Inhibition Of Ornithine Decarboxylase Activity Research Articles

Characterization of an Arginine Decarboxylase from Streptococcus pneumoniae by Ultrahigh-Performance Liquid Chromatography-Tandem Mass Spectrometry.

Polyamines are polycations derived from amino acids that play an important role in proliferation and growth in almost all living cells. In Streptococcus pneumoniae (the pneumococcus), modulation of polyamine metabolism not only plays an important regulatory role in central metabolism, but also impacts virulence factors such as the capsule and stress responses that affect survival in the host. However, functional annotation of enzymes from the polyamine biosynthesis pathways in the pneumococcus is based predominantly on computational prediction. In this study, we cloned SP_0166, predicted to be a pyridoxal-dependent decarboxylase, from the Orn/Lys/Arg family pathway in S. pneumoniae TIGR4 and expressed and purified the recombinant protein. We performed biochemical characterization of the recombinant SP_0166 and confirmed the substrate specificity. For polyamine analysis, we developed a simultaneous quantitative method using hydrophilic interaction liquid chromatography (HILIC)-based liquid chromatography-tandem mass spectrometry (LC-MS/MS) without derivatization. SP_0166 has apparent Km, kcat, and kcat/Km values of 11.3 mM, 715,053 min-1, and 63,218 min-1 mM-1, respectively, with arginine as a substrate at pH 7.5. We carried out inhibition studies of SP_0166 enzymatic activity with arginine as a substrate using chemical inhibitors DFMO and DFMA. DFMO is an irreversible inhibitor of ornithine decarboxylase activity, while DFMA inhibits arginine decarboxylase activity. Our findings confirm that SP_0166 is inhibited by DFMA and DFMO, impacting agmatine production. The use of arginine as a substrate revealed that the synthesis of putrescine by agmatinase and N-carbamoylputrescine by agmatine deiminase were both affected and inhibited by DFMA. This study provides experimental validation that SP_0166 is an arginine decarboxylase in pneumococci.

Pharmacological targeting of polyamine and hypusine biosynthesis reduces tumour activity of endometrial cancer

Endometrial cancer (EC) is a common and deadly cancer in women and novel therapeutic approaches are urgently needed. Polyamines (putrescine, spermidine, spermine) are critical for mammalian cell proliferation and MYC coordinately regulates polyamine metabolism through ornithine decarboxylase (ODC). ODC is a MYC target gene and rate-limiting enzyme of polyamine biosynthesis and the FDA-approved anti-protozoan drug α-difluoromethylornithine (DFMO) inhibits ODC activity and induces polyamine depletion that leads to tumour growth arrest. Spermidine is required for the hypusine-dependent activation of eukaryotic translation initiation factors 5A1 (eIF5A1) and 5A2 (eIF5A2) and connects the MYC/ODC-induced deregulation of spermidine to eIF5A1/2 protein translation, which is increased during cancer cell proliferation. We show that eIF5A1 is significantly upregulated in EC cells compared to control cells (p=.000038) and that combined pharmacological targeting of ODC and eIF5A hypusination with cytostatic drugs DFMO and N1-guanyl-1,7-diaminoheptane (GC7), respectively, reduces eIF5A1 activation and synergistically induces apoptosis in EC cells. In vivo, DFMO/GC7 suppressed xenografted EC tumour growth in mice more potently than each drug alone compared to control (p=.002) and decreased putrescine (p=.045) and spermidine levels in tumour tissues. Our data suggest DFMO and GC7 combination therapy may be useful in the treatment or prevention of EC.

Optimization of a cucurbit[6]uril-based real-time label-free method for analyzing the activity of ornithine decarboxylase

Ornithine decarboxylase (ODC) is a key enzyme in the biosynthetic pathway of polyamines and catalyzes the decarboxylation of ornithine to produce putrescine. Inhibition of ODC activity is a potential approach for the prevention and treatment of many diseases including cancer, as the expression levels and the activities of ODC in many abnormal cells and tumor cells are generally higher than those of normal cells. The discovery and evaluation of ODC inhibitors rely on the monitoring of the reaction processes catalyzed by ODC. There are several commonly used methods for analyzing the activity of ODC, such as measuring the yield of putrescine by high performance liquid chromatography, or quantifying the yield of isotope labelled carbon dioxide. However, the cumbersome operation and cost of these assays, as well as the difficulty to achieve high-throughput and real-time detection, hampered their applications. In this work, we optimized a real-time label-free method for analyzing the activity of ODC based on the macromolecule cucurbit[6]uril (CB6) and a fluorescent dye, DSMI (trans-4-[4-(dimethylamino) styryl]-1-methylpyridinium iodide). Finally, the optimized method was used to determine the activities of different ODC inhibitors with different inhibition mechanisms.

Refractive Index Sensing for Measuring Single Cell Growth.

Accessing cell growth on adhesive substrates is critical for identifying biophysical properties of cells and their therapeutic response to drug therapies. However, optical techniques have low sensitivity, and their reliability varies with cell type, whereas microfluidic technologies rely on cell suspension. In this paper, we introduced a plasmonic functional assay platform that can precisely measure cell weight and the dynamic change in real-time for adherent cells. Possessing this ability, our platform can determine growth rates of individual cells within only 10 min to map the growth profile of populations in short time intervals. The platform could successfully determine heterogeneity within the growth profile of populations and assess subpopulations exhibiting distinct growth profiles. As a proof of principle, we investigated the growth profile of MCF-7 cells and the effect of two intracellular metabolisms critical for their proliferation. We first investigated the negative effect of serum starvation on cell growth. We then studied ornithine decarboxylase (ODC) activity, a key enzyme which is involved in proliferation, and degraded under low osmolarity that inhibits cell growth. We successfully determined the significant distinction between growth profiles of MCF-7 cells and their ODC-overproducing variants that possess strong resistance to the negative effects of low osmolarity. We also demonstrated that an exogenous parameter, putrescine, could rescue cells from ODC inhibition under hypoosmotic conditions. In addition to the ability of accessing intracellular activities through ex vivo measurements, our platform could also determine therapeutic behaviors of cancer cells in response to drug treatments. Here, we investigated difluoromethylornithine (DFMO), which has antitumor effects on MCF-7 cells by inhibiting ODC activity. We successfully demonstrated the susceptibility of MCF-7 cells to such drug treatment, while its DFMO-resistant subpopulation could survive in the presence of this antigrowth agent. By rapidly determining cell growth kinetics in small samples, our plasmonic platform may be of broad use to basic research and clinical applications.

Arginine Metabolism and Its Potential in Treatment of Colorectal Cancer.

Colorectal cancer is the leading cause of death from cancer globally. The current treatment protocol still heavily relies on early detection and surgery. The molecular mechanisms underlying development of colorectal cancer are clinically important and determine the prognosis and treatment response. The arginine metabolism pathway is hyperactive in colorectal cancer and several molecules involved in the pathway are potential targets for chemoprevention and targeted colorectal cancer therapy. Endothelial nitric oxide synthase (eNOS), argininosuccinate synthetase and ornithine decarboxylase (ODC) are the main enzymes for arginine metabolism. Limiting arginine-rich meat consumption and inhibiting ODC activity largely reduces polyamine synthesis and the incidence of colorectal cancer. Arginine transporter CAT-1 and Human member 14 of the solute carrier family 6 (SLC6A14) are overexpressed in colorectal cancer cells and contributes to intracellular arginine levels. Human member 9 of the solute carrier family 38 (SLC38A9) serves as a component of the lysosomal arginine-sensing machinery. Pharmaceutical inhibition of single enzyme or arginine transporter is hard to meet requirement of restoring of abnormal arginine metabolic network. Apart from application in early screening for colorectal cancer, microRNA-based therapeutic strategy that simultaneously manipulating multiple targets involved in arginine metabolism brings promising future in the treatment of colorectal cancer.

Abstract 464: Allicin, a potent new ornithine decarboxylase inhibitor in neuroblastoma

Abstract The purpose of this study was to test if allicin inhibits ornithine decarboxylase (ODC) in pediatric neuroblastoma (NB). The natural product allicin is a reactive sulfur species (RSS) from garlic (Allium sativum L.). NB is an early childhood cancer arising from the developing peripheral nervous system. In up to 25% of cases MYCN gene amplification is correlated to high tumor stage and poor patient prognosis. High stage NB relapses frequently despite multimodal therapy and relapsed tumors are virtually untreatable. Therefore, there is a clear need for specific, novel therapeutics. MYCN transactivates the E-box gene ODC1, and the gene product ODC is a rate-limiting enzyme in polyamine biosynthesis. The increase of polyamines (putrescine, spermidine, spermine) triggers cell hyperproliferation in NB and other MYC-driven cancers through the activation of Rb-regulated cell cycle progression. ODC is a validated drug target and α-difluoromethylornithine (DFMO) is an ODC inhibitor under investigation in phase II NB trials. Although a safe drug with clinical promise, DFMO has various challenges including the need for exceptionally high treatment doses and rapid renal clearance in the urine. In an effort to identify pharmacologically superior ODC inhibitors, we identified allicin as a potent ODC inhibitor using a specific radioactive in vitro activity assay with purified ODC. Allicin also inhibited ODC activity in actively growing NB cells, reduced polyamine levels, and induced apoptosis in cell cultures. ODC is a homodimer with 12 cysteines per monomer and allicin S-thioallylates cysteines. Allicin reacts with, and is titrated-out by, low molecular weight thiols like dithiothreitol (DTT). Removal of DTT from the ODC activity assay reaction resulted in significantly higher allicin potency (IC50 at 11 nM). Although allicin has multiple cellular targets, our data reveal that one mode of action is ODC inhibition, which suppresses polyamine accumulation and induces apoptosis. The natural product allicin could be used in conjunction with other anticancer treatments, the latter at a lower than usual dosage, to achieve drug synergism with good prognosis and less side-effects. Citation Format: Chad R. Schultz, Martin C. Gruhlke, Alan J. Slusarenko, Andre S. Bachmann. Allicin, a potent new ornithine decarboxylase inhibitor in neuroblastoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 464.

Putrescine mitigates intestinal atrophy through suppressing inflammatory response in weanling piglets

BackgroundPolyamines are essential for cell growth and beneficial for intestinal maturation. To evaluate the effects of putrescine on alleviating intestinal atrophy and underlying molecular mechanisms, both in vivo feeding trial and in vitro cell culture were conducted. Weanling pigs were fed a diet supplemented with 0, 0.1%, 0.2% or 0.3% putrescine dihydrochloride, whereas porcine intestinal epithelial cells (IPEC-J2) were challenged with lipopolysaccharide (LPS) in the presence of 200 μmol/L putrescine.ResultsDietary supplementation with 0.2% putrescine dihydrochloride decreased the incidence of diarrhea with an improvement in intestinal integrity. Inhibition of ornithine decarboxylase activity decreased the proliferation and migration of IPEC-J2 cells, and this effect was alleviated by the supplementation with putrescine. The phosphorylation of extracellular signal regulated kinase and focal adhesion kinase was enhanced by putrescine. LPS increased the expression of inflammatory cytokines [tumor necrosis factor α (TNF-α), interleukin 6 (IL-6) and IL-8], and inhibited cell proliferation and migration in IPEC-J2 cells. Adding exogenous putrescine suppressed the expression of TNF-α, IL-6 and IL-8, and recovered cell migration and proliferation in LPS-treated IPEC-J2 cells. Dietary putrescine supplementation also reduced the mRNA levels of TNF-α, IL-6 and IL-8 and their upstream regulator nuclear receptor kappa B p65 subunit in the jejunal mucosa of piglets.ConclusionsDietary supplementation with putrescine mitigated mucosal atrophy in weanling piglets through improving anti-inflammatory function and suppressing inflammatory response. Our results have important implications for nutritional management of intestinal integrity and health in weanling piglets and other neonates.

IN VITRO ANALYSIS OF ORNITHINE DECARBOXYLASE INHIBITORY ACTIVITY OF THE EXTRACTS OF EXCOECARIA AGALLOCHA L.

Objective: Ornithine decarboxylase (ODC) is one of the key enzymes involved in polyamine synthesis that has a prominent regulatory role in cell growth in prokaryotes and eukaryotes. Its regulation/inhibition is a potential means of controlling cell growth. The purpose of the study was to check the effect of the methanol extracts of leaves and seeds of the mangrove plant Excoecaria agallocha in controlling/inhibiting ODC enzyme produced by Escherichia coli.Methods: The methanol extracts of leaves and seeds of the mangrove plant E. agallocha were made. The inhibitory effect of the extracts on ODC enzyme produced by E. coli was tested by a modified spectrophotometric assay (measured in terms of µM of putrescine produced), and the antibacterial effect of the extract was determined by well diffusion assay.Results: It was found that both the leaf and seed extracts inhibited ODC activity thereby preventing the conversion of ornithine to putrescine (the prominent by-product of ODC activity) in a concentration-dependent manner. The leaf extract exhibited 50% inhibition of ODC activity at a concentration of 17.5 µg/ml whereas seed extract inhibited 50% of the activity at a concentration of 16.1 µg/ml. In the growth inhibition assay by well diffusion method, the effect of the extracts on the growth of E. coli was studied and a concentration-dependent growth inhibition was found - 100 µl of leaf extract showed a zone of inhibition of 12.31 mm (diameter) and seed extract showed 12.64 mm, confirming the antibacterial activity of the extracts.Conclusion: The ODC inhibition-mediated growth inhibition of E. coli is a novel finding as far as the extracts of E. agallocha are concerned. The extract was potent enough to be suggested as an antibacterial agent.Keywords: Ornithine decarboxylase, Excoecaria agallocha, Escherichia coli, Ornithine decarboxylase inhibition, Putrescine, Ornithine, Antibacterial effect.

Antizyme inhibitor 1: a potential carcinogenic molecule.

Polyamines are multivalent and organic cations essential for cellular growth, proliferation, differentiation, and apoptosis. Increased levels of polyamines are closely associated with numerous forms of cancer. An autoregulatory circuit composed of ornithine decarboxylase (ODC), antizyme (AZ) and antizyme inhibitor (AZI) govern the intracellular level of polyamines. Antizyme binds with ODC to inhibit ODC activity and to promote the ubiquitin‐independent degradation of ODC. Antizyme inhibitor binds to AZ with a higher affinity than ODC. Consequently, ODC is released from the ODC–AZ complex to rescue its activity. Antizyme inhibitor increases the ODC activity to accelerate the formation of intracellular polyamines, triggering gastric and breast carcinogenesis as well as hepatocellular carcinoma and esophageal squamous cell carcinoma development. Antizyme inhibitor 1 (AZIN1), a primary member of the AZI family, has aroused more attention because of its contribution to cancer. Even though its conformation is changed by adenosine‐to‐inosine (A→I) RNA editing, it plays an important role in tumorigenesis through regulating intracellular polyamines. Encouragingly, AZIN1 has been revealed to have an additional function outside the polyamine pathway so as to bypass the deficiency of targeting the polyamine biosynthetic pathway, promising to become a critical target for cancer therapy. Here, we review the latest research advances into AZIN1 and its potential contribution to carcinogenesis.

Genistein as antioxidant and antibrowning agents in in vivo and in vitro: A review.

Genistein is a phytoestrogen with diverse biological activities. It is a potent antioxidant and antibrowning agent in in vivo and in vitro. Genistein acts as a preventative and therapeutic effects for cancers, postmenopausal syndrome, osteoporosis and cardiovascular diseases in animals and humans. Gensitein possesses cancer related enzyme-inhibitory effect and substantially inhibits skin carcinogenesis and cutaneous aging induced by ultraviolet (UV) light in mice and photodamage in humans. Two-stage skin carcinogenesis showed genistein exhibited a moderate inhibition of ornithine decarboxylase activity through blockage of DNA adducts formation. The anticancer, anti-inflammatory, cardio-protective and enzyme-inhibitory effects of genistein might be related to their antioxidant activities. Genistein also altered the Maillard reaction pathway by trapping the advanced glycation end products (AGEs) both in biological and protein-lactose suspensions. As a result, soy isoflavone can be used to enrich or fortify different types of food products.

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.

Chemoprotective Effect of Elettaria Cardamomum against Chemically induced Hepatocellular Carcinoma in Rats by Inhibiting NF-κB, Oxidative Stress, and Activity of Ornithine Decarboxylase

The chemoprotective effect of Elettaria cardamomum (cardamom) extract was studied against DENA (200mg/kg) induced hepatocellular carcinoma (HCC) in comparison to the effect of geraniol in rats. Cardamom extract or geraniol was orally administered on a daily basis (100 and 200mg/kg) for 1week before HCC induction with DENA and CCl4, with the daily supply of the plant extract up to the end of the experiment. Cardamom or geraniol decreased the levels of TNF, IL-1 and NF-κB, which were induced by the DENA treatment. The levels of ornithine decarboxylase (ODC), hepatic malondialdehyde, and liver injury markers; such as alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP), and γ-glutamyl transferase (GGT), were substantially increased following DENA treatment. However, cardamom or geraniol treatment reduced liver injury and inhibited the elevation effect of DENA of the liver cancer marker (ODC). Cardamom or geraniol also significantly decreased hepatic malondialdehyde (MDA) formation and increased reduced glutathione (GSH) in DENA-treated rats. Additionally, cardamom/geraniol also increased the activities of antioxidant enzymes viz., catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione reductase, and glutathione-S-transferase (GST) in the liver of DENA-treated rats. Histological observations of the liver confirmed the biochemical observations. HPLC analysis of cardamom showed the presence of gallic, tannic, caffeic and 4,5-dicaffeoyl quinic acid. The results supported the chemo preventive anticancer effect of cardamom through its antioxidant effect and the inhibition of ODC activity and inflammatory markers.

Chlamydia trachomatis inhibits inducible NO synthase in human mesenchymal stem cells by stimulating polyamine synthesis.

Chlamydia trachomatis is considered the most common agent of sexually transmitted disease worldwide. As an obligate intracellular bacterium, it relies on the host for survival. Production of NO is an effective antimicrobial defense mechanism of the innate immune system. However, whether NO is able to arrest chlamydial growth remains unclear. Similarly, little is known about the mechanisms underlying subversion of cellular innate immunity by C. trachomatis. By analyzing protein and mRNA expression in infected human mesenchymal stem cells, combined with RNA interference and biochemical assays, we observed that infection with C. trachomatis led to downregulated expression of inducible NO synthase (iNOS) in human mesenchymal stem cells in vitro. Furthermore, infection upregulated the expression of the rate-limiting enzyme in the polyamine biosynthetic pathway, ornithine decarboxylase, diverting the iNOS substrate l-arginine toward the synthesis of polyamines. Inhibition of ornithine decarboxylase activity using small interfering RNA or the competitive inhibitor difluoromethylornithine restored iNOS protein expression and activity in infected cells and inhibited chlamydial growth. This inhibition was mediated through tyrosine nitration of chlamydial protein by peroxynitrite, an NO metabolite. Thus, Chlamydia evades innate immunity by inhibiting NO production through induction of the alternative polyamine pathway.

The effect of a mealybug infestation on the activity of amino acid decarboxylases in orchid leaves

Changes in the activity of lysine decarboxylase (LDC), tyrosine decarboxylase (TyDC), and ornithine decarboxylase (ODC) within orchid (Phalaenopsis × hybridum ‘Innocence’) leaves, infested by two mealybug species: Pseudococcus longispinus (Targ. Tozz.) and Pseudococcus maritimus (Ehrh.) were quantified. The pattern of changes was dependent on the insect species and duration of infestation. P. longispinus feeding increased LDC and TyDC activity after one week during the total period of observations. This species inhibited ODC activity after one week but increased later. P. maritimus decreased LDC activity in orchid leaves at all studied terms. TyDC action also went up during the first week of the infestation and was reduced after two weeks, while ODC was decreased after one day and induced later. The mechanism for the participation of analysed amino acid decarboxylases in local and/or systemic steps of orchid responses to mealybug infestation is discussed.

Regulation of intestinal mucosal growth by amino acids

Amino acids, especially glutamine (GLN) have been known for many years to stimulate the growth of small intestinal mucosa. Polyamines are also required for optimal mucosal growth, and the inhibition of ornithine decarboxylase (ODC), the first rate-limiting enzyme in polyamine synthesis, blocks growth. Certain amino acids, primarily asparagine (ASN) and GLN stimulate ODC activity in a solution of physiological salts. More importantly, their presence is also required before growth factors and hormones such as epidermal growth factor and insulin are able to increase ODC activity. ODC activity is inhibited by antizyme-1 (AZ) whose synthesis is stimulated by polyamines, thus, providing a negative feedback regulation of the enzyme. In the absence of amino acids mammalian target of rapamycin complex 1 (mTORC1) is inhibited, whereas, mTORC2 is stimulated leading to the inhibition of global protein synthesis but increasing the synthesis of AZ via a cap-independent mechanism. These data, therefore, explain why ASN or GLN is essential for the activation of ODC. Interestingly, in a number of papers, AZ has been shown to inhibit cell proliferation, stimulate apoptosis, or increase autophagy. Each of these activities results in decreased cellular growth. AZ binds to and accelerates the degradation of ODC and other proteins shown to regulate proliferation and cell death, such as Aurora-A, Cyclin D1, and Smad1. The correlation between the stimulation of ODC activity and the absence of AZ as influenced by amino acids is high. Not only do amino acids such as ASN and GLN stimulate ODC while inhibiting AZ synthesis, but also amino acids such as lysine, valine, and ornithine, which inhibit ODC activity, increase the synthesis of AZ. The question remaining to be answered is whether AZ inhibits growth directly or whether it acts by decreasing the availability of polyamines to the dividing cells. In either case, evidence strongly suggests that the regulation of AZ synthesis is the mechanism through which amino acids influence the growth of intestinal mucosa. This brief article reviews the experiments leading to the information presented above. We also present evidence from the literature that AZ acts directly to inhibit cell proliferation and increase the rate of apoptosis. Finally, we discuss future experiments that will determine the role of AZ in the regulation of intestinal mucosal growth by amino acids.

Polyamines regulate intestinal epithelial restitution through TRPC1-mediated Ca²+ signaling by differentially modulating STIM1 and STIM2.

Early epithelial restitution occurs as a consequence of intestinal epithelial cell (IEC) migration after wounding, and its defective regulation is implicated in various critical pathological conditions. Polyamines stimulate intestinal epithelial restitution, but their exact mechanism remains unclear. Canonical transient receptor potential-1 (TRPC1)-mediated Ca(2+) signaling is crucial for stimulation of IEC migration after wounding, and induced translocation of stromal interaction molecule 1 (STIM1) to the plasma membrane activates TRPC1-mediated Ca(2+) influx and thus enhanced restitution. Here, we show that polyamines regulate intestinal epithelial restitution through TRPC1-mediated Ca(2+) signaling by altering the ratio of STIM1 to STIM2. Increasing cellular polyamines by ectopic overexpression of the ornithine decarboxylase (ODC) gene stimulated STIM1 but inhibited STIM2 expression, whereas depletion of cellular polyamines by inhibiting ODC activity decreased STIM1 but increased STIM2 levels. Induced STIM1/TRPC1 association by increasing polyamines enhanced Ca(2+) influx and stimulated epithelial restitution, while decreased formation of the STIM1/TRPC1 complex by polyamine depletion decreased Ca(2+) influx and repressed cell migration. Induced STIM1/STIM2 heteromers by polyamine depletion or STIM2 overexpression suppressed STIM1 membrane translocation and inhibited Ca(2+) influx and epithelial restitution. These results indicate that polyamines differentially modulate cellular STIM1 and STIM2 levels in IECs, in turn controlling TRPC1-mediated Ca(2+) signaling and influencing cell migration after wounding.

Ornithine decarboxylase is upregulated by the androgen receptor in skeletal muscle and regulates myoblast proliferation

The aim of this study is to determine if the Odc1 gene, which encodes ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine biosynthesis, is directly regulated by the androgen receptor (AR) in skeletal muscle myoblasts and if Odc1 regulates myoblast proliferation and differentiation. We previously showed that expression of Odc1 is decreased in muscle from AR knockout male mice. In this study, we show in vivo that Odc1 expression is also decreased >60% in muscle from male muscle-specific AR knockout mice. In normal muscle homeostasis, Odc1 expression is regulated by age and sex, reflecting testosterone levels, as muscle of adult male mice expresses high levels of Odc1 compared with age-matched females and younger males. In vitro, expression of Odc1 is 10- and 1.5-fold higher in proliferating mouse C(2)C(12) and human skeletal muscle myoblasts, respectively, than in differentiated myotubes. Dihydrotestosterone increases Odc1 levels 2.7- and 1.6-fold in skeletal muscle cell myoblasts after 12 and 24 h of treatment, respectively. Inhibition of ODC activity in C(2)C(12) myoblasts by α-difluoromethylornithine decreases myoblast number by 40% and 66% following 48 and 72 h of treatment, respectively. In contrast, overexpression of Odc1 in C(2)C(12) myoblasts results in a 27% increase in cell number vs. control when cells are grown under differentiation conditions for 96 h. This prolonged proliferation is associated with delayed differentiation, with reduced expression of the differentiation markers myogenin and Myf6 in Odc1-overexpressing cells. In conclusion, androgens act via the AR to upregulate Odc1 in skeletal muscle myoblasts, and Odc1 promotes myoblast proliferation and delays differentiation.

Arginine–Nitric Oxide–Polyamine Metabolism in Periodontal Disease

Arginine is converted to nitric oxide (NO) via NO synthase and to ornithine via arginase. Ornithine decarboxylase (ODC) catalyzes the conversion of ornithine to polyamines. Arginase can inhibit NO production, and NO can inhibit ODC activity as part of an early inflammatory response. This study examines the arginine-NO-polyamine pathway alteration in saliva and gingival biopsy samples of patients with gingivitis or periodontitis and healthy controls and evaluates the response to periodontal treatment. This study includes nine gingivitis patients, 15 chronic periodontitis patients, and 11 healthy age-matched controls. Periodontal clinical measurements, gingival biopsies, and saliva samples were obtained before treatment (BT) and 1 month after periodontal treatment (AT). Arginase and ODC activities and NO levels were determined spectrophotometrically. The BT salivary and gingival NO levels were found to be highest in the gingivitis group, followed by the healthy and the periodontitis groups, respectively. Salivary NO levels significantly increased in the periodontitis group and decreased in the gingivitis group AT (P <0.05). Gingival NO levels decreased significantly in the periodontitis and the gingivitis groups AT (P <0.05). Arginase levels were detected highest in the gingivitis group and lowest in the periodontitis group, both in saliva and gingiva. Only gingival arginase levels significantly increased AT (P <0.05). ODC activity was highest in saliva, and lowest in the gingiva of the periodontitis patients BT. It was found to be significantly higher in the periodontitis group AT (P <0.05). In this study, regarding arginine-NO-polyamine metabolism, gingival tissue seems to be more informative about periodontal pathogenesis than saliva. At early phase of periodontal inflammation, NO arginase and ODC levels were measured as higher than at an established lesion of periodontitis.

Channelling of arginine in NO and polyamine pathways in colonocytes and consequences

Colon epithelium is renewed within few days through asymmetric mitosis of pluripotent cells followed by differentiation and exfoliation. Absorptive colonocytes do not transport amino acids from lumen to bloodstream but import amino acids from plasma. Among amino acids, colonocytes can use L-arginine as a precursor for nitric oxide (NO) synthesis and also for polyamine synthesis through the stepwise conversion of L-arginine into L-ornithine and urea, conversion of L-ornithine into putrescine and then synthesis of spermidine and spermine. Colonic epithelial cells can transport polyamines produced exogenously by the microbiota. Polyamines are strictly necessary for undifferentiated colonic epithelial cell proliferation but NO exerts anti-proliferative effect on these cells raising the view that the channelling of arginine in the nitric oxide and polyamine pathways is involved in the control of cellular proliferation. Furthermore, NO has been shown to be a potent inhibitor of ornithine decarboxylase activity in colonic epithelial cells. Unbalance of the chanelling of arginine in the NO and polyamine pathways in colonic cancerous epithelial cells as a determinant promoting their proliferation is discussed.

Polyamines and hair: a couple in search of perfection

Polyamines (spermidine, putrescine and spermine) are multifunctional cationic amines that are indispensable for cellular proliferation; of key significance in the growth of rapidly regenerating tissues and tumors. Given that the hair follicle (HF) is one of the most highly proliferative organs in mammalian biology, it is not surprising that polyamines are crucial to HF growth. Indeed, growing (anagen) HFs show the highest activity of ornithine decarboxylase (ODC), the rate-limiting enzyme of polyamine biosynthesis, while inhibition of ODC, using eflornithine, results in a decreased rate of excessive facial hair growth in vivo and inhibits human scalp hair growth in organ culture. In sheep, manipulation of dietary intake of polyamines also results in altered wool growth. Polyamine-containing nutraceuticals have therefore been proposed as promoters of human hair growth. Recent progress in polyamine research, coupled with renewed interest in the role of polyamines in skin biology, encourages one to revisit their potential roles in HF biology and highlights the need for a systematic evaluation of their mechanisms of action and clinical applications in the treatment of hair disorders. The present viewpoint essay outlines the key frontiers in polyamine-related hair research and defines the major open questions. Moreover, it argues that a renaissance in polyamine research in hair biology, well beyond the inhibition of ODC activity in hirsutism therapy, is important for the development of novel therapeutic strategies for the manipulation of human hair growth. Such targets could include the manipulation of polyamine biosynthesis and the topical administration of selected polyamines, such as spermidine.