Enzyme In Polyamine Synthesis Research Articles

Effect of cryopreservation on Odc1 and RhoA genes expression in diapausing mouse blastocysts.

The possibility of embryo cryopreservation is important for applying the genome resource banking (GRB) concept to those mammalian species that exhibit embryonal diapause in their early development. Odc1 encodes ODC1, which is a key enzyme in polyamine synthesis. RhoA is an essential part of Rho/ROCK system. Both Odc1 and RhoA play an important role in preimplantation embryo development. Studying these systems in mammalian species with obligate or experimentally designed embryonic diapause may provide insight into the molecular machinery underlying embryo dormancy and re-activation. The effect of cryopreservation procedures on the expression of the Odc1 and RhoA in diapausing embryos has not been properly studied yet. The purpose of this work is to address the possibility of cryopreservation diapausing embryos and to estimate the expression of the Odc1 and RhoA genes in diapausing and non-diapausing embryos before and after freeze-thaw procedures using ovariectomized progesterone treated mice as a model. Both diapausing and non-diapausing invivo-derived embryos continued their development invitro after freezing-thawing as evidenced by blastocoel re-expansion. Although cryopreservation dramatically decreased the expression of the Odc1 and RhoA genes in non-diapausing embryos, no such effects have been observed in diapausing embryos where these genes were already at the low level before freeze-thaw procedures. Future studies may attempt to facilitate the re-activation of diapausing embryos, for example frozen-thawed ones, specifically targeting Odc1 or Rho/ROCK system.

Polyamine Catabolism and Its Role in Renal Injury and Fibrosis in Mice Subjected to Repeated Low-Dose Cisplatin Treatment.

Cisplatin, a chemotherapeutic agent, can cause nephrotoxic and ototoxic injuries. Using a mouse model of repeated low dose cisplatin (RLDC), we compared the kidneys of cisplatin- and vehicle-treated mice on days 3 (early injury phase) and 35 (late injury/recovery phase) after the final treatment. RNA-seq analyses revealed increases in the expression of markers of kidney injury (e.g., lipocalin 2 and kidney injury molecule 1) and fibrosis (e.g., collagen 1, fibronectin, and vimentin 1) in RLDC mice. In addition, we observed increased expression of polyamine catabolic enzymes (spermidine/spermine N1-acetyltransferase, Sat1, and spermine oxidase, Smox) and decreased expression of ornithine decarboxylase (Odc1), a rate-limiting enzyme in polyamine synthesis in mice subjected to RLDC. Upon confirmation of the RNA-seq results, we tested the hypothesis that enhanced polyamine catabolism contributes to the onset of renal injury and development of fibrosis. To test our hypothesis, we compared the severity of RLDC-induced renal injury and fibrosis in wildtype (WT), Sat1-KO, and Smox-KO mice. Our results suggest that the ablation of polyamine catabolic enzymes reduces the severity of renal injury and that modulation of the activity of these enzymes may protect against kidney damage and fibrosis caused by cisplatin treatment.

Ornithine aminotransferase supports polyamine synthesis in pancreatic cancer.

There is a need to develop effective therapies for pancreatic ductal adenocarcinoma (PDA), a highly lethal malignancy with increasing incidence1 and poor prognosis2. Although targeting tumour metabolism has been the focus of intense investigation for more than a decade, tumour metabolic plasticity and high risk of toxicity have limited this anticancer strategy3,4. Here we use genetic and pharmacological approaches in human and mouse in vitro and in vivo models to show that PDA has a distinct dependence on de novo ornithine synthesis from glutamine. We find thatthis process, which is mediated through ornithine aminotransferase (OAT), supports polyamine synthesis and is required for tumour growth. This directional OAT activity is usually largely restricted to infancy and contrasts with the reliance of most adult normal tissues and other cancer types on arginine-derived ornithine for polyamine synthesis5,6. This dependency associates with arginine depletion in the PDA tumour microenvironment and is driven by mutant KRAS. ActivatedKRAS induces the expression of OAT and polyamine synthesis enzymes, leading to alterations in the transcriptome and open chromatin landscape in PDA tumour cells. The distinct dependence of PDA, but not normal tissue, on OAT-mediated de novo ornithine synthesis provides an attractive therapeutic window for treating patients with pancreatic cancer with minimal toxicity.

Effects of polyamine synthesis enzymes on angiogenesis and apoptosis during endometriosis

Objectives: Since we assumed that endometriosis is a benign cell division disorder, our study was conducted to investigate the effects of the relationships between polyamine synthesis and angiogenesis in the formation of endometriosis. Material and methods: Thirty-five patients with endometriosis and 35 healthy female women were included in the study. The patient and the control groups were compared regarding the blood levels of agmatine, argininecarboxylase (ADC), ornithinecarboxylase (ODC), agmatinase, arginase, ornithine, and the vascular endothelial growth factor (VEGF). Results: There is a statistically significant difference between the patient and the control groups regarding the agmatinase, arginase and VEGF levels (higher in the patient group) (p < 0.05). There is no statistically significant difference between the patient and the control groups regarding the ODC, ornithine and the ADC levels (p > 0.05). There is a statistically significant difference between the patient and the control groups regarding the agmatine levels (higher in the control group) (p < 0.05). Conclusions: The increase in the serum levels of polyamine synthesis enzymes may contribute to the formation of endometriosis. It is anticipated that the study of the relationship between enzymes and molecules in the polyamine synthesis pathway and angiogenesis in patients with endometriosis will contribute to the literature.

Polyamine synthesis enzyme AMD1 is closely related to the tumorigenesis and prognosis of human breast cancer

Adenosylmethionine decarboxylase 1 (AMD1) has been implicated in carcinogenesis and tumor progression. However, the potential biomechanism and biological implications of AMD1 in breast cancer (BC) remain unclear. The purpose of this study was to investigate the effect of abnormal expression of AMD1 in BC. The expression of AMD1 in different human BC cell lines was studied by using western blotting and qRT–PCR. In vitro cell proliferation, clone formation, cell cycle and apoptosis assays were performed to explore the effect of AMD1 on cellular proliferation. Xenograft mouse models were established to elucidate the role of AMD1 in BC growth. The expression profiles of AMD1 in 28 pairs of BC tissues and adjacent noncancerous tissues (ANTs) were investigated by using western blotting and immunohistochemistry. The clinical implication and prognostic evaluation of AMD1 in BC were examined by excavating the online database. We found that the expression levels of AMD1 in BC cell lines were significantly higher than those in the normal human breast epithelial cell line MCF-10A. In addition, AMD1 potentiated proliferation, induced cell cycle progression and inhibited apoptosis in BC cells. Subcutaneous tumor xenografts also supported the promotive role of AMD1 in BC growth. We discovered that the level of AMD1 in BC tissues was significantly higher than that in ANTs. Using the online database, increased AMD1 was found to be associated with clinical indicators and predicted a poor prognosis in patients with BC. Our findings indicate that AMD1 elicits potent oncogenic effects on the malignant progression of BC. AMD1 might serve as a promising diagnostic biomarker and therapeutic target for patients with BC.

Abstract PO-028: Pancreatic ductal adenocarcinoma is dependent on an unconventional pathway for polyamine synthesis

Abstract Targeting altered metabolism in pancreatic ductal adenocarcinoma (PDAC) has been an area of extensive investigation for over a decade now. A major hurdle however, for most anti-tumor metabolic strategies, is the high risk for toxicity, given the essential roles of metabolic pathways in the maintenance of normal tissue homeostasis. Indeed, this has been the case for targeting polyamines in cancers. Polyamines are small, highly positively charged molecules involved in multiple fundamental processes of cell growth and survival, including the synthesis of nucleic acids, modifications of chromatin structure, gene transcription and mRNA translation. Polyamine levels are significantly increased in many cancers, including PDAC. Prior anti-tumor strategies focused on pharmacological inhibition of the rate-limiting enzyme of polyamine synthesis, ornithine decarboxylase (ODC1) with little success, partially due to risk of harming normal tissues at higher drug doses. In this project, using both in vitro and in vivo mouse models of PDAC, we identified a dependency of PDAC on an unconventional way for the synthesis of the polyamine precursor ornithine, specifically from glutamine via ornithine aminotransferase (OAT); this is in contrast to its synthesis in most adult normal tissues from arginine via arginase (ARG) activity. We also identified potential key players mediating the induction of this metabolic pathway via KRAS, the main oncogenic driver in PDAC and are currently characterizing the downstream effects of polyamines on pancreatic tumor cells. The high dependency of PDAC, compared to normal tissue, on de novo ornithine synthesis via OAT provides an attractive therapeutic window for treating pancreatic cancer patients with minimal toxicity. Citation Format: Min-Sik Lee, Insia Naqvi, Courtney Dennis, Lucas Dailey, Alireza Lorzadeh, Tamara Zaytouni, Ashley Adler, Daniel S. Hitchcock, Lin Lin, Unmesh Jadhav, Clary B. Clish, Nada Y. Kalaany. Pancreatic ductal adenocarcinoma is dependent on an unconventional pathway for polyamine synthesis [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-028.

Polyamine metabolism is a central determinant of helper T cell lineage fidelity

SummaryPolyamine synthesis represents one of the most profound metabolic changes during T cell activation, but the biological implications of this are scarcely known. Here, we show that polyamine metabolism is a fundamental process governing the ability of CD4+ helper T cells (TH) to polarize into different functional fates. Deficiency in ornithine decarboxylase, a crucial enzyme for polyamine synthesis, results in a severe failure of CD4+ T cells to adopt correct subset specification, underscored by ectopic expression of multiple cytokines and lineage-defining transcription factors across TH cell subsets. Polyamines control TH differentiation by providing substrates for deoxyhypusine synthase, which synthesizes the amino acid hypusine, and mice in which T cells are deficient for hypusine develop severe intestinal inflammatory disease. Polyamine-hypusine deficiency caused widespread epigenetic remodeling driven by alterations in histone acetylation and a re-wired tricarboxylic acid (TCA) cycle. Thus, polyamine metabolism is critical for maintaining the epigenome to focus TH cell subset fidelity.

Pre-implantation exogenous progesterone and pregnancy in sheep: I. polyamines, nutrient transport, and progestamedins

BackgroundAdministration of exogenous progesterone (P4) to ewes during the pre-implantation period advances conceptus development and implantation. This study determined effects of exogenous P4 on transport of select nutrients and pathways that enhance conceptus development. Pregnant ewes (n = 38) were treated with either 25 mg P4 in 1 mL corn oil (P4, n = 18) or 1 mL corn oil alone (CO, n = 20) from day 1.5 through day 8 of pregnancy and hysterectomized on either day 9 or day 12 of pregnancy. Endometrial expression of genes encoding enzymes for synthesis of polyamines, transporters of glucose, arginine, and glycine, as well as progestamedins was determined by RT-qPCR.ResultsOn day 12 of pregnancy, conceptuses from P4-treated ewes had elongated while those from CO-treated ewes were spherical. The mRNA expression of AZIN2, an arginine decarboxylase, was lower in endometria of P4-treated than CO-treated ewes on day 9 of pregnancy. Expression of FGF10, a progestamedin, was greater in endometria of CO and P4-treated ewes on day 12 of gestation in addition to P4-treated ewes necropsied on day 9 of gestation. Treatment with P4 down-regulated endometrial expression of amino acid transporter SLC1A4 on day 12 of pregnancy.ConclusionsResults indicated that administration of exogenous P4 during the pre-implantation period advanced the expression of FGF10, which may accelerate proliferation of trophectoderm cells, but also was correlated with decreased expression of glycine and serine transporters and polyamine synthesis enzyme AZIN2. Further research with increased sample sizes may determine how differential expression affects endometrial functions and potentially embryonic loss.

Dual targeting of polyamine synthesis and uptake in diffuse intrinsic pontine gliomas

Diffuse intrinsic pontine glioma (DIPG) is an incurable malignant childhood brain tumor, with no active systemic therapies and a 5-year survival of less than 1%. Polyamines are small organic polycations that are essential for DNA replication, translation and cell proliferation. Ornithine decarboxylase 1 (ODC1), the rate-limiting enzyme in polyamine synthesis, is irreversibly inhibited by difluoromethylornithine (DFMO). Herein we show that polyamine synthesis is upregulated in DIPG, leading to sensitivity to DFMO. DIPG cells compensate for ODC1 inhibition by upregulation of the polyamine transporter SLC3A2. Treatment with the polyamine transporter inhibitor AMXT 1501 reduces uptake of polyamines in DIPG cells, and co-administration of AMXT 1501 and DFMO leads to potent in vitro activity, and significant extension of survival in three aggressive DIPG orthotopic animal models. Collectively, these results demonstrate the potential of dual targeting of polyamine synthesis and uptake as a therapeutic strategy for incurable DIPG.

Exogenous spermine attenuates diabetic kidney injury inrats by inhibiting AMPK/mTOR signaling pathway.

Diabetic nephropathy (DN) is the primary cause of end-stage renal disease, which is closely associated with dysfunction of the podocytes, the main component of the glomerular filtration membrane; however, the exact underlying mechanism is unknown. Polyamines, including spermine, spermidine and putrescine, have antioxidant and anti-aging properties that are involved in the progression of numerous diseases, but their role in DN has not yet been reported. The present study aimed to explore the role of polyamines in DN, particularly in podocyte injury, and to reveal the molecular mechanism underlying the protective effect of exogenous spermine. Streptozotocin intraperitoneal injection-induced type 1 diabetic (T1D) rat models and high glucose (HG)-stimulated podocyte injury models were established. It was found that in T1D rat kidneys and HG-induced podocytes, ornithine decarboxylase (a key enzyme for polyamine synthesis) was downregulated, while spermidine/spermine N1-acetyltransferase (a key enzyme for polyamines degradation) was upregulated, which suggested that reduction of the polyamine metabolic pool particularly decreased spermine content, is a major factor in DN progression. In addition, hyperglycemia can induce an increased rat kidney weight ratio, serum creatinine, urea, urinary albumin excretion and glomerular cell matrix levels, and promote mesangial thickening and loss or fusion of podocytes. The expression levels of podocyte marker proteins (nephrin, CD2-associated protein and podocin) and autophagy-related proteins [autophagy protein 5, microtube-associated proteins 1A/1B light chain 3 (LC3)II/LC3I, Beclin 1 and phosphorylated (p)-AMPK] were downregulated, while cleaved caspase-3, P62 and p-mTOR were increased. These changes could be improved by pretreatment with exogenous spermine or rapamycin (autophagic agonist). In conclusion, spermine may have the potential to prevent diabetic kidney injury in rats by promoting autophagy via regulating the AMPK/mTOR signaling pathway.

AMD1 is required for the maintenance of leukemic stem cells and promotes chronic myeloid leukemic growth.

Polyamines are critical elements in mammals, but it remains unknown whether adenosyl methionine decarboxylase (AMD1), a rate-limiting enzyme in polyamine synthesis, is required for myeloid leukemia. Here, we found that leukemic stem cells (LSCs) were highly differentiated, and leukemia progression was severely impaired in the absence of AMD1 in vivo. AMD1 was highly upregulated as chronic myeloid leukemia (CML) progressed from the chronic phase to the blast crisis phase, and was associated with the poor prognosis of CML patients. In addition, the pharmacological inhibition of AMD1 by AO476 treatment resulted in a robust reduction of the progression of leukemic cells both in vitro and in vivo. Mechanistically, AMD1 depletion induced loss of mitochondrial membrane potential and accumulation of reactive oxygen species (ROS), resulting in the differentiation of LSCs via oxidative stress and aberrant activation of unfolded protein response (UPR) pathway, which was partially rescued by the addition of polyamine. These results indicate that AMD1 is an essential element in the progression of myeloid leukemia and could be an attractive target for the treatment of the disease.

The rational discovery of multipurpose inhibitors of the ornithine decarboxylase

Metabolic reprograming is a hallmark of cancer, and the polyamine metabolic network is dysregulated in many cancers. Ornithine decarboxylase (ODC) is a rate-limiting enzyme for polyamine synthesis in the polyamine metabolic network. In many cancer cells, ODC is over-expressed, so this enzyme has been an attracting anti-cancer drug target. In the catalysis axis (pathway), ODC converts ornithine to putrescine. Meanwhile, ODC's activity is regulated by protein-protein interactions (PPIs), including the ODC-OAZ1-AZIN1 PPI axis and its monomer-dimer equilibrium. Previous studies showed that when ODC's activity is inhibited, the PPIs might counteract the inhibition efficiency. Therefore, we proposed that multipurpose inhibitors that can simultaneously inhibit ODC's activity and perturb the PPIs would be very valuable as drug candidates and molecular tools. To discover multipurpose ODC inhibitors, we established a computational pipeline by combining positive screening and negative screening. We used this pipeline for the forward screening of multipurpose ligands that might inhibit ODC's activity, block ODC-OAZ1 interaction and enhance ODC non-functional dimerization. With a combination of different experimental assays, we identified three multipurpose ODC inhibitors. At last, we showed that one of these inhibitors is a promising drug candidate. This work demonstrated that our computational pipeline is useful for discovering multipurpose ODC inhibitors, and multipurpose inhibitors would be very valuable. Similar with ODC, there are a lot of proteins in human proteome that act as both enzymes and PPI components. Therefore, this work is not only presenting new molecular tools for polyamine study, but also providing potential insights and protocols for discovering multipurpose inhibitors to target more important protein targets.

Ornithine decarboxylase, the rate-limiting enzyme of polyamine synthesis, modifies brain pathology in a mouse model of tuberous sclerosis complex.

Tuberous sclerosis complex (TSC) is a rare autosomal dominant neurodevelopmental disorder characterized by variable expressivity. TSC results from inactivating variants within the TSC1 or TSC2 genes, leading to constitutive activation of mechanistic target of rapamycin complex 1 signaling. Using a mouse model of TSC (Tsc2-RG) in which the Tsc2 gene is deleted in radial glial precursors and their neuronal and glial descendants, we observed increased ornithine decarboxylase (ODC) enzymatic activity and concentration of its product, putrescine. To test if increased ODC activity and dysregulated polyamine metabolism contribute to the neurodevelopmental defects of Tsc2-RG mice, we used pharmacologic and genetic approaches to reduce ODC activity in Tsc2-RG mice, followed by histologic assessment of brain development. We observed that decreasing ODC activity and putrescine levels in Tsc2-RG mice worsened many of the neurodevelopmental phenotypes, including brain growth and neuronal migration defects, astrogliosis and oxidative stress. These data suggest a protective effect of increased ODC activity and elevated putrescine that modify the phenotype in this developmental Tsc2-RG model.

Inhibition of the polyamine synthesis enzyme ornithine decarboxylase sensitizes triple-negative breast cancer cells to cytotoxic chemotherapy

Treatment of patients with triple-negative breast cancer (TNBC) is limited by a lack of effective molecular therapies targeting this disease. Recent studies have identified metabolic alterations in cancer cells that can be targeted to improve responses to standard-of-care chemotherapy regimens. Using MDA-MB-468 and SUM-159PT TNBC cells, along with LC-MS/MS and HPLC metabolomics profiling, we found here that exposure of TNBC cells to the cytotoxic chemotherapy drugs cisplatin and doxorubicin alter arginine and polyamine metabolites. This alteration was because of a reduction in the levels and activity of a rate-limiting polyamine biosynthetic enzyme, ornithine decarboxylase (ODC). Using gene silencing and inhibitor treatments, we determined that the reduction in ODC was mediated by its negative regulator antizyme, targeting ODC to the proteasome for degradation. Treatment with the ODC inhibitor difluoromethylornithine (DFMO) sensitized TNBC cells to chemotherapy, but this was not observed in receptor-positive breast cancer cells. Moreover, TNBC cell lines had greater sensitivity to single-agent DFMO, and ODC levels were elevated in TNBC patient samples. The alterations in polyamine metabolism in response to chemotherapy, as well as DFMO-induced preferential sensitization of TNBC cells to chemotherapy, reported here suggest that ODC may be a targetable metabolic vulnerability in TNBC.

Exogenous spermine attenuates myocardial fibrosis in diabetic cardiomyopathy by inhibiting endoplasmic reticulum stress and the canonical Wnt signaling pathway.

Myocardial fibrosis is one of the main pathological manifestations of diabetic cardiomyopathy (DCM). Spermine (SPM), a product of polyamine metabolism, plays an important role in many cardiac diseases including hypertrophy, ischemia, and infarction, but its role in diabetic myocardial fibrosis has not been clarified. This study aimed to investigate the role of polyamine metabolism, specifically SPM, in diabetic myocardial fibrosis and to explore the related mechanisms. We used intraperitoneal injection of streptozotocin (STZ, 60 mg/kg) in Wistar rats and high glucose (HG, 40 mM) stimulated cardiac fibroblasts (CFs) to established a type 1 diabetes (T1D) model in vivo and in vitro, which were pretreated with exogenous SPM (5 mg/kg per day and 5 μM). The results showed that hyperglycemia induced the expression of the key polyamine synthesis enzyme ornithine decarboxylase (ODC) decreased and the key catabolic enzyme spermidine/spermine N1 -acetyltransferase (SSAT) increased compared with those in the control group. The body weight, blood insulin level, and cardiac ejection function were decreased, while blood glucose, heart weight, the ratio of heart weight to body weight, myocardial interstitial collagen deposition, and endoplasmic reticulum stress (ERS)-related protein (glucose-regulated protein-78, glucose-regulated protein-94, activating transcription factor-4, and C/EBP homology protein) expression in the T1D group were all significantly increased. HG also caused an increased expression of Wnt3, β-catenin (in cytoplasm and nucleus), while Axin2 and phosphorylated β-catenin decreased. Exogenous SPM improved the above changes caused by polyamine metabolic disorders. In conclusion, polyamine metabolism disorder occurs in the myocardial tissue of diabetic rats, causing myocardial fibrosis and ERS. Exogenous SPM plays a myocardial protective role via inhibiting of ERS and the canonical Wnt/β-catenin signaling pathway.

The Role of Reduced Polyamine Synthesis in Ischemic Stroke

Our aim was to determine the levels of arginine decarboxylase (ADC), ornithine decarboxylase (ODC) and agmatine, ornithine, nitric oxide (NO) and arginase activity in the acute and subacute phases of acute ischemic stroke (AIS), by revealing the possible role of these levels in neuroinflammation and neurogenesis that are postulated to begin after AIS. 35 patients diagnosed with AIS and 35 controls were included in the study. ADC, ODC, agmatinase and NO levels were determined by ELISA kits. Arginase activity and ornithine levels were measured spectrophotometrically. There was no statistically significant difference between the levels of agmatinase, ornithine and arginase activity on days 1 and 7 in the AIS group and the levels measured in the control group (p > 0.05). High ADC and low ODC and NO values were found in the patients compared to the controls in both acute and subacute periods (p < 0.05). It was shown that polyamine synthesis decreased in both acute and subacute periods after AIS; and this was due to the decrease in ODC levels, the rate-regulating step of the major pathway. This reduction may be associated with both the inadequate stimulation of neurogenesis and the immune system with reduced NO synthesis. polyamine synthesis enzymes, ornithine, ornitine decarboxylase, nitric oxide, arginine decarboxylase, agmatine, agmatinase, arginase activity, neuroinflammation, neurogenesis, immune system, acute ischemic stroke.

Polyamine Regulation in Diabetic Breast Cancer Cells

Polyamines (spermine, spermidine, and putrescine) are involved in basic cellular processes such as cell growth, replication, and transcription. They are highly regulated cations that are elevated in cancer. Upregulation of polyamines can thus develop tumors due to hyper‐proliferation of cells as observed in cancers of the colon, skin, prostate and breast. Inhibitors of polyamine pathway could prevent tumor growth as shown in animal models of colon and skin cancer. Intracellular polyamine is regulated by biosynthesis, intestinal uptake and microbial production. Thus inhibitors of synthesis alone have not been very effective as cytotoxic agents but exhibit cytostatic properties. Diabetic conditions further accelerate the growth and metastasis of cancers such as pancreas, breast, liver, and colorectal, however the role of polyamines in such states has not been investigated. We hypothesized that polyamine regulation affects proliferation of breast cancer cells and normal breast epithelial cells in diabetic conditions. Breast cancer cells (MDA‐MB‐231) and normal breast epithelial cells (MCF‐10A) were treated with normal glucose (NG, 5mM) or high glucose (HG, 25 mM) in the presence/absence of polyamine pathway inhibitors. Cells were then treated with polyamine supplements, following depletion with polyamine pathway inhibitors. There was a time dependent increase in cell proliferation with HG from 48–72 hr compared to NG. This was correlated with an increase in polyamines (putrescine, spermidine) though spermine levels were not changed considerably. Simultaneous treatment with an ornithine decarboxylase (polyamine synthesis enzyme) inhibitor, difluoromethylornithine (DFMO) could prevent cell proliferation, restore polyamines, and normalize polyamine catabolic enzymes with high glucose exposure. In conclusion, polyamine regulation is responsible for proliferation of breast cancer cells and normal breast epithelial cells in diabetic states, which can be prevented using inhibitors affecting polyamine levels in cells.Support or Funding Information Funding: Grants from the UNK Undergraduate Research Fellows Program and UNK Research Services Council Seed Grant Equipment: Microplate Reader (Dr. Nuxoll’s lab), Grant from the National Institute for General Medical Science (NIGMS) (5P20GM103427), a component of the National Institutes of Health (NIH)

Searching for Drug Synergy Against Cancer Through Polyamine Metabolism Impairment: Insight Into the Metabolic Effect of Indomethacin on Lung Cancer Cells.

Non-small cell lung cancer (NSCLC) is the most lethal and prevalent type of lung cancer. In almost all types of cancer, the levels of polyamines (putrescine, spermidine, and spermine) are increased, playing a pivotal role in tumor proliferation. Indomethacin, a non-steroidal anti-inflammatory drug, increases the abundance of an enzyme termed spermidine/spermine-N1-acetyltransferase (SSAT) encoded by the SAT1 gene. This enzyme is a key player in the export of polyamines from the cell. The aim of this study was to compare the effect of indomethacin on two NSCLC cell lines, and their combinatory potential with polyamine-inhibitor drugs in NSCLC cell lines. A549 and H1299 NSCLC cells were exposed to indomethacin and evaluations included SAT1 expression, SSAT levels, and the metabolic status of cells. Moreover, the difference in polyamine synthesis enzymes among these cell lines as well as the synergistic effect of indomethacin and chemical inhibitors of the polyamine pathway enzymes on cell viability were investigated. Indomethacin increased the expression of SAT1 and levels of SSAT in both cell lines. In A549 cells, it significantly reduced the levels of putrescine and spermidine. However, in H1299 cells, the impact of treatment on the polyamine pathway was insignificant. Also, the metabolic features upstream of the polyamine pathway (i.e., ornithine and methionine) were increased. In A549 cells, the increase of ornithine correlated with the increase of several metabolites involved in the urea cycle. Evaluation of the levels of the polyamine synthesis enzymes showed that ornithine decarboxylase is increased in A549 cells, whereas S-adenosylmethionine-decarboxylase and polyamine oxidase are increased in H1299 cells. This observation correlated with relative resistance to polyamine synthesis inhibitors eflornithine and SAM486 (inhibitors of ornithine decarboxylase and S-adenosyl-L-methionine decarboxylase, respectively), and MDL72527 (inhibitor of polyamine oxidase and spermine oxidase). Finally, indomethacin demonstrated a synergistic effect with MDL72527 in A549 cells and SAM486 in H1299 cells. Collectively, these results indicate that indomethacin alters polyamine metabolism in NSCLC cells and enhances the effect of polyamine synthesis inhibitors, such as MDL72527 or SAM486. However, this effect varies depending on the basal metabolic fingerprint of each type of cancer cell.

Arginine, ornithine and citrulline supplementation in rainbow trout: Free amino acid dynamics and gene expression responses to bacterial infection

Supplementing the diet with functional ingredients is a key strategy to improve fish performance and health in aquaculture. The amino acids of the urea and nitric oxide (NO) cycles - arginine, ornithine and citrulline - perform crucial roles in the immune response through the generation of NO and the synthesis of polyamine used for tissue repair. We previously found that citrulline supplementation improves and maintains circulating free arginine levels in rainbow trout more effectively than arginine supplementation. Here, to test whether supplementation of urea cycle amino acids modulates the immune response in rainbow trout (Oncorhynchus mykiss), we supplemented a commercial diet with high levels (2% of total diet) of either arginine, ornithine or citrulline during a 7-week feeding trial, before challenging fish with the bacterium Aeromonas salmonicida. We carried out two separate experiments to investigate fish survival and 24 h post-infection to investigate the immediate response of free amino acid levels, and transcriptional changes in genes encoding urea cycle, NO cycle and polyamine synthesis enzymes. There were no differences in percentage fish mortality between diets, however there were numerous highly significant changes in free amino acid levels and gene expression to both dietary supplementation and infection. Out of 26 amino acids detected in blood plasma, 8 were significantly changed by infection and 9 by dietary supplementation of either arginine, ornithine or citrulline. Taurine, glycine and aspartic acid displayed the largest decreases in circulating levels in infected fish, while ornithine and isoleucine were the only amino acids that increased in concentration. We investigated transcriptional responses of the enzymes involved in arginine metabolism in liver and head kidney; transcripts for polyamine synthesis enzymes showed highly significant increases in both tissues across all diets following infection. The paralogous arginase-encoding genes, Arg1a, Arg1b, Arg2a and Arg2b, displayed complex responses across tissues and also due to diet and infection. Overall, these findings improve our understanding of amino acid metabolism following infection and suggests new potential amino acid targets for improving the immune response in salmonids.

Supplementation of arginine, ornithine and citrulline in rainbow trout (Oncorhynchus mykiss): Effects on growth, amino acid levels in plasma and gene expression responses in liver tissue

Functional amino acids (FAA) regulate metabolic pathways directly linked to health, survival, growth and development. Arginine is a FAA with crucial roles in protein deposition and the immune response. In mammals, supplementation of arginine's precursor amino acid, citrulline, is known to increase circulating arginine to levels beyond direct arginine supplementation, however, citrulline supplementation is poorly studied in fish. To address this knowledge gap, we supplemented the diet of rainbow trout with arginine and its precursor amino acids, ornithine and citrulline, at 3 levels (0.5%, 1% and 2% of the total diet) during a 14-week experiment. We sampled fish at 3 h and 24 h post-feeding to investigate immediate and steady-state effects, respectively. There were no differences in fish growth for any of the diets across a range of indicators. In blood plasma, out of 26 amino acids detected, 11 and 6 displayed significant changes 24 h and 3 h post-prandial, respectively. Arginine, ornithine and citrulline levels were all significantly increased by the citrulline supplemented diets. In muscle, 8 amino acids were significantly altered by supplemented diets, while there were no significant changes in liver. Arginine was increased by 2% citrulline supplementation in muscle tissue. We also investigated the transcriptional responses of urea cycle, nitric oxide cycle and rate-limiting polyamine synthesis enzymes, related to arginine's metabolism, in liver. At both time points, only 2 enzymes were significantly altered by the supplemented diets, however several significant changes were observed comparing 3 h and 24 h post-prandial expression levels. Of these, the paralogous polyamine synthesis enzyme encoding genes ODC1 and ODC2 displayed the largest increases in 3 h post-prandial fish. These findings demonstrate that endogenous synthesis of arginine is possible from a citrulline supplemented diet and improve our understanding of arginine metabolism in fish.