Intracellular Polyamine Content Research Articles

Polyamine Inhibitor SAM486A Augments Cytarabine Cytotoxicity in Methylthioadenosine Phosphorylase-deficient Leukemia Cells.

Methionine metabolism contributes to supplying sulfur-containing amino acids, controlling the methyl group transfer reaction, and producing polyamines in cancer cells. Polyamines play important roles in various cellular functions. Methylthioadenosine phosphorylase (MTAP), the key enzyme of the methionine salvage pathway, is reported to be deficient in 15-62% of cases of hematological malignancies. MTAP-deficient cancer cells accumulate polyamines, resulting in enhanced cell proliferation. The aim of this study was to investigate the combined effects of the polyamine synthesis inhibitor SAM486A and the anticancer antimetabolite cytarabine in MTAP-deficient leukemic cells in vitro. The leukemia cell line U937 and the subline, U937/MTAP(-), in which MTAP was knocked down by shRNA, were used. The experiments were performed in media supplemented with 20% methionine (low methionine), which was the minimum concentration for maintaining cellular viability. The knockdown efficiency test confirmed a 70% suppression of the expression of the MTAP gene in U937/MTAP(-) cells. Even in the media with low methionine, the intracellular methionine concentration was not reduced in U937/MTAP(-) cells, suggesting that the minimum supply of methionine was sufficient to maintain intracellular levels of methionine. Both U937/MTAP(+) and U937/MTAP(-) cells were comparably sensitive to anticancer drugs (cytarabine, methotrexate, clofarabine and 6-thioguanine). The combination of SAM486A and cytarabine was demonstrated to have synergistic cytotoxicity in U937/MTAP(-) cells with regard to cell growth inhibition and apoptosis induction, but not in U937/MTAP(+) cells. Mechanistically, SAM486A altered the intracellular polyamine concentrations and reduced the antiapoptotic proteins. Methionine metabolism and polyamine synthesis can be attractive therapeutic targets in leukemia.

Spermidine improves angiogenic capacity of senescent endothelial cells, and enhances ischemia-induced neovascularization in aged mice

Aging is closely associated with the increased morbidity and mortality of ischemic cardiovascular disease, at least partially through impaired angiogenic capacity. Endothelial cells (ECs) play a crucial role in angiogenesis, and their angiogenic capacity declines during aging. Spermidine is a naturally occurring polyamine, and its dietary supplementation has exhibited distinct anti-aging and healthy lifespan-extending effects in various species such as yeast, worms, flies, and mice. Here, we explore the effects of spermidine supplementation on the age-related decline in angiogenesis both in vitro and in vivo. Intracellular polyamine contents were reduced in replicative senescent ECs, which were subsequently recovered by spermidine supplementation. Our findings reveal that spermidine supplementation improved the declined angiogenic capacity of senescent ECs, including migration and tube-formation, without affecting the senescence phenotypes. Mechanistically, spermidine enhanced both autophagy and mitophagy, and improved mitochondrial quality in senescent ECs. Ischemia-induced neovascularization was assessed using the hind-limb ischemia model in mice. Limb blood flow recovery and neovascularization in the ischemic muscle were considerably impaired in aged mice compared to young ones. Of note, dietary spermidine significantly enhanced ischemia-induced angiogenesis, and improved the blood flow recovery in the ischemic limb, especially in aged mice. Our results reveal novel proangiogenic functions of spermidine, suggesting its therapeutic potential against ischemic disease.

Abstract 13491: Spermidine Improves Angiogenic Capacity in Senescent Endothelial Cells and Enhances Ischemia Induced Neovascularization in Aged Mice

Aging is closely associated with high morbidity and mortality of various diseasesincluding cardiovascular disease. Cellular senescence has been cruciallyinvolved in the mechanisms underlying age-related diseases, and vascularendothelial cells <ECs> become senescent during aging. Spermidine, a naturallyoccurring polyamine, is an emerging organic compound that shows various anti-aging effects. Serum spermidine concentration decreases during aging inhuman, and dietary spermidine has been reported to extend healthy lifespanand delay the age-related decline in cognitive and physical performance inmany species such as yeast, worms, flies, and mice. Here, we explored aneffect of spermidine on age-associated decline in angiogenesis. Replicativesenescent ECs were prepared by culturing human ECs for an extended perioduntil passage 18-20, and cellular senescence was confirmed by loss ofproliferation capacity, increased CDK inhibitors expression, and senescence-associated beta-Gal activity. Intracellular polyamine contents were significantlyreduced in replicative senescent ECs, which was recovered by spermidinesupplementation. Spermidine supplementation improved the declinedangiogenic capacities including tube-formation on Matrigel in senescent ECs,while senescence features such as reduced proliferation and senescence-associated beta-Gal activity were not affected. Mechanistically, spermidinesupplementation accelerated the static autophagy flux, and improvedmitochondrial quality, e.g. reduced mitochondrial ROS and preservedmitochondrial membrane potential, in senescent ECs. Ischemia-inducedneovascularization was assessed using the hind-limb ischemia model in youngand aged mice. Blood flow recovery and neovascularization in ischemic musclewere considerably impaired in aged mice comparing to those in young mice. Ofnote, dietary spermidine significantly enhanced ischemia-induced angiogenesis,and improved the blood flow recovery in the ischemic limb, especially in agedmice. Our results reveal new mechanistic insights in anti-aging property ofspermidine, and suggest its therapeutic potential against ischemiccardiovascular disease, particularly in elderly population.

Understanding the Polyamine and mTOR Pathway Interaction in Breast Cancer Cell Growth.

The polyamines putrescine, spermidine and spermine are nutrient-like polycationic molecules involved in metabolic processes and signaling pathways linked to cell growth and cancer. One important pathway is the PI3K/Akt pathway where studies have shown that polyamines mediate downstream growth effects. Downstream of PI3K/Akt is the mTOR signaling pathway, a nutrient-sensing pathway that regulate translation initiation through 4EBP1 and p70S6K phosphorylation and, along with the PI3K/Akt, is frequently dysregulated in breast cancer. In this study, we investigated the effect of intracellular polyamine modulation on mTORC1 downstream protein and general translation state in two breast cancer cell lines, MCF-7 and MDA-MB-231. The effect of mTORC1 pathway inhibition on the growth and intracellular polyamines was also measured. Results showed that polyamine modulation alters 4EBP1 and p70S6K phosphorylation and translation initiation in the breast cancer cells. mTOR siRNA gene knockdown also inhibited cell growth and decreased putrescine and spermidine content. Co-treatment of inhibitors of polyamine biosynthesis and mTORC1 pathway induced greater cytotoxicity and translation inhibition in the breast cancer cells. Taken together, these data suggest that polyamines promote cell growth in part through interaction with mTOR pathway. Similarly intracellular polyamine content appears to be linked to mTOR pathway regulation. Finally, dual inhibition of polyamine and mTOR pathways may provide therapeutic benefits in some breast cancers.

The Effects of Indole on Intracellular Polyamine Content and Antibiotic Susceptibility of Escherichia coli

The Effects of Indole on Intracellular Polyamine Content and Antibiotic Susceptibility of Escherichia coli

Tpo3 and dur3, Aspergillus fumigatus Plasma Membrane Regulators of Polyamines, Regulate Polyamine Homeostasis and Susceptibility to Itraconazole.

Aspergillus fumigatus is a well-known opportunistic pathogen that causes invasive aspergillosis (IA) infections, which have high mortality rates in immunosuppressed individuals. Long-term antifungal drug azole use in clinical treatment and agriculture results in loss of efficacy or drug resistance. Drug resistance is related to cellular metabolites and the corresponding gene transcription. In this study, through untargeted metabolomics and transcriptomics under itraconazole (ITC) treatment, we identified two plasma membrane-localized polyamine regulators tpo3 and dur3, which were important for polyamine homeostasis and susceptibility to ITC in A. fumigatus. In the absence of tpo3 and/or dur3, the levels of cytoplasmic polyamines had a moderate increase, which enhanced the tolerance of A. fumigatus to ITC. In comparison, overexpression of tpo3 or dur3 induced a drastic increase in polyamines, which increased the sensitivity of A. fumigatus to ITC. Further analysis revealed that polyamines concentration-dependently affected the susceptibility of A. fumigatus to ITC by scavenging reactive oxygen species (ROS) at a moderate concentration and promoting the production of ROS at a high concentration rather than regulating drug transport. Moreover, inhibition of polyamine biosynthesis reduced the intracellular polyamine content, resulted in accumulation of ROS and enhanced the antifungal activity of ITC. Interestingly, A. fumigatus produces much lower levels of ROS under voriconazole (VOC) treatment than under ITC-treatment. Accordingly, our study established the link among the polyamine regulators tpo3 and dur3, polyamine homeostasis, ROS content, and ITC susceptibility in A. fumigatus.

Upregulation of Polyamine Transport in Human Colorectal Cancer Cells

Polyamines are essential growth factors that have a positive role in cancer cell growth. Their metabolic pathway and the diverse enzymes involved have been studied in depth in multiple organisms and cells. Polyamine transport also contributes to the intracellular polyamine content but this is less well-studied in mammalian cells. As the polyamine transporters could provide a means of selective drug delivery to cancer cells, a greater understanding of polyamine transport and its regulation is needed. In this study, transport of polyamines and polyamine content was measured and the effect of modulating each was determined in human colorectal cancer cells. The results provide evidence that upregulation of polyamine transport depends on polyamine depletion and on the rate of cell growth. Polyamine transport occurred in all colorectal cancer cell lines tested but to varying extents. The cell lines with the lowest basal uptake showed the greatest increase in response to polyamine depletion. Kinetic parameters for putrescine and spermidine suggest the existence of two separate transporters. Transport was shown to be a saturable but non-polarised process that can be regulated both positively and negatively. Using the polyamine transporter to deliver anticancer drugs more selectively is now a reality, and the ability to manipulate the polyamine transport process increases the possibility of using these transporters therapeutically.

РОЛЬ ПОЛИАМИНОВ В ОГРАНИЧЕНИИ ПРОНИЦАЕМОСТИ ВНЕШНЕЙ МЕМБРАНЫ ESCHERICHIA COLI ДЛЯ АНТИБИОТИКОВ

Various hydrophilic antibiotics enter the cells of gram-negative bacteria through porin channels, which can be blocked by polyamine cadaverine. In this paper, we studied the rate of porin-mediated transport and the intracellular concentration of the main biogenic polyamines of Escherichia coli cells subjected to fluoroquinolone antibiotics depending on the exposure time. Porin permeability sharply decreased in the first two hours of antibiotic exposure, after that the transport rate continued to decline, but at a lower rate. Free polyamines accumulated by the second hour of the exposure, then putrescine and spermidine de-clined, and the concentration of cadaverine continued to grow. Activation of micF gene, a product of which is involved in the negative control of ompF porin gene expression, was observed after the two hours of antibiotic exposure. It could be supposed that the inhibition of porin-mediated transport of anti-biotics by blocking pores precedes the mechanisms regulating the number of porins. The paper discusses the probable specialization of polyamines in the regulation of porin-mediated transport of antibiotics.

Polyamines Deficient Diet is a Complementary Strategy in Fighting Cancer: Analysis on Selected Medicinal Fruits

Polyamines are one of the oldest substances known in biochemistry consist of putrescine, spermidine and spermine. They are essential components of all living cells primarily involve in cell growth and proliferation. Upregulation of polyamines in human body is highly associated with various diseases, including cancer. Therefore, polyamines deficient diet (PDD) has become one of the strategies to inhibit carcinogenesis. This study was aimed to determine the polyamines content in selected anticancer fruits and their effect on polyamine polyamine metabolic enzymes was explored in human lung adenocarcinoma cells (A549). In this study, the selected medicinal fruits are Phoenix dactylifera (ajwa dates),Ficus auriculata (fig),Beta vulgaris (beetroot), Ziziphus jujube (jujube) and Vitis vinifera (raisin). The selected medicinal fruits were initially grouped into classes based on total polyamines content using High performance Liquid Chromatography (HPLC). The ability of selected fruits to reduce A549 intracellular polyamines was determined using the same technique. Accordingly, changes in polyamine metabolic enzymes activities; ornithine decarboxylase (ODC) and spermidine/spermine N-acetyltransferase (SSAT) were measured correspondingly using quantitative polymerase chain reactions (qPCR). Results suggested Beetroot, ajwa dates, fig and raisin are classified as low polyamines fruits while jujube demonstrates high content of polyamines. Significant decreased of total intracellular polyamine content after 24 h of treatments with these fruits were identified when compared with untreated A549 cells.Ornithine decarboxylase (ODC) activity showed downregulation upon treatment with ajwa dates, jujube and raisin while SSAT activity displayed alterations in beetroot and jujube treated A549 cells. Thus, we concluded that beetroot, ajwa dates, fig and raisins are the promising candidates for PDD strategy for their low polyamines, ability to reduce A549 intracellular polyamines and modification on ODC and SSAT activities.

Polyamines: The possible missing link between mental disorders and epilepsy (Review).

Polyamines are small positively charged alkylamines that are essential in a number of crucial eukaryotic processes, like normal cell growth and development. In normal physiological conditions, intracellular polyamine content is tightly regulated through a fine regulated network of biosynthetic and catabolic enzymes and a transport system. The dysregulation of this network is frequently associated to different tumors, where high levels of polyamines has been detected. Polyamines also modulate ion channels and ionotropic glutamate receptors and altered levels of polyamines have been observed in different brain diseases, including mental disorders and epilepsy. The goal of this article is to review the role of polyamines in mental disorders and epilepsy within a frame of the possible link between these two brain pathologies. The high comorbidity between these two neurological illnesses is strongly suggestive that they share a common background in the central nervous system. This review proposes an additional association between the noradrenalin/serotonin and glutamatergic neuronal circuits with polyamines. Polyamines can be considered supplementary defensive shielding molecules, important to protect the brain from the development of epilepsy and mental illnesses that are caused by different types of neurons. In this contest, the modulation of polyamine metabolism may be a novel important target for the prevention and therapeutic treatment of these diseases that have a high impact on the costs of public health and considerably affect quality of life.

Changes in intracellular and extracellular free polyamines during the growth cycle of Prorocentrum donghaiense

We used HPLC to measure the concentrations of free putrescine, spermidine, and spermine in cells and culture medium during the growth of Prorocentrum donghaiense. Spermine was the main intracellular free polyamines in Prorocentrum donghaiense. The intracellular concentration of free polyamines kept increasing during the growth cycle and the ratio of spermine to putrescine increased with cell density. Changes in the ratios of spermine/spermidine in Prorocentrum donghaiense, however, occurred before changes in cell density, suggesting that polyamines can regulate cell growth and replication. Putrescine was the most abundant free polyamine in the culture medium, followed by spermine and spermidine. Changes to the media polyamines contents implied that Prorocentrum donghaiense could absorb free polyamines from the culture solution as well as release free polyamines into the medium during different growth periods, especially during the decline phase. These results suggest that masses of dead algae in the population decline phase would release abundant free polyamines into the environment.

Agmatine as a potential therapeutic intervention in bipolar depression: the preclinical landscape

ABSTRACTIntroduction: Present antidepressant treatments are only helpful in a quarter of patients with bipolar depression, and new strategies are warranted. Increasing evidence suggests that accelerated polyamine metabolism is associated with the pathophysiology of depression. Polyamines regulate stress responses, inflammation, and neuronal signaling in the central and enteric nervous system. Agmatine is a promising target of altered polyamine metabolism considering its unique ability to regulate intracellular polyamine content and neuroprotective effects.Areas covered: This review discusses the polyamine system and its relationship to the central and enteric nervous system, focusing on results from preclinical studies supporting the relationship between agmatine and the pathophysiology of depression. We also discussed the main mechanisms underlying the antidepressant and neuroprotective effects of agmatine.Expert opinion: Our review points out the possible relationship between polyamines and the pathophysiology of depression. It discusses the efficacy of agmatine in several models of depressive-like behaviour, and suggests that it may prove to be an efficacious adjunctive treatment in bipolar depression. Furthermore, it discusses a proposed pathway linking systemic inflammation, observed in a subset of bipolar disorder patients, to abnormal polyamine metabolism and associated changes in the epithelial gut barrier and blood-brain barrier.

Knocking out Ornithine Decarboxylase Antizyme 1 (OAZ1) Improves Recombinant Protein Expression in the HEK293 Cell Line.

Creating efficient cell lines is a priority for the biopharmaceutical industry, which produces biologicals for various uses. A recent approach to achieving this goal is the use of non-coding RNAs, microRNA (miRNA) and small interfering RNA (siRNA), to identify key genes that can potentially improve production or growth. The ornithine decarboxylase antizyme 1 (OAZ1) gene, a negative regulator of polyamine biosynthesis, was identified in a genome-wide siRNA screen as a potential engineering target, because its knock down by siRNA increased recombinant protein expression from human embryonic kidney 293 (HEK293) cells by two-fold. To investigate this further, the OAZ1 gene in HEK293 cells was knocked out using CRISPR genome editing. The OAZ1 knockout cell lines displayed up to four-fold higher expression of both stably and transiently expressed proteins, with comparable growth and metabolic activity to the parental cell line; and an approximately three-fold increase in intracellular polyamine content. The results indicate that genetic inactivation of OAZ1 in HEK293 cells is an effective strategy to improve recombinant protein expression in HEK293 cells.

Reduction of Spermidine Content Resulting from Inactivation of Two Arginine Decarboxylases Increases Biofilm Formation in Synechocystis sp. Strain PCC 6803.

The phototropic bacterium Synechocystis sp. strain PCC 6803 is able to adapt its morphology in order to survive in a wide range of harsh environments. Under conditions of high salinity, planktonic cells formed cell aggregates in culture. Further observations using crystal violet staining, confocal laser scanning microscopy, and field emission-scanning electron microscopy confirmed that these aggregates were Synechocystis biofilms. Polyamines have been implicated in playing a role in biofilm formation, and during salt stress the content of spermidine, the major polyamine in Synechocystis, was reduced. Two putative arginine decarboxylases, Adc1 and Adc2, in Synechocystis were heterologously expressed in Escherichia coli and purified. Adc2 had high arginine decarboxylase activity, whereas Adc1 was much less active. Disruption of the adc genes in Synechocystis resulted in decreased spermidine content and formation of biofilms even under nonstress conditions. Based on the characterization of the adc mutants, Adc2 was the major arginine decarboxylase whose activity led to inhibition of biofilm formation, and Adc1 contributed only minimally to the process of polyamine synthesis. Taken together, in Synechocystis the shift from planktonic lifestyle to biofilm formation was correlated with a decrease in intracellular polyamine content, which is the inverse relationship of what was previously reported in heterotroph bacteria.IMPORTANCE There are many reports concerning biofilm formation in heterotrophic bacteria. In contrast, studies on biofilm formation in cyanobacteria are scarce. Here, we report on the induction of biofilm formation by salt stress in the model phototrophic bacterium Synechocystis sp. strain PCC 6803. Two arginine decarboxylases (Adc1 and Adc2) possess function in the polyamine synthesis pathway. Inactivation of the adc1 and adc2 genes leads to biofilm formation even in the absence of salt. The shift from planktonic culture to biofilm formation is regulated by a decrease in spermidine content in Synechocystis This negative correlation between biofilm formation and polyamine content, which is the opposite of the relationship reported in other bacteria, is important not only in autotrophic but also in heterotrophic bacteria.

The Role of Cadaverine Synthesis on Pneumococcal Capsule and Protein Expression.

Invasive infections caused by Streptococcus pneumoniae, a commensal in the nasopharynx, pose significant risk to human health. Limited serotype coverage by the available polysaccharide-based conjugate vaccines coupled with increasing incidence of antibiotic resistance complicates therapeutic strategies. Bacterial physiology and metabolism that allows pathogens to adapt to the host are a promising avenue for the discovery of novel therapeutics. Intracellular polyamine concentrations are tightly regulated by biosynthesis, transport and degradation. We previously reported that deletion of cadA, a gene that encodes for lysine decarboxylase, an enzyme that catalyzes cadaverine synthesis results in an attenuated phenotype. Here, we report the impact of cadA deletion on pneumococcal capsule and protein expression. Our data show that genes for polyamine biosynthesis and transport are downregulated in ∆cadA. Immunoblot assays show reduced capsule in ∆cadA. Reduced capsule synthesis could be due to reduced transcription and availability of precursors for synthesis. The capsule is the predominant virulence factor in pneumococci and is critical for evading opsonophagocytosis and its loss in ∆cadA could explain the reported attenuation in vivo. Results from this study show that capsule synthesis in pneumococci is regulated by polyamine metabolism, which can be targeted for developing novel therapies.

Biochemical characterization and homology modeling of polyamine oxidase from cyanobacterium Synechocystis sp. PCC 6803

The intracellular polyamine contents are regulated not only by polyamine biosynthesis and transport but also by polyamine degradation catalyzed by copper-dependent amine oxidase (DAO) and FAD-dependent polyamine oxidase (PAO). The genome sequence of Synechocystis sp. PCC 6803 reveals the presence of at least one putative polyamine oxidase gene, slr5093. The open reading frame of slr5093 encoding Synechocystis polyamine oxidase (SynPAO, E.C. 1.5.3.17) was expressed in Escherichia coli. The purified recombinant enzyme had the characteristic absorption spectrum of a flavoprotein with absorbance peaks at 380 and 450 nm. The optimum pH and temperature for the oxidation of both spermidine and spermine are 8.5 and 30 °C, respectively. The enzyme catalyzed the conversion of spermine and spermidine to spermidine and putrescine, respectively, with higher catalytic efficiency when spermine served as substrate. These results suggest that SynPAO is a polyamine oxidase involved in a polyamine back-conversion pathway. Based on the structural analysis, Gln94, Tyr403 and Thr440 in SynPAO are predicted to be important residues in the active site.

Abstract 5485: The combined effects of polyamine and GSH inhibition on the viability of human cancer cell lines

Abstract Polyamines play essential roles in DNA stability and cell proliferation, making polyamine metabolism a potential target for cancer therapeutics. The spermine analog N1,N11- Diethylnorspermine (DenSpm) reduces levels of ODC, the enzyme responsible for polyamine biosynthesis, and also induces the enzyme SSAT, responsible for the catabolism of polyamines. Polyamines have been shown to inactivate reactive oxygen species (ROS), including peroxides, protecting mammalian cells from oxidative damage to nucleic acids. The catabolism of polyamines by SSAT, in contrast, leads to the production of ROS, which results in cellular death and limits the progression of cancer. Buthionine sulfoximine (BSO) selectively depletes stores of intracellular glutathione GSH, an antioxidant that neutralizes ROS. We hypothesized that treating cancer cell lines with both DenSpm and BSO will have a synergistic cytotoxic effect on the cells, due to the depletion of ROS-scavenging polyamines, the generation of ROS caused by DenSpm and the absence of GSH. Treatment of the four cell lines, MCF-7, MDA-MD-231, Caco-2 and PANC-1, with DenSpm resulted in moderate reductions in cell viability as measured using the Cell Titer Blue assay (Promega, Inc.). As has been widely reported in other mammalian systems, DenSpm (10 µM) reduced intracellular polyamine concentrations (HPLC of benzoylated polyamine derivatives) and induced SSAT (Western Blots). Treatment of cells with BSO (500 µM) caused a significant decrease in intracellular GSH levels. Similar findings have been reported in previous studies with human neuroblastoma lines (Anderson, et al. 1999). The combination of BSO and DenSpm had effects markedly greater than either compound alone. Pretreatment of cells with BSO (500 µM) for 24 hours with subsequent removal of BSO before DenSpm (10, 100, 1000 µM) addition resulted in significantly reduced viability in all cell lines. DenSpm was found to be most effective in the continued presence of BSO as compared to a 24-hour BSO pretreatment. The compounds used in combination also enhanced ROS production, further demonstrating their synergistic effects. Citation Format: John Grady, Davey Holzer, Kaci Keleher, Emma Meetz, Russ Feirer. The combined effects of polyamine and GSH inhibition on the viability of human cancer cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5485. doi:10.1158/1538-7445.AM2017-5485

Trypanocidal Effect of Isotretinoin through the Inhibition of Polyamine and Amino Acid Transporters in Trypanosoma cruzi.

Polyamines are essential compounds to all living organisms and in the specific case of Trypanosoma cruzi, the causative agent of Chagas disease, they are exclusively obtained through transport processes since this parasite is auxotrophic for polyamines. Previous works reported that retinol acetate inhibits Leishmania growth and decreases its intracellular polyamine concentration. The present work describes a combined strategy of drug repositioning by virtual screening followed by in vitro assays to find drugs able to inhibit TcPAT12, the only polyamine transporter described in T. cruzi. After a screening of 3000 FDA-approved drugs, 7 retinoids with medical use were retrieved and used for molecular docking assays with TcPAT12. From the docked molecules, isotretinoin, a well-known drug used for acne treatment, showed the best interaction score with TcPAT12 and was selected for further in vitro studies. Isotretinoin inhibited the polyamine transport, as well as other amino acid transporters from the same protein family (TcAAAP), with calculated IC50 values in the range of 4.6–10.3 μM. It also showed a strong inhibition of trypomastigote burst from infected cells, with calculated IC50 of 130 nM (SI = 920) being significantly less effective on the epimastigote stage (IC50 = 30.6 μM). The effect of isotretinoin on the parasites plasma membrane permeability and on mammalian cell viability was tested, and no change was observed. Autophagosomes and apoptotic bodies were detected as part of the mechanisms of isotretinoin-induced death indicating that the inhibition of transporters by isotretinoin causes nutrient starvation that triggers autophagic and apoptotic processes. In conclusion, isotretinoin is a promising trypanocidal drug since it is a multi-target inhibitor of essential metabolites transporters, in addition to being an FDA-approved drug largely used in humans, which could reduce significantly the requirements for its possible application in the treatment of Chagas disease.

Arabidopsis Polyamine oxidase-2 uORF is required for downstream translational regulation

In eukaryotic mRNAs, small upstream open reading frames (uORFs) located in the 5′-untranslated region control the translation of the downstream main ORF. Polyamine oxidase (PAO) enzymes catalyze the oxidation of higher polyamines such as spermidine and spermine, and therefore contribute to the maintenance of intracellular polyamine content and to the regulation of physiological processes through their catabolic products. Recently, we reported that the Arabidopsis thaliana Polyamine Oxidase 2 (AtPAO2) is post-transcriptionally regulated by its 5′-UTR region through an uORF. In the present study, we analyzed whether the translation of the uORF is needed for the translational repression of the main ORF, and whether the inactivation of the uORF had an effect on the translational control mediated by polyamines. To this aim, we generated diverse single mutations in the uORF sequence; these mutant 5′-UTRs were fused to the GUS reporter gene, and tested in onion monolayer cells and A. thaliana transgenic seedlings. Removal of the start codon or introduction of a premature stop codon in the AtPAO2 uORF sequence abolished the negative regulation of the GUS expression exerted by the wild-type AtPAO2 uORF. An artificial uORF (32 amino acids in length) generated by the addition of a single nucleotide in AtPAO2 uORF proved to be less repressive than the wild-type uORF. Thus, our findings suggest that translation of the AtPAO2 uORF is necessary for the translational repression of the main ORF.

Intracellular sources of ornithine for polyamine synthesis in endothelial cells.

Polyamines are essential for proliferation of endothelial cells (EC)and angiogenesis. This study was conducted to identify the metabolic source(s) of ornithine for polyamine synthesis in EC, using N(ω)-hydroxy-nor-L-arginine (Nor-NOHA, an inhibitor of arginase) and gabaculine (an inhibitor of ornithine aminotransferase; OAT). Nor-NOHA inhibited arginase with an IC50 value of 10µM for intact EC. Nor-NOHA (0.5mM) alone inhibited arginase activity in EC by 98%, increased total cellular concentrations of arginine by 14%, and decreased total cellular concentrations of ornithine, putrescine and spermidine by 17, 65 and 74%, respectively. Arginine and glutamine contributed to 73 and 26% of the ornithine produced by EC, respectively. Gabaculine (1mM) alone decreased the total cellular concentrations of arginine, ornithine, putrescine, and spermidine by 14, 96, 32, and 42%, respectively. A combination of both Nor-NOHA and gabaculine completely blocked ornithine production in EC, resulting in no detectable cellular ornithine and almost complete depletion of cellular putrescine and spermidine. Addition of 0.5mM ornithine restored the intracellular concentrations of polyamines in EC treated with Nor-NOHA plus gabaculine, indicating that Nor-NOHA and gabaculine did not inhibit ornithine decarboxylase activity. Our results suggest that the arginase and OAT pathways are the exclusive sources of ornithine in EC when there is little extracellular ornithine and that there is intracellular compartmentalization of arginine and ornithine for endothelial synthesis of polyamines. These novel findings may have important implications for improving placental vascular growth, wound healing, and cancer therapy.