir, Adam Karami, Anbin Mu, Don-Gerard Conde, Kelly A. Whelan. Diclofenac inhibits esophageal cancer cell growth by depleting mitochondrial functions [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 5808.
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Park, Bohye, Ji Yeon Kim, Olivia F. Riffey, Presley Dowker-Key, Antje Bruckbauer, James McLoughlin, Ahmed Bettaieb, and Dallas R. Donohoe. "Pyruvate kinase M1 regulates butyrate metabolism in cancerous colonocytes." Scientific Reports 12, no. 1 (May 24, 2022). http://dx.doi.org/10.1038/s41598-022-12827-9.
Анотація:
AbstractColorectal cancer (CRC) cells shift metabolism toward aerobic glycolysis and away from using oxidative substrates such as butyrate. Pyruvate kinase M1/2 (PKM) is an enzyme that catalyzes the last step in glycolysis, which converts phosphoenolpyruvate to pyruvate. M1 and M2 are alternatively spliced isoforms of the Pkm gene. The PKM1 isoform promotes oxidative metabolism, whereas PKM2 enhances aerobic glycolysis. We hypothesize that the PKM isoforms are involved in the shift away from butyrate oxidation towards glycolysis in CRC cells. Here, we find that PKM2 is increased and PKM1 is decreased in human colorectal carcinomas as compared to non-cancerous tissue. To test whether PKM1/2 alter colonocyte metabolism, we created a knockdown of PKM2 and PKM1 in CRC cells to analyze how butyrate oxidation and glycolysis would be impacted. We report that butyrate oxidation in CRC cells is regulated by PKM1 levels, not PKM2. Decreased butyrate oxidation observed through knockdown of PKM1 and PKM2 is rescued through re-addition of PKM1. Diminished PKM1 lowered mitochondrial basal respiration and decreased mitochondrial spare capacity. We demonstrate that PKM1 suppresses glycolysis and inhibits hypoxia-inducible factor-1 alpha. These data suggest that reduced PKM1 is, in part, responsible for increased glycolysis and diminished butyrate oxidation in CRC cells.
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Li, Lin, Siyuan Cheng, Yunshin Yeh, Yingli Shi, Nikayla Henderson, David Price, Xin Gu, and Xiuping Yu. "The expression of PKM1 and PKM2 in developing, benign, and cancerous prostatic tissues." Frontiers in Oncology 14 (April 12, 2024). http://dx.doi.org/10.3389/fonc.2024.1392085.
Анотація:
BackgroundNeuroendocrine prostate cancer (NEPCa) is the most aggressive type of prostate cancer (PCa). However, energy metabolism, one of the hallmarks of cancer, in NEPCa has not been well studied. Pyruvate kinase M (PKM), which catalyzes the final step of glycolysis, has two main splicing isoforms, PKM1 and PKM2. The expression pattern of PKM1 and PKM2 in NEPCa remains unknown.MethodsIn this study, we used immunohistochemistry, immunofluorescence staining, and bioinformatics analysis to examine the expression of PKM1 and PKM2 in mouse and human prostatic tissues.ResultsWe found that PKM2 was the predominant isoform expressed throughout prostate development and PCa progression, with slightly reduced expression in murine NEPCa. PKM1 was mostly expressed in stromal cells but low-level PKM1 was also detected in prostate basal epithelial cells. Its expression was absent in the majority of prostate adenocarcinoma (AdPCa) specimens but present in a subset of NEPCa. Additionally, we evaluated the mRNA levels of ten PKM isoforms that express exon 9 (PKM1-like) or exon 10 (PKM2-like). Some of these isoforms showed notable expression levels in PCa cell lines and human PCa specimens.DiscussionOur study characterized the expression pattern of PKM1 and PKM2 in prostatic tissues including developing, benign, and cancerous prostate. These findings lay the groundwork for understanding the metabolic changes in different PCa subtypes.
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Davidson, Shawn M., Daniel R. Schmidt, Julia E. Heyman, James P. O'Brien, Amy C. Liu, William J. Israelsen, Talya L. Dayton, et al. "Pyruvate kinase M1 suppresses development and progression of prostate adenocarcinoma." Cancer Research, May 18, 2022. http://dx.doi.org/10.1158/0008-5472.can-21-2352.
Анотація:
Abstract Altered metabolisms helps sustain cancer cell proliferation and survival. Most cancers, including prostate cancers, express the M2 splice isoform of pyruvate kinase (Pkm2), which can support anabolic metabolism to support cell proliferation. However, Pkm2 expression is dispensable for the formation and growth of many cancers in vivo. Expression of pyruvate kinase isoform M1 (Pkm1) is restricted to relatively few tissues and has been reported to promote growth of select tumors, but the role of Pkm1 in cancer has been less studied than Pkm2. To test how differential expression of pyruvate kinase isoforms affects cancer initiation and progression, we generated mice harboring a conditional allele of Pkm1, and crossed these mice or those with a Pkm2 conditional allele with a Pten loss-driven prostate cancer model. Pkm1 loss led to increased Pkm2 expression and accelerated prostate cancer development, while Pkm2 deletion of led to increased Pkm1 expression and suppressed tumor progression. Metabolic profiling revealed altered nucleotide levels in tumors with high Pkm1 expression, and failure of these tumors to progress was associated with DNA replication stress and senescence. Consistent with these data, a small molecule pyruvate kinase activator that mimics a high activity Pkm1-like state suppressed progression of established prostate tumors. Analysis of human specimens showed PKM2 expression is retained in most human prostate cancers. Overall, this study uncovers a role for pyruvate kinase isoforms in prostate cancer initiation and progression, and argues that pharmacological pyruvate kinase activation may be beneficial for treating prostate cancer.
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Olea‐Flores, Monserrat, Tapan Sharma, Odette Verdejo‐Torres, Imaru DiBartolomeo, Paul R. Thompson, Teresita Padilla‐Benavides, and Anthony N. Imbalzano. "Muscle‐specific pyruvate kinase isoforms, PKM1 and PKM2, regulate mammalian SWI/SNF proteins and histone 3 phosphorylation during myoblast differentiation." FASEB Journal 38, no. 11 (June 4, 2024). http://dx.doi.org/10.1096/fj.202400784r.
Анотація:
AbstractPyruvate kinase is a glycolytic enzyme that converts phosphoenolpyruvate and ADP into pyruvate and ATP. There are two genes that encode pyruvate kinase in vertebrates; Pkm and Pkl encode muscle‐ and liver/erythrocyte‐specific forms, respectively. Each gene encodes two isoenzymes due to alternative splicing. Both muscle‐specific enzymes, PKM1 and PKM2, function in glycolysis, but PKM2 also has been implicated in gene regulation due to its ability to phosphorylate histone 3 threonine 11 (H3T11) in cancer cells. Here, we examined the roles of PKM1 and PKM2 during myoblast differentiation. RNA‐seq analysis revealed that PKM2 promotes the expression of Dpf2/Baf45d and Baf250a/Arid1A. DPF2 and BAF250a are subunits that identify a specific sub‐family of the mammalian SWI/SNF (mSWI/SNF) of chromatin remodeling enzymes that is required for the activation of myogenic gene expression during differentiation. PKM2 also mediated the incorporation of DPF2 and BAF250a into the regulatory sequences controlling myogenic gene expression. PKM1 did not affect expression but was required for nuclear localization of DPF2. Additionally, PKM2 was required not only for the incorporation of phosphorylated H3T11 in myogenic promoters but also for the incorporation of phosphorylated H3T6 and H3T45 at myogenic promoters via regulation of AKT and protein kinase C isoforms that phosphorylate those amino acids. Our results identify multiple unique roles for PKM2 and a novel function for PKM1 in gene expression and chromatin regulation during myoblast differentiation.
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McNally, Lindsey A., Parul Mehra, Andrew Gibb, Kenneth Brittian, Anna Gumpert, Sujith Dassanayaka, Steven P. Jones, and Bradford G. Hill. "Abstract 14268: PKM2 to PKM1 Isoform Switching in Fibroblasts Attenuates Cardiac Dysfunction in Infarcted Mice." Circulation 144, Suppl_1 (November 16, 2021). http://dx.doi.org/10.1161/circ.144.suppl_1.14268.
Анотація:
Introduction: The muscle isoform of pyruvate kinase, PKM, has two splice variants, with PKM2 having important roles in cardiomyocytes during development. The goal of this study was to understand the role of PKM2 in the adult failing heart. Methods: To determine how PKM2 expression changes after myocardial infarction (MI), wild-type (WT) mice were subjected to permanent coronary ligation followed by measurement of PKM2 abundance and localization by Western blotting and immunofluorescence imaging. To delineate the role of PKM2 in fibroblasts after MI, PKM2 fl/fl mice were bred with a fibroblast-specific, tamoxifen-inducible Cre driver (Col1a2-CreER; Cre). The Cre + and Cre - PKM2 fl/fl mice of both sexes were treated with tamoxifen (i.p. 20 mg/kg/d; 5 d), followed by tamoxifen washout and MI surgery. Four weeks after MI, cardiac function was examined by echocardiography. Results: Hearts from infarcted WT mice had 10-fold higher levels of PKM2 compared with sham WT hearts (n=8/gp, p<0.05). Immunofluorescence staining revealed PKM2 localized to areas of replacement fibrosis in infarcted hearts, and immunoblotting of isolated, adult cell populations showed PKM2 expressed in fibroblasts, smooth muscle cells, and endothelial cells, but not in cardiomyocytes. Furthermore, PKM2 levels were higher in cardiac fibroblasts isolated from infarcted mice compared with sham mice (n=3/group; p=0.005). Cardiac fibroblasts isolated from tamoxifen-treated Cre + /PKM2 fl/fl mice had decreased PKM2 expression and increased PKM1 expression. In naïve mice, PKM2 to PKM1 isoform switching did not affect cardiac function for up to 5 wk after tamoxifen administration. Although survival was not different between Cre - and Cre + PKM2 fl/fl mice after MI (n=18-20/gp; p=0.6383), PKM2 to PKM1 switching in Cre + PKM2 fl/fl male mice improved cardiac function compared with Cre - PKM2 fl/fl controls (n=7-12/gp), with a 15% improvement in ejection fraction (p=0.03), a decrease in LVIDd (p=0.04), EDV (p=0.04), and ESV (p=0.03), and an increase in LVPWd (p=0.01) and LVPWs (p=0.01); however, in female mice, PKM isoform switching did not improve cardiac function. Conclusions: Fibroblast-specific switching of PKM2 to PKM1 prior to MI prevents cardiac functional decline in male mice.
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Yu, Lei, Shao Thing Teoh, Elliot Ensink, Martin P. Ogrodzinski, Che Yang, Ana I. Vazquez, and Sophia Y. Lunt. "Cysteine catabolism and the serine biosynthesis pathway support pyruvate production during pyruvate kinase knockdown in pancreatic cancer cells." Cancer & Metabolism 7, no. 1 (December 2019). http://dx.doi.org/10.1186/s40170-019-0205-z.
Анотація:
Abstract Background Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer with limited treatment options. Pyruvate kinase, especially the M2 isoform (PKM2), is highly expressed in PDAC cells, but its role in pancreatic cancer remains controversial. To investigate the role of pyruvate kinase in pancreatic cancer, we knocked down PKM2 individually as well as both PKM1 and PKM2 concurrently (PKM1/2) in cell lines derived from a KrasG12D/-; p53-/- pancreatic mouse model. Methods We used liquid chromatography tandem mass spectrometry (LC-MS/MS) to determine metabolic profiles of wildtype and PKM1/2 knockdown PDAC cells. We further used stable isotope-labeled metabolic precursors and LC-MS/MS to determine metabolic pathways upregulated in PKM1/2 knockdown cells. We then targeted metabolic pathways upregulated in PKM1/2 knockdown cells using CRISPR/Cas9 gene editing technology. Results PDAC cells are able to proliferate and continue to produce pyruvate despite PKM1/2 knockdown. The serine biosynthesis pathway partially contributed to pyruvate production during PKM1/2 knockdown: knockout of phosphoglycerate dehydrogenase in this pathway decreased pyruvate production from glucose. In addition, cysteine catabolism generated ~ 20% of intracellular pyruvate in PDAC cells. Other potential sources of pyruvate include the sialic acid pathway and catabolism of glutamine, serine, tryptophan, and threonine. However, these sources did not provide significant levels of pyruvate in PKM1/2 knockdown cells. Conclusion PKM1/2 knockdown does not impact the proliferation of pancreatic cancer cells. The serine biosynthesis pathway supports conversion of glucose to pyruvate during pyruvate kinase knockdown. However, direct conversion of serine to pyruvate was not observed during PKM1/2 knockdown. Investigating several alternative sources of pyruvate identified cysteine catabolism for pyruvate production during PKM1/2 knockdown. Surprisingly, we find that a large percentage of intracellular pyruvate comes from cysteine. Our results highlight the ability of PDAC cells to adaptively rewire their metabolic pathways during knockdown of a key metabolic enzyme.
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FOSTER, HANNAH R., THUONG HO, EVGENIY POTAPENKO, REBECCA L. CARDONE, RICHARD KIBBEY, and MATTHEW J. MERRINS. "316-OR: Genetic Deletion of Beta-Cell Pkm1, Pkm2, and Pck2 Identifies PEP as an Essential Signal for Compartmentalized KATP Closure and Cycling of the Insulin Secretory Pathway." Diabetes 71, Supplement_1 (June 1, 2022). http://dx.doi.org/10.2337/db22-316-or.
Анотація:
β-cells use pyruvate kinase to sense nutrients and respond with appropriate insulin secretion. Here, we used β-cell deletion to identify the functions of constitutively active PKm1 and allosterically recruitable PKm2, and assess their regulation by mitochondrial PEP from PCK2. We demonstrate that both PKm isoforms are present in the KATP channel microcompartment. Compartmentation nullifies the disparities in PK isoform expression and activity, allowing the minor but recruitable PKm2 isoform to fully participate in KATP regulation. However, this shared control does not extend to β-cell response to amino acids, which requires PKm1 for the initiation of Ca2+ influx and insulin secretion and PKm2 for their termination. Mitochondrial PEP is required for this cycling between “on” and “off” states, as demonstrated by β-cell-specific deletion of PCK2. PCK2 was revealed to be an essential mechanism of β-cell cataplerosis, possessing metabolic control over cytosolic ATP/ADP generation in response to anaplerotic fuels. Further, PCK2 is required for amino acid-stimulated Ca2+ influx (via PKm1) and efflux (via PKm2) . These data provide strong genetic evidence for a revised oscillatory model of β-cell metabolism in which PKM1- and PKM2-driven PEP cycles play unique roles in the initiation and termination of nutrient-stimulated insulin secretion. Disclosure H.R.Foster: None. T.Ho: None. E.Potapenko: None. R.L.Cardone: n/a. R.Kibbey: Consultant; Agios, Inc. M.J.Merrins: None. Funding NIH/NIDDK (R01DK113103) HRSA (T32HP10010) NIH/NIA (T32AG000213)
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Foster, Hannah R., Thuong Ho, Evgeniy Potapenko, Sophia M. Sdao, Shih Ming Huang, Sophie L. Lewandowski, Halena R. VanDeusen та ін. "β-cell deletion of the PKm1 and PKm2 isoforms of pyruvate kinase in mice reveal their essential role as nutrient sensors for the KATP channel". eLife 11 (23 серпня 2022). http://dx.doi.org/10.7554/elife.79422.
Анотація:
Pyruvate kinase (PK) and the phosphoenolpyruvate (PEP) cycle play key roles in nutrient-stimulated KATP channel closure and insulin secretion. To identify the PK isoforms involved, we generated mice lacking β-cell PKm1, PKm2, and mitochondrial PEP carboxykinase (PCK2) that generates mitochondrial PEP. Glucose metabolism generates both glycolytic and mitochondrially-derived PEP, which triggers KATP closure through local PKm1 and PKm2 signaling at the plasma membrane. Amino acids, which generate mitochondrial PEP without producing glycolytic fructose 1,6-bisphosphate to allosterically activate PKm2, signal through PKm1 to raise ATP/ADP, close KATP channels, and stimulate insulin secretion. Raising cytosolic ATP/ADP with amino acids is insufficient to close KATP channels in the absence of PK activity or PCK2, indicating that KATP channels are primarily regulated by PEP that provides ATP via plasma membrane-associated PK, rather than mitochondrially-derived ATP. Following membrane depolarization, the PEP cycle is also involved in an 'off-switch' that facilitates KATP channel reopening and Ca2+ extrusion, as shown by PK activation experiments and β-cell PCK2 deletion, which prolongs Ca2+ oscillations and increases insulin secretion. In conclusion, the differential response of PKm1 and PKm2 to the glycolytic and mitochondrial sources of PEP influences the β-cell nutrient response, and controls the oscillatory cycle regulating insulin secretion.
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He, Shuo, Yan Liang, Yiyi Tan, Qing Liu, Tao Liu, Xiaomei Lu, and Shutao Zheng. "Positioning determines function: Wandering PKM2 performs different roles in tumor cells." Cell Biology International, November 17, 2023. http://dx.doi.org/10.1002/cbin.12103.
Анотація:
AbstractShort for pyruvate kinase M2 subtype, PKM2 can be said of all‐round player that is notoriously known for its metabolic involvement in glycolysis. Holding a dural role as a metabolic or non‐metabolic (kinase) enzyme, PKM2 has drawn extensive attention over its biological roles implicated in tumor cells, including proliferation, migration, invasion, metabolism, and so on. wandering PKM2 can be transboundary both intracellularly and extracellularly. Specifically, PKM2 can be nuclear, cytoplasmic, mitochondrial, exosomal, or even circulate within the body. Importantly, PKM2 can function as an RNA‐binding protein (RBP) to self‐support its metabolic function. Despite extensive investigations or reviews available surrounding the biological roles of PKM2 from different angles in tumor cells, little has been described regarding some novel role of PKM2 that has been recently found, including, for example, acting as RNA‐binding protein, protection of Golgi apparatus, and remodeling of microenvironment, and so forth. Given these findings, in this review, we summarize the recent advancements made in PKM2 research, mainly from non‐metabolic respects. By the way, PKM1, another paralog of PKM2, seems to have been overlooked or under‐investigated since its discovery. Some recent discoveries made about PKM1 are also preliminarily mentioned and discussed.
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"Pyruvate kinase M2 isozyme (PKM2); PKM1." Science-Business eXchange 6, no. 3 (January 2013): 54. http://dx.doi.org/10.1038/scibx.2013.54.
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Yadav, Sandhya, Somnath D. Bhagat, Amit Gupta, Atul Samaiya, Aasheesh Srivastava, and Sanjeev Shukla. "Dietary-phytochemical mediated reversion of cancer-specific splicing inhibits Warburg effect in head and neck cancer." BMC Cancer 19, no. 1 (November 1, 2019). http://dx.doi.org/10.1186/s12885-019-6257-1.
Анотація:
Abstract Background The deregulated alternative splicing of key glycolytic enzyme, Pyruvate Kinase muscle isoenzyme (PKM) is implicated in metabolic adaptation of cancer cells. The splicing switch from normal PKM1 to cancer-specific PKM2 isoform allows the cancer cells to meet their energy and biosynthetic demands, thereby facilitating the cancer cells growth. We have investigated the largely unexplored epigenetic mechanism of PKM splicing switch in head and neck cancer (HNC) cells. Considering the reversible nature of epigenetic marks, we have also examined the utility of dietary-phytochemical in reverting the splicing switch from PKM2 to PKM1 isoform and thereby inhibition of HNC tumorigenesis. Methods We present HNC-patients samples, showing the splicing-switch from PKM1-isoform to PKM2-isoform analyzed via immunoblotting and qRT-PCR. We performed methylated-DNA-immunoprecipitation to examine the DNA methylation level and chromatin-immunoprecipitation to assess the BORIS (Brother of Regulator of Imprinted Sites) recruitment and polII enrichment. The effect of dietary-phytochemical on the activity of denovo-DNA-methyltransferase-3b (DNMT3B) was detected by DNA-methyltransferase-activity assay. We also analyzed the Warburg effect and growth inhibition using lactate, glucose uptake assay, invasion assay, cell proliferation, and apoptosis assay. The global change in transcriptome upon dietary-phytochemical treatment was assayed using Human Transcriptome Array 2.0 (HTA2.0). Results Here, we report the role of DNA-methylation mediated recruitment of the BORIS at exon-10 of PKM-gene regulating the alternative-splicing to generate the PKM2-splice-isoform in HNC. Notably, the reversal of Warburg effect was achieved by employing a dietary-phytochemical, which inhibits the DNMT3B, resulting in the reduced DNA-methylation at exon-10 and hence, PKM-splicing switch from cancer-specific PKM2 to normal PKM1. Global-transcriptome-analysis of dietary-phytochemical-treated cells revealed its effect on alternative splicing of various genes involved in HNC. Conclusion This study identifies the epigenetic mechanism of PKM-splicing switch in HNC and reports the role of dietary-phytochemical in reverting the splicing switch from cancer-specific PKM2 to normal PKM1-isoform and hence the reduced Warburg effect and growth inhibition of HNC. We envisage that this approach can provide an effective way to modulate cancer-specific-splicing and thereby aid in the treatment of HNC.
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Lee, Katie C., Allison L. Williams, and Ralph V. Shohet. "Abstract 10730: Pkm2 Ablation Dysregulates Cardiac Glucose Metabolism in Mice." Circulation 146, Suppl_1 (November 8, 2022). http://dx.doi.org/10.1161/circ.146.suppl_1.10730.
Анотація:
Introduction: The predominant isoform of pyruvate kinase (PKM1) directs pyruvate to the Krebs cycle for oxidative metabolism in the healthy heart. Our lab described a hypoxia-mediated switch to the alternatively spliced isoform PKM2. The lower PKM activity of dimerized PKM2 redirects glycolytic metabolites to the pentose phosphate pathway (PPP) and lactate production. Recently, we have found that mice with deletion of Pkm2 had profound depletion of cardiac basal glucose and higher ROS levels compared to controls. Hypothesis: We hypothesize that glucose metabolism is dysregulated in the absence of Pkm2, where the remaining isoform PKM1 promotes oxidative metabolism, leading to accumulation of ROS. Methods: Global Pkm2 KO mice were used for this study. Transcript abundance was determined by RNA-seq and RT-qPCR. Cardiomyocytes were isolated according to an established Langendorff-free method, and metabolites were traced using [U- 13 C] glucose with LC/MS (n=3). ROS concentration was measured using DCFDA in cardiomyocytes incubated at 1% O 2 (n=4). Inflammatory marker C-reactive protein (CRP) was measured in plasma of mice before and after myocardial infarction (n=12). Results: Although glucose uptake was similar, glucose was redirected to fatty acid synthesis, the polyol pathway, and the PPP, and away from oxidative metabolism in Pkm2 KO cardiomyocytes. Despite the increased flux into the PPP, NADPH production was low. Assessment of ROS levels in normoxia revealed that Pkm2 KO cardiomyocytes had higher levels of ROS, possibly stimulating the inflammatory response in Pkm2 KO mice. CRP levels were higher in Pkm2 KO plasma at baseline. ROS remained high in Pkm2 KO cardiomyocytes after hypoxic treatment and CRP remained high in Pkm2 KO plasma after MI. Conclusions: Increased expression of Pkm1 in the Pkm2 KO mice increases flux of metabolites through oxidative metabolism, exacerbating mitochondrial ROS and activating the PPP. Although NADPH levels should have correlated with increased PPP flux, the low NADPH production in Pkm2 KO cardiomyocytes suggest rapid consumption of NADPH by ROS and fatty acid synthesis. This study suggests a role for hypoxia-stimulated PKM2 accumulation in stabilizing glucose flux to mitigate oxidative stress in the ischemic heart.