Dictyostelium Differentiation-Inducing Factor 1 Promotes Glucose Uptake via Direct Inhibition of Mitochondrial Malate Dehydrogenase in Mouse 3T3-L1 Cells.

Differentiation-inducing factor 1 (DIF-1), found in Dictyostelium discoideum, has antiproliferative and glucose-uptake-promoting activities in mammalian cells. DIF-1 is a potential lead for the development of antitumor and/or antiobesity/antidiabetes drugs, but the mechanisms underlying its actions have not been fully elucidated. In this study, we searched for target molecules of DIF-1 that mediate the actions of DIF-1 in mammalian cells by identifying DIF-1-binding proteins in human cervical cancer HeLa cells and mouse 3T3-L1 fibroblast cells using affinity chromatography and liquid chromatography-tandem mass spectrometry and found mitochondrial malate dehydrogenase (MDH2) to be a DIF-1-binding protein in both cell lines. Since DIF-1 has been shown to directly inhibit MDH2 activity, we compared the effects of DIF-1 and the MDH2 inhibitor LW6 on the growth of HeLa and 3T3-L1 cells and on glucose uptake in confluent 3T3-L1 cells in vitro. In both HeLa and 3T3-L1 cells, DIF-1 at 10-40 µM dose-dependently suppressed growth, whereas LW6 at 20 µM, but not at 2-10 µM, significantly suppressed growth in these cells. In confluent 3T3-L1 cells, DIF-1 at 10-40 µM significantly promoted glucose uptake, with the strongest effect at 20 µM DIF-1, whereas LW6 at 2-20 µM significantly promoted glucose uptake, with the strongest effect at 10 µM LW6. Western blot analyses showed that LW6 (10 µM) and DIF-1 (20 µM) phosphorylated and, thus, activated AMP kinase in 3T3-L1 cells. Our results suggest that MDH2 inhibition can suppress cell growth and promote glucose uptake in the cells, but appears to promote glucose uptake more strongly than it suppresses cell growth. Thus, DIF-1 may promote glucose uptake, at least in part, via direct inhibition of MDH2 and a subsequent activation of AMP kinase in 3T3-L1 cells.

Inhibition of Malate Dehydrogenase-2 Protects Renal Tubular Epithelial Cells from Anoxia-Reoxygenation-Induced Death or Senescence.

Ischemia-reperfusion injury is the leading cause of acute kidney injury. Reactive oxygen species (ROS) production causes cell death or senescence. In cultures of primary human renal tubular epithelial cells (RPTECs) subjected to anoxia-reoxygenation, inhibition of the Krebs cycle at the level of malate dehydrogenase-2 (MDH-2) decreases hypoxia-inducible factor-1α and oxidative stress and protects from apoptotic or ferroptotic cell death. Inhibition of MDH-2 decreased reoxygenation-induced upregulation of p53 and p21, restored the levels of the proliferation marker Ki-67, and prevented the upregulation of the senescence marker beta-galactosidase and interleukin-1ß production. MDH-2 inhibition reduced the reoxygenation-induced upregulation of ATP, but the alterations of critical cell metabolism enzymes allowed enough ATP production to prevent cell energy collapse. Thus, inhibition of the Krebs cycle at the level of MDH-2 protects RPTECs from anoxia-reoxygenation-induced death or senescence. MDH-2 may be a promising pharmaceutical target against ischemia-reperfusion injury.

Analysis of Hsp90 allosteric modulators interactome reveals a potential dual action mode involving mitochondrial MDH2.

Hsp90 (i.e., Heat shock protein 90) is a well-established therapeutic target for several diseases, ranging from misfolding-related disfunctions to cancer. In this framework, we have developed in recent years a family of benzofuran compounds that act as Hsp90 allosteric modulators. Such molecules can interfere with the stability of some relevant Hsp90 client oncoproteins, showing a low µM cytotoxic activity in vitro in cancer cell lines. Here we identify the target profile of these chemical probes by means of chemical proteomics, which established MDH2 (mitochondrial malate dehydrogenase) as an additional relevant cellular target that might help elucidate the molecular mechanism of their citotoxicity. Western blotting, DARTS (i.e., Drug Affinity Responsive Target Stability) and enzymatic assays data confirmed a dose-dependent interaction of MDH2 with several members of the benzofuran Hsp90 modulators family and a computational model allowed to interpret the observed interactions.

Malic enzyme 2 connects the Krebs cycle intermediate fumarate to mitochondrial biogenesis.

Mitochondria have an independent genome (mtDNA) and protein synthesis machinery that coordinately activate for mitochondrial generation. Here, we report that the Krebs cycle intermediate fumarate links metabolism to mitobiogenesis through binding to malic enzyme 2 (ME2). Mechanistically, fumarate binds ME2 with two complementary consequences. First, promoting the formation of ME2 dimers, which activate deoxyuridine 5'-triphosphate nucleotidohydrolase (DUT). DUT fosters thymidine generation and an increase of mtDNA. Second, fumarate-induced ME2 dimers abrogate ME2 monomer binding to mitochondrial ribosome protein L45, freeing it for mitoribosome assembly and mtDNA-encoded protein production. Methylation of the ME2-fumarate binding site by protein arginine methyltransferase-1 inhibits fumarate signaling to constrain mitobiogenesis. Notably, acute myeloid leukemia is highly dependent on mitochondrial function and is sensitive to targeting of the fumarate-ME2 axis. Therefore, mitobiogenesis can be manipulated in normal and malignant cells through ME2, an unanticipated governor of mitochondrial biomass production that senses nutrient availability through fumarate.

Synthesis, biological evaluation and molecular docking studies of indeno [1, 2-c] pyrazol derivatives as inhibitors of mitochondrial malate dehydrogenase 2 (MDH2).

Hypoxia inducible factor-1 (HIF-1) is a pivotal transcription factor, which is strongly correlated with the induction of angiogenesis, tumor survival, metastasis, and cell proliferation, making it a pivotal therapeutic target for solid tumor therapeutic agents. Herein, a new series of multi-functional chemical probes were designed including principal groups, viz. adamantyl and indene, at various locations of the parent compound LW6. Molecular docking studies were performed on the designed compounds and their relationship with HIF-1α and malate dehydrogenase 2 (MDH2). Inhibition of MDH2 by our compounds was expected to decrease the NADH level. Indeed, treatment of the breast cancer cell line 4T1 led to a strong reduction of the NADH concentration. The greatest reduction in NADH production in mitochondria was observed with (E)-3-(4-((3r, 5r, 7r)-adamantan-1-yl) phenoxy)-N-(5-(piperidine-1-carbonyl)-1, 4-dihydroindeno [1, 2-c] pyrazol-3-yl) acrylamide (18: IC50 = 59 nM), and has the best inhibitory potential under hypoxic conditions (MCF-7: IC50 = 57 nM). This compound also gave one of the highest docking "higher than the score obtained with LW6 in parallel (-31.63 kcal/mol) in the initial docking runs (PDB Code: 4WLO). Other related compounds with good yields were also synthesized from docking results, and all the synthesized compounds (14, 18, 22, 26, 29, 30) were evaluated in vitro on human adenocarcinoma cell lines.

Malic Enzyme 1 Is Associated with Tumor Budding in Oral Squamous Cell Carcinomas.

Budding at the tumor invasive front has been correlated with the malignant properties of many cancers. Malic enzyme 1 (ME1) promotes the Warburg effect in cancer cells and induces epithelial-mesenchymal transition (EMT) in oral squamous cell carcinoma (OSCC). Therefore, we investigated the role of ME1 in tumor budding in OSCC. Tumor budding was measured in 96 human OSCCs by immunostaining for an epithelial marker (AE1/AE3), and its expression was compared with that of ME1. A significant correlation was observed between tumor budding and ME1 expression. The correlation increased with the progression of cancer. In human OSCC cells, lactate secretion decreased when lactate fermentation was suppressed by knockdown of ME1 and lactate dehydrogenase A or inhibition of pyruvate dehydrogenase (PDH) kinase. Furthermore, the extracellular pH increased, and the EMT phenotype was suppressed. In contrast, when oxidative phosphorylation was suppressed by PDH knockdown, lactate secretion increased, extracellular pH decreased, and the EMT phenotype was promoted. Induction of chemical hypoxia in OSCC cells by CoCl2 treatment resulted in increased ME1 expression along with HIF1α expression and promotion of the EMT phenotype. Hypoxic conditions also increased matrix metalloproteinases expression and decreased mitochondrial membrane potential, mitochondrial oxidative stress, and extracellular pH. Furthermore, the hypoxic treatment resulted in the activation of Yes-associated protein (YAP), which was abolished by ME1 knockdown. These findings suggest that cancer cells at the tumor front in hypoxic environments increase their lactate secretion by switching their energy metabolism from oxidative phosphorylation to glycolysis owing to ME1 overexpression, decrease in extracellular pH, and YAP activation. These alterations enhance EMT and the subsequent tumor budding. Tumor budding and ME1 expression are thus considered useful markers of OSCC malignancy, and ME1 is expected to be a relevant target for molecular therapy.

ddhCTP produced by the radical-SAM activity of RSAD2 (viperin) inhibits the NAD+ -dependent activity of enzymes to modulate metabolism.

Radical S-adenosylmethionine (SAM) domain-containing protein 2 (RSAD2; viperin) is a key enzyme in innate immune responses that is highly expressed in response to viral infection and inflammatory stimuli in many cell types. Recently, it was found that RSAD2 catalyses transformation of cytidine triphosphate (CTP) to its analogue 3'-deoxy-3',4'-didehydro-CTP (ddhCTP). The cellular function of this metabolite is unknown. Here, we analysed the extra- and intracellular metabolite levels in human induced pluripotent stem cell (hiPSC)-derived macrophages using high-resolution LC-MS/MS. The results together with biochemical assays and molecular docking simulations revealed that ddhCTP inhibits the NAD+ -dependent activity of enzymes including that of the housekeeping enzyme glyceraldehyde 3-phosphate dehydrogenase (GAPDH). We propose that ddhCTP regulates cellular metabolism in response to inflammatory stimuli such as viral infection, pointing to a broader function of RSAD2 than previously thought.

MDH1 and MPP7 Regulate Autophagy in Pancreatic Ductal Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is driven by metabolic changes in pancreatic cells caused by oncogenic mutations and dysregulation of p53. PDAC cell lines and PDAC-derived xenografts grow as a result of altered metabolic pathways, changes in stroma, and autophagy. Selective targeting and inhibition of one of these may open avenues for the development of new therapeutic strategies. In this study, we performed a genome-wide siRNA screen in a PDAC cell line using endogenous autophagy as a readout and identified several regulators of autophagy that were required for autophagy-dependent PDAC cell survival. Validation of two promising candidates, MPP7 (MAGUK p55 subfamily member 7, a scaffolding protein involved in cell-cell contacts) and MDH1 (cytosolic Malate dehydrogenase 1), revealed their role in early stages of autophagy during autophagosome formation. MPP7 was involved in the activation of YAP1 (a transcriptional coactivator in the Hippo pathway), which in turn promoted autophagy, whereas MDH1 was required for maintenance of the levels of the essential autophagy initiator serine-threonine kinase ULK1, and increased in the activity upon induction of autophagy. Our results provide a possible explanation for how autophagy is regulated by MPP7 and MDH1, which adds to our understanding of autophagy regulation in PDAC. SIGNIFICANCE: This study identifies and characterizes MPP7 and MDH1 as novel regulators of autophagy, which is thought to be responsible for pancreatic cancer cell survival.

Malic Enzyme 1 (ME1) is pro-oncogenic in ApcMin/+ mice.

Cytosolic Malic Enzyme (ME1) provides reduced NADP for anabolism and maintenance of redox status. To examine the role of ME1 in tumor genesis of the gastrointestinal tract, we crossed mice having augmented intestinal epithelial expression of ME1 (ME1-Tg mice) with ApcMin/+ mice to obtain male ApcMin/+/ME1-Tg mice. ME1 protein levels were significantly greater within gut epithelium and adenomas of male ApcMin/+/ME1-Tg than ApcMin/+ mice. Male ApcMin/+/ME1-Tg mice had larger and greater numbers of adenomas in the small intestine (jejunum and ileum) than male ApcMin/+ mice. Male ApcMin/+/ME1-Tg mice exhibited greater small intestine crypt depth and villus length in non-adenoma regions, correspondent with increased KLF9 protein abundance in crypts and lamina propria. Small intestines of male ApcMin/+/ME1-Tg mice also had enhanced levels of Sp5 mRNA, suggesting Wnt/ß-catenin pathway activation. A small molecule inhibitor of ME1 suppressed growth of human CRC cells in vitro, but had little effect on normal rat intestinal epithelial cells. Targeting of ME1 may add to the armentarium of therapies for cancers of the gastrointestinal tract.

Malic enzyme 2 as a potential therapeutic drug target for cancer.

Reprogrammed metabolic profile is a biochemical fingerprint of cancerous cells, which represents one of the "hallmarks of cancer." The aberrant expression pattern of enzymatic machineries orchestrates metabolic activities into a platform that ultimately promotes cellular growth, survival, and proliferation. The NADP(+)-dependent mitochondrial malic enzyme 2 (ME2) has been widely appreciated due to its function as a provider of pyruvate and reducing power to the cell for biosynthesis of fatty acids and nucleotides along with maintenance of redox balance. Multiple lines of evidences have indicated that ME2 is a bonafide therapeutic target and novel biomarker which plays critical role during tumorigenesis. The objective of this review is to provide an update on the cancer-specific role of ME2 in order to explore its potential for therapeutic opportunities. Furthermore, we have discussed the potential of genetic and pharmacological inhibitors of ME2 in the light of previous research work for therapeutic advancements in cancer treatment. It is contemplated that additional investigations should focus on the use of ME2 inhibitors in combinational therapies as rational combinations of metabolic inhibitors and chemotherapy may have the ability to cure cancer. © 2018 IUBMB Life, 70(11):1076-1083, 2018.

Identification of multiple-derived peptides produced by Saccharomyces cerevisiae involved in malolactic fermentation inhibition.

An oenological strain of Saccharomyces cerevisiae was previously shown to produce a 5-10 kDa peptidic fraction responsible for the inhibition of malolactic fermentation (MLF). In the present study, we aim to further purify the anti-MLF peptides of this fraction. The yeast fermented synthetic grape juice medium was fractionated by ammonium sulfate precipitation combined with ultrafiltration. The 5-10 kDa fraction recovered at a saturation degree of 60%-80% was the only fraction that inhibited both the bacterial growth and the malate consumption in vivo. It also inhibited the malolactic enzyme activity in vitro at a pH range between 3.5 and 6.7. Therefore, it was purified by both anion and cation exchange chromatography. The eluates that inhibited the malolactic enzyme activity in vitro were migrated on Tricine SDS-PAGE and the protein bands were excised and sequenced by LC-MS/MS. The sequencing revealed nine peptides originating from eight proteins of S. cerevisiae. Two GAPDH cationic fragments of 0.9 and 1.373 kDa having a pI of 10.5 and 11 respectively, Wtm2p and Utr2p anionic fragments of 2.42 kDa with a pI of 3.5 and 4 respectively were thought to contribute the most to the MLF inhibition.

Tumor-suppressing effects of microRNA-612 in bladder cancer cells by targeting malic enzyme 1 expression.

The present study investigated the possible tumor-suppressing function of microRNA (miR)-612 and the underlying molecular mechanism of its action in bladder cancer in vitro and in vivo. Reverse transcription­quantitative polymerase chain reaction (RT­qPCR) was carried out to quantify the expression levels of miR­612 in bladder cancer tissues and cell lines. The data demonstrated that the level of miR­612 expression was significantly reduced in bladder cancer tissues and cell lines, as compared with that in non­cancerous tissues and cells. Reduced miR­612 expression was associated with advanced tumor, lymph node and metastasis stages, and with distant metastasis of bladder cancer. A functional study revealed that transfection of cells with an miR­612 mimic suppressed bladder cancer cell growth, colony formation, migration, invasion and epithelial-mesenchymal transition. Bioinformatics analysis identified that miR­612 targeted the expression of malic enzyme 1 (ME1), and this was confirmed by western blot and luciferase reporter assay results. Furthermore, the ME1 expression levels were inversely associated with miR­612 expression in bladder cancer tissue specimens. In addition, knockdown of ME1 expression using ME1 siRNA mimicked the effect of ectopic miR­612 overexpression in bladder cancer cells in terms of tumor cell growth, migration and invasion. By contrast, ME1 overexpression weakened the inhibitory effect of the miR­612 mimic in bladder cancer cells. In conclusion, the present study demonstrated that miR­612 may function as a tumor suppressor in bladder cancer by targeting ME1 expression.

Expression of cytosolic malic enzyme (ME1) is associated with disease progression in human oral squamous cell carcinoma.

Malic enzyme 1 (ME1) is a multifunctional protein involved in glycolysis, the citric acid cycle, NADPH production, glutamine metabolism, and lipogenesis. It is overexpressed in various cancers. We examined the expression of ME1 in 119 oral squamous cell carcinomas (OSCCs) using immunohistochemistry. Malic enzyme 1 expression was moderate to strong in 57 (48%) OSCCs and correlated with pT, pN, clinical stage, and histological grade. In 37 cases with prognostic evaluation, moderate to strong ME1 expression indicated a worse prognosis than did weak ME1 expression. Malic enzyme 1 knockdown or inactivation by lanthanide inhibited cell proliferation and motility and suppressed the epithelial-mesenchymal transition in HSC3 human OSCC cells. Knockdown of ME1 also shifted energy metabolism from aerobic glycolysis and lactate fermentation to mitochondrial oxidative phosphorylation, and the redox status from reductive to oxidative. In a mouse tumor model, lanthanide suppressed tumor growth and increased survival time. These findings reveal that ME1 is a valid target for molecular therapy in OSCC.

Methyl 3-(3-(4-(2,4,4-Trimethylpentan-2-yl)phenoxy)-propanamido)benzoate as a Novel and Dual Malate Dehydrogenase (MDH) 1/2 Inhibitor Targeting Cancer Metabolism.

Previously, we reported a hypoxia-inducible factor (HIF)-1 inhibitor LW6 containing an (aryloxyacetylamino)benzoic acid moiety inhibits malate dehydrogenase 2 (MDH2) using a chemical biology approach. Structure-activity relationship studies on a series of (aryloxyacetylamino)benzoic acids identified selective MDH1, MDH2, and dual inhibitors, which were used to study the relationship between MDH enzyme activity and HIF-1 inhibition. We hypothesized that dual inhibition of MDH1 and MDH2 might be a powerful approach to target cancer metabolism and selected methyl-3-(3-(4-(2,4,4-trimethylpentan-2-yl)phenoxy)propanamido)-benzoate (16c) as the most potent dual inhibitor. Kinetic studies revealed that compound 16c competitively inhibited MDH1 and MDH2. Compound 16c inhibited mitochondrial respiration and hypoxia-induced HIF-1α accumulation. In xenograft assays using HCT116 cells, compound 16c demonstrated significant in vivo antitumor efficacy. This finding provides concrete evidence that inhibition of both MDH1 and MDH2 may provide a valuable platform for developing novel therapeutics that target cancer metabolism and tumor growth.

In vitro evaluation of the effect of botanical formulations used in the control of Aedes aegypti L. (Diptera: Culicidae) on liver enzymes.

Abstract: INTRODUCTION: Dengue fever is a viral disease transmitted by the Aedes aegypti Linn. (1792) (Diptera: Culicidae) mosquito, which is endemic in several regions of Brazil. Alternative methods for the control of the vector include botanical insecticides, which offer advantages such as lower environmental contamination levels and less likelihood of resistant populations. Thus, in this study, the ability of botanical insecticide formulations to inhibit the activity of the liver enzymes serum cholinesterase and malate dehydrogenase was evaluated. METHODS: Inhibition profiles were assessed using in vitro assays for cholinesterase and malate dehydrogenase activity and quantitated by ultraviolet-visible spectroscopy at 410nm to 340nm. RESULTS Insecticide products formulated from cashew nutshell liquid [A] and ricinoleic acid [B] showed cholinesterase activity levels of 6.26IU/mL and 6.61IU/mL, respectively, while the control level for cholinesterase was 5-12IU/mL. The products did not affect the level of 0.44IU/mL established for malate dehydrogenase, as the levels produced by [A] and [B] were 0.43IU/mL and 0.45IU/mL, respectively. CONCLUSIONS Our findings show that in vitro testing of the formulated products at concentrations lethal to A. aegypti did not affect the activity of cholinesterase and malate dehydrogenase, indicating the safety of these products.

A Novel Malate Dehydrogenase 2 Inhibitor Suppresses Hypoxia-Inducible Factor-1 by Regulating Mitochondrial Respiration.

We previously reported that hypoxia-inducible factor (HIF)-1 inhibitor LW6, an aryloxyacetylamino benzoic acid derivative, inhibits malate dehydrogenase 2 (MDH2) activity during the mitochondrial tricarboxylic acid (TCA) cycle. In this study, we present a novel MDH2 inhibitor compound 7 containing benzohydrazide moiety, which was identified through structure-based virtual screening of chemical library. Similar to LW6, compound 7 inhibited MDH2 activity in a competitive fashion, thereby reducing NADH level. Consequently, compound 7 reduced oxygen consumption and ATP production during the mitochondrial respiration cycle, resulting in increased intracellular oxygen concentration. Therefore, compound 7 suppressed the accumulation of HIF-1α and expression of its target genes, vascular endothelial growth factor (VEGF) and glucose transporter 1 (GLUT1). Moreover, reduction in ATP content activated AMPK, thereby inactivating ACC and mTOR the downstream pathways. As expected, compound 7 exhibited significant growth inhibition of human colorectal cancer HCT116 cells. Compound 7 demonstrated substantial anti-tumor efficacy in an in vivo xenograft assay using HCT116 mouse model. Taken together, a novel MDH2 inhibitor, compound 7, suppressed HIF-1α accumulation via reduction of oxygen consumption and ATP production, integrating metabolism into anti-cancer efficacy in cancer cells.

The mechanisms of malic enzyme 2 in the tumorigenesis of human gliomas.

The high level of resistance of glioblastoma multiforme (GBM) to currently used chemotherapies and other conventional therapies, its invasive characteristics and the presence of stem-like cells are the major factors that make the treatment of GBM difficult. Recent studies have demonstrated that the homeostasis of energy metabolism, glycolysis and mitochondrial oxidation of glucose are important for GBM cell growth and chemo-resistance. However, it is not clear which specific gene(s) are involved in the homeostasis of energy metabolism and invasiveness of GBM cells. We performed a preliminary analysis of data obtained from Gene Expression Omnibus profiles and determined that malic enzyme 2 (ME2) expression was positively associated with WHO grade in human primary gliomas. Hence, we evaluated the detailed working mechanisms of ME2 in human GBM cell processes, including proliferation, cell cycle, invasion, migration, ROS, and ATP production. Our data demonstrated that ME2 was involved in GBM growth, invasion and migration. ME2 has two cofactors, NAD+ or NADP+, which are used to produce NADH and NADPH for ATP production and ROS clearance, respectively. If the catalytic activity of ME2 is determined to be critical for its roles in GBM growth, invasion and migration, small molecule inhibitors of ME2 may be valuable drugs for GBM therapy. We hope that our current data provides a candidate treatment strategy for GBM.

Development of a Clickable Probe for Profiling of Protein Glutathionylation in the Central Cellular Metabolism of E. coli and Drosophila.

Protein glutathionylation is an important post-translational modification that regulates many cellular processes, including energy metabolism, signal transduction, and protein homeostasis. Global profiling of glutathionylated proteins (denoted as glutathionylome) is crucial for understanding redox-regulated signal transduction. Here, we developed a novel method based on click reaction and proteomics to enrich and identify the glutathionylated peptides in Escherichia coli and Drosophila lysates, in which 937 and 1,930 potential glutathionylated peptides were identified, respectively. Bioinformatics analysis showed that the cysteine residue next to negatively charged amino acid residues has a higher frequency of glutathionylation. Importantly, we found that most proteins associated with metabolic pathways were glutathionylated and that the glutathionylation sites of metabolic enzymes were highly conserved among different species. Our results indicate that the glutathione analog is a useful tool to characterize protein glutathionylation, and glutathionylation of metabolic enzymes, which play important roles in regulating cellular metabolism, is conserved.

A small-molecule inhibitor suppresses the tumor-associated mitochondrial NAD(P)+-dependent malic enzyme (ME2) and induces cellular senescence.

Here, we found a natural compound, embonic acid (EA), that can specifically inhibit the enzymatic activity of mitochondrial NAD(P)+-dependent malic enzyme (m-NAD(P)-ME, ME2) either in vitro or in vivo. The in vitro IC50 value of EA for m-NAD(P)-ME was 1.4 ± 0.4 µM. Mutagenesis and binding studies revealed that the putative binding site of EA on m-NAD(P)-ME is located at the fumarate binding site or at the dimer interface near the site. Inhibition studies reveal that EA displayed a non-competitive inhibition pattern, which demonstrated that the binding site of EA was distinct from the active site of the enzyme. Therefore, EA is thought to be an allosteric inhibitor of m-NAD(P)-ME. Both EA treatment and knockdown of m-NAD(P)-ME by shRNA inhibited the growth of H1299 cancer cells. The protein expression and mRNA synthesis of m-NAD(P)-ME in H1299 cells were not influenced by EA, suggesting that the EA-inhibited H1299 cell growth occurs through the suppression of in vivo m-NAD(P)-ME activity EA treatment further induced the cellular senescence of H1299 cells. However, down-regulation of the enzyme-induced cellular senescence was not through p53. Therefore, the EA-evoked senescence of H1299 cells may occur directly through the inhibition of ME2 or a p53-independent pathway.