Functional identification of bacterial spermine, thermospermine, norspermine, norspermidine, spermidine, and N1-aminopropylagmatine synthases.

Spermine synthase is an aminopropyltransferase that adds an aminopropyl group to the essential polyamine spermidine to form tetraamine spermine, needed for normal human neural development, plant salt and drought resistance, and yeast CoA biosynthesis. We functionally identify for the first time bacterial spermine synthases, derived from phyla Bacillota, Rhodothermota, Thermodesulfobacteriota, Nitrospirota, Deinococcota, and Pseudomonadota. We also identify bacterial aminopropyltransferases that synthesize the spermine same mass isomer thermospermine, from phyla Cyanobacteriota, Thermodesulfobacteriota, Nitrospirota, Dictyoglomota, Armatimonadota, and Pseudomonadota, including the human opportunistic pathogen Pseudomonas aeruginosa. Most of these bacterial synthases were capable of synthesizing spermine or thermospermine from the diamine putrescine and so possess also spermidine synthase activity. We found that most thermospermine synthases could synthesize tetraamine norspermine from triamine norspermidine, that is, they are potential norspermine synthases. This finding could explain the enigmatic source of norspermine in bacteria. Some of the thermospermine synthases could synthesize norspermidine from diamine 1,3-diaminopropane, demonstrating that they are potential norspermidine synthases. Of 18 bacterial spermidine synthases identified, 17 were able to aminopropylate agmatine to form N1-aminopropylagmatine, including the spermidine synthase of Bacillus subtilis, a species known to be devoid of putrescine. This suggests that the N1-aminopropylagmatine pathway for spermidine biosynthesis, which bypasses putrescine, may be far more widespread than realized and may be the default pathway for spermidine biosynthesis in species encoding L-arginine decarboxylase for agmatine production. Some thermospermine synthases were able to aminopropylate N1-aminopropylagmatine to form N12-guanidinothermospermine. Our study reveals an unsuspected diversification of bacterial polyamine biosynthesis and suggests a more prominent role for agmatine.

Spermine: Its Emerging Role in Regulating Drought Stress Responses in Plants.

In recent years, research on spermine (Spm) has turned up a lot of new information about this essential polyamine, especially as it is able to counteract damage from abiotic stresses. Spm has been shown to protect plants from a variety of environmental insults, but whether it can prevent the adverse effects of drought has not yet been reported. Drought stress increases endogenous Spm in plants and exogenous application of Spm improves the plants' ability to tolerate drought stress. Spm's role in enhancing antioxidant defense mechanisms, glyoxalase systems, methylglyoxal (MG) detoxification, and creating tolerance for drought-induced oxidative stress is well documented in plants. However, the influences of enzyme activity and osmoregulation on Spm biosynthesis and metabolism are variable. Spm interacts with other molecules like nitric oxide (NO) and phytohormones such as abscisic acid, salicylic acid, brassinosteroids, and ethylene, to coordinate the reactions necessary for developing drought tolerance. This review focuses on the role of Spm in plants under severe drought stress. We have proposed models to explain how Spm interacts with existing defense mechanisms in plants to improve drought tolerance.

Polyamines and Their Biosynthesis/Catabolism Genes Are Differentially Modulated in Response to Heat Versus Cold Stress in Tomato Leaves (Solanum lycopersicum L.).

Polyamines (PAs) regulate growth in plants and modulate the whole plant life cycle. They have been associated with different abiotic and biotic stresses, but little is known about the molecular regulation involved. We quantified gene expression of PA anabolic and catabolic pathway enzymes in tomato (Solanum lycopersicum cv. Ailsa Craig) leaves under heat versus cold stress. These include arginase1 and 2, arginine decarboxylase 1 and 2, agmatine iminohydrolase/deiminase 1, N-carbamoyl putrescine amidase, two ornithine decarboxylases, three S-adenosylmethionine decarboxylases, two spermidine synthases; spermine synthase; flavin-dependent polyamine oxidases (SlPAO4-like and SlPAO2) and copper dependent amine oxidases (SlCuAO and SlCuAO-like). The spatiotemporal transcript abundances using qRT-PCR revealed presence of their transcripts in all tissues examined, with higher transcript levels observed for SAMDC1, SAMDC2 and ADC2 in most tissues. Cellular levels of free and conjugated forms of putrescine and spermidine were found to decline during heat stress while they increased in response to cold stress, revealing their differential responses. Transcript levels of ARG2, SPDS2, and PAO4-like increased in response to both heat and cold stresses. However, transcript levels of ARG1/2, AIH1, CPA, SPDS1 and CuAO4 increased in response to heat while those of ARG2, ADC1,2, ODC1, SAMDC1,2,3, PAO2 and CuPAO4-like increased in response to cold stress, respectively. Transcripts of ADC1,2, ODC1,2, and SPMS declined in response to heat stress while ODC2 transcripts declined under cold stress. These results show differential expression of PA metabolism genes under heat and cold stresses with more impairment clearly seen under heat stress. We interpret these results to indicate a more pronounced role of PAs in cold stress acclimation compared to that under heat stress in tomato leaves.

Prevention of Atherosclerosis by the Induction of Microbial Polyamine Production in the Intestinal Lumen.

Low molecular weight metabolites produced by the intestinal microbiome that have been associated with health and disease as metabolites need to be constantly absorbed from the intestinal lumen and transported to intestinal epithelial cells and blood. Polyamines, especially spermidine and spermine, are bioactive chemicals which promote autophagy and suppress inflammation. The main source of exogenous polyamines is the intestinal lumen, where they are produced by intestinal microbiome. Considering the intestinal microbiome as a manufacturing plant for bioactive substances, we developed a novel hybrid putrescine biosynthesis system strategy, in which the simultaneous intake of Bifidobacterium animalis ssp. lactis LKM512 (Bifal) and arginine (Arg) upregulates the production of the putrescine, a precursor of spermidine and spermine, in the gut by controlling the bacterial metabolism beyond its vast diversity and inter-individual differences. In a clinical trial, healthy individuals with a body mass index near the maximum "healthy" range (25 kg/m3; n = 44) were randomized to consume either normal yogurt containing Bifal and Arg (Bifal + Arg YG) or placebo (normal yogurt) for 12 weeks. The change in reactive hyperemia index determined by EndoPAT from week 0 to 12 in the Bifal + Arg YG group was significantly higher than that in the placebo group, indicating that Bifal + Arg YG intake improved vascular endothelial function. In addition, the concentrations of fecal putrescine and serum spermidine in the Bifal+ Arg YG group were significantly higher than those in the placebo group. These findings suggest that consuming Bifal + Arg YG prevents or reduces atherosclerosis risk by upregulating blood spermidine levels, which subsequently induces autophagy.

Association Between Plasma Diacetylspermine and Tumor Spermine Synthase With Outcome in Triple-Negative Breast Cancer.

BACKGROUND: MYC is an oncogenic driver of development and progression in triple-negative breast cancer (TNBC). Ornithine decarboxylase, the rate-limiting enzyme in polyamine metabolism, is a transcriptional target of MYC. We therefore hypothesized that a plasma polyamine signature may be predictive of TNBC development and progression. METHODS: Using liquid chromatography mass spectrometry, polyamine levels were determined in plasma samples from newly diagnosed patients with TNBC (n = 87) and cancer-free controls (n = 115). Findings were validated in plasma samples from an independent prospective cohort of 54 TNBC, 55 estrogen receptor negative (ER-) and progesterone receptor negative (PR-) and HER2 positive (HER2+), and 73 ER+ case patients, and 30 cancer-free control subjects. Gene expression data and clinical data for 921 and 2359 breast cancer tumors were obtained from The Cancer Genome Atlas repository and the Oncomine database, respectively. Relationships between plasma diacetylspermine (DAS) and tumor spermine synthase (SMS) mRNA expression with metastasis-free survival and overall survival were determined using Cox proportional hazard models; Fisher exact tests were used to assess risk of distant metastasis in relation to tumor SMS mRNA expression. RESULTS: An increase in plasma DAS, a catabolic product of spermine mediated through SMS, was observed in the TNBC subtype of breast cancer. Plasma levels of DAS in TNBC associated with increased risk of metastasis (plasma DAS value ≥ 1.16, hazard ratio = 3.06, 95% confidence interval [CI] = 1.15 to 8.13, two-sided P = .03). SMS mRNA expression in TNBC tumor tissue was also found to be predictive of poor overall survival (top 25th percentile hazard ratio = 2.06, 95% CI = 1.04 to 4.08, one-sided P = .04) and increased risk of distant metastasis in TNBC (comparison of lowest SMS quartile [reference] to highest SMS quartile relative risk = 1.90, 95% CI = 0.97 to 4.06, one-sided Fisher exact test P=.03). CONCLUSIONS: Metabolomic profiling identified plasma DAS as a predictive marker for TNBC progression and metastasis.

Effect of exogenous spermidine on floral induction, endogenous polyamine and hormone production, and expression of related genes in 'Fuji' apple (Malus domestica Borkh.).

Flower bud formation in 'Fuji' apple (Malus domestica Borkh.) is difficult, which severely constrains commercial production. Spermidine (Spd) plays an important role in floral induction, but the mechanism of its action is incompletely understood. To investigate the effect of Spd on flowering, 6-year-old 'Fuji' apple trees were treated with 1 × 10-5 mol L-1 Spd to study the responses of polyamines [putrescine (Put), Spd and spermine (Spm)], hormones [gibberellins (GA3) and abscisic acid (ABA)], and polyamine-, hormone- and flowering-related genes. Spd application promoted flowering during floral induction by increasing MdGA2ox2 (gibberellin 2-oxidase) through GA3 reduction and increasing MdNCED1 and MdNCED3 (9-cis-epoxycarotenoid dioxygenase) through ABA enrichment during 60 to 80 days after full bloom. The flowering rate as well as the expressions of flower-related genes, except for MdLEY (LEAFY), also increased, thereby promoting flowering. In addition, spraying with Spd significantly increased the contents of endogenous polyamines except for Spm in terminal buds by increasing the expressions of polyamine-associated genes. We hypothesize that the contribution of Spd to flowering is related to crosstalk among polyamines, hormone signals, and related gene expressions, which suggests that Spd participates in the apple floral induction process.

Spermine on Endothelial Extracellular Vesicles Mediates Smoking-Induced Pulmonary Hypertension Partially Through Calcium-Sensing Receptor.

Objective- This study aims to determine whether and how the enriched metabolites of endothelial extracellular vesicles (eEVs) are critical for cigarette smoke-induced direct injury of endothelial cells and the development of pulmonary hypertension, rarely explored in contrast to long-investigated mechanisms secondary to chronic hypoxemia. Approach and Results- Metabonomic screen of eEVs from cigarette-smoking human subjects reveals prominent elevation of spermine-a polyamine metabolite with potent agonist activity for the extracellular CaSR (calcium-sensing receptor). CaSR inhibition with the negative allosteric modulator Calhex231 or CaSR knockdown attenuates cigarette smoke-induced pulmonary hypertension in rats without emphysematous changes in lungs or chronic hypoxemia. Cigarette smoke exposure increases the generation of spermine-positive eEVs and their spermine content. Immunocytochemical staining and immunogold electron microscopy recognize the spermine enrichment not only within the cytosol but also on the outer surface of eEV membrane. The repression of spermine synthesis, the inhibitory analog of spermine, N1-dansyl-spermine, Calhex231, or CaSR knockdown profoundly suppresses eEV exposure-mobilized cytosolic calcium signaling, pulmonary artery constriction, and smooth muscle cell proliferation. Confocal imaging of immunohistochemical staining demonstrates the migration of spermine-positive eEVs from endothelium into smooth muscle cells in pulmonary arteries of cigarette smoke-exposed rats. The repression of spermine synthesis or CaSR knockout results in attenuated development of pulmonary hypertension induced by an intravascular administration of eEVs. Conclusions- Cigarette smoke enhances eEV generation with spermine enrichment at their outer surface and cytosol, which activates CaSR and subsequently causes smooth muscle cell constriction and proliferation, therefore, directly leading to the development of pulmonary hypertension.

Analysis of polyamine biosynthetic- and transport ability of human indigenous Bifidobacterium.

Bifidobacteria are members of the human intestinal microbiota, being numerically dominant in the colon of infants, and also being prevalent in the large intestine of adults. In this study, we measured the concentrations of major polyamines (putrescine, spermidine, and spermine) in cells and culture supernatant of 13 species of human indigenous Bifidobacterium at growing and stationary phase. Except for Bifidobacterium bifidum and Bifidobacterium gallicum, 11 species contained spermidine and/or spermine when grown in Gifu-anaerobic medium (GAM). However, Bifidobacterium scardovii and Bifidobacterium longum subsp. infantis, which contain spermidine when grown in GAM, did not contain spermidine when grown in polyamine-free 199 medium. Of the tested 13 Bifidobacterium species, 10 species showed polyamine transport ability. Combining polyamine concentration analysis in culture supernatant and in cells, with basic local alignment search tool analysis suggested that novel polyamine transporters are present in human indigenous Bifidobacterium. ABBREVIATIONS: Put: putrescine; Spd: spermidine; Spm: spermine; GAM: Gifu anaerobic medium; BHI: brain-heart infusion.

Expression and distribution patterns of spermine, spermidine, and putrescine in rat hair follicle.

No expression and distribution patterns of polyamines (PAs), spermine, spermidine, and their precursor putrescine in mammalian hair follicle are available, although polyamines are known to correlate well with hair growth and epidermal tumor genesis. Immunohistochemistry (IHC) using our original two monoclonal antibodies (mAbs) ASPM-29 specific for spermine or spermidine, and APUT-32 specific for putrescine allowed us to detect immunoreactivity for polyamines in hair follicles from normal adult rats. A wide range of immunoreactivity for the total spermine and spermidine was observed in the compartments of hair follicle: The highest degree of immunoreactivity for polyamines was observed in the matrix, in the Huxley's layer, in the deeper Henle's layer, and in the cuticle of the inner root sheath/the hair cuticle, while moderate immunoreactivity existed in the lower-to-mid cortex and the companion layer, followed by lower immunoreactivity in the outer root sheath, including the bulge region and in the deeper medulla, in which the immunoreactivity was also evident in their nuclei. In addition, somewhat surprisingly, with IHC by APUT-32 mAb, we detected significant levels of putrescine in the compartments, in which the immunostaining pattern was the closely similar to that of the total spermine and spermidine. Thus, among these compartments, the cell types of the matrix, the Huxley's layer, the deeper Henle's layer, and the cuticle of the inner root sheath/the hair cuticle seem to have the biologically higher potential in compartments of anagen hair follicle, maybe suggesting that they are involved more critically in the biological event of hair growth. In addition, we noted sharp differences of immunostaining by IHCs between ASPM-29 mAb and APUT-32 mAb in the epidermis cells and fibroblast. ASPM-29 mAb resulted in strong staining in both the cell types, but APUT-32 mAb showed only very light staining in both types. Consequently, the use of the two IHCs could be extremely useful in further studies on hair cycle and epidermal tumor genesis experimentally or clinically.

Determination of S-Adenosylmethionine Decarboxylase Activity in Plants.

The synthesis of spermidine, spermine and thermospermine requires the addition of aminopropyl groups from decarboxylated S-adenosyl-methionine (dSAM). The synthesis of dSAM is catalyzed by S-adenosylmethionine decarboxylase. dSAM levels are usually low, which constitutes a rate-limiting factor in the synthesis of polyamines. In this chapter, we provide a protocol for the determination of SAMDC activity in plants through the detection of radiolabelled CO2 released during the SAMDC reaction.

Effects of probiotic bacteria on mucosal polyamines levels in dogs with IBD and colonic polyps: a preliminary study.

Spermine (SPM) and its precursor putrescine (PUT), regulated by ornithine decarboxylase (ODC) and diamino-oxidase (DAO), are polyamines required for cell growth and proliferation. Only a few studies have investigated the anti-inflammatory and tumour inhibitory properties of probiotics on mucosal polyamine levels. We investigated the effects of a high concentration multistrain probiotic for human use on colonic polyamine biosynthesis in dogs. Histological sections (inflammatory bowel disease, n=10; polyposis, n=5) were assessed after receiving 112 to 225×109 lyophilised bacteria daily for 60 days at baseline (T0) and 30 days after treatment end (T90). Histology scores, expression of PUT, SPM, ODC and DAO, and a clinical activity index (CIBDAI) were compared at T0 and T90. In polyps, cellular proliferation (Ki-67 expression), and apoptosis (caspase-3 protein expression) were also evaluated. After treatment, in inflammatory bowel disease significant decreases were observed for CIBDAI (P=0.006) and histology scores (P<0.001); PUT, SPM and ODC expression increased (P<0.01). In polyps, a significant decrease in polyamine levels, ODC activity, and Ki-67, and a significant increase in caspase-3 positivity and DAO expression (P=0.005) was noted. Our results suggest potential anti-proliferative and anti-inflammatory effects of the probiotic mixture in polyps and inflammation, associated with reduced mucosal infiltration and up-regulation of PUT, SPM, and ODC levels.

[Low-molecular-weight regulators of biogenic polyamine metabolism affect cytokine production and expression of hepatitis С virus proteins in Huh7.5 human hepatocarcinoma cells].

Hepatitis C virus (HCV) induces the expression of the genes of proinflammatory cytokines, the excessive production of which may cause cell death, and contribute to development of liver fibrosis and hepatocarcinoma. The relationship between cytokine production and metabolic disorders in HCV-infected cells remains obscure. The levels of biogenic polyamines, spermine, spermidine, and their precursor putrescine, may be a potential regulator of these processes. The purpose of the present work was to study the effects of the compounds which modulate biogenic polyamines metabolism on cytokine production and HCV proteins expression. Human hepatocarcinoma Huh7.5 cells have been transfected with the plasmids that encode HCV proteins and further incubated with the following low-molecular compounds that affect different stages of polyamine metabolism: (1) difluoromethylornithine (DFMO), the inhibitor of ornithine decarboxylase, the enzyme that catalyzes the biosynthesis of polyamines; (2) N,N'-bis(2,3-butane dienyl)-1,4-diaminobutane (MDL72.527), the inhibitor of proteins involved in polyamine degradation; and (3) synthetic polyamine analog N^(I),N^(II)-diethylnorspermine (DENSpm), an inducer of polyamine degradation enzyme. The intracellular accumulation and secretion of cytokines (IL-6, IL-1ß, TNF-α, and TGF-ß) was assessed by immunocytochemistry and in the immunoenzyme assay, while the cytokine gene expression was studied using reverse transcription and PCR. The effects of the compounds under analysis on the expression of HCV proteins were analyzed using the indirect immunofluorescence with anti-HCV monoclonal antibodies. It has been demonstrated that, in cells transfected with HCV genes, DFMO reduces the production of three out of four tested cytokines, namely, TNF-α and TGF-ß in cells that express HCV core, Е1Е2, NS3, NS5A, and NS5B proteins, and IL-1ß in the cells that express HCV core, Е1Е2, and NS3 proteins. MDL72527 and DENSpm decreased cytokine production to a lesser extent. Incubation with DFMO led to a 28-32% decrease in the number of cells expressing NS5B or NS5A, both of which are key components of the HCV replication complex. The results obtained in the work indicate that a further detailed study of the antiviral activity of DFMO is required in order to assess its potential as an anti-hepatitis C therapeutic agent.

Sensitivity to Enterocins of Biogenic Amine-Producing Faecal Enterococci from Ostriches and Pheasants.

Enterococci are widespread bacteria forming the third largest genus among lactic acid bacteria. Some possess probiotic properties or they can produce beneficial proteinaceous antimicrobial substances called enterocins. On the other hand, some enterococci produce biogenic amines (BAs), so this study is focused on the sensitivity to enterocins of biogenic amine-producing faecal enterococci from ostriches and pheasants. Altogether, 60 enterococci isolated from faeces of ostriches and pheasants were tested for production of BAs. This target of the identified enterococci involved 46 strains selected from 140 ostriches and 17 from 60 pheasants involving the species Enterococcus hirae, E. faecium, E. faecalis, and E. mundtii. Although BAs histamine, cadaverine, putrescine, and tryptamine were not detected in the enterococci tested, in general high BA production by the tested enterococci was noted. The species E. hirae formed the majority of the enterococcal strains from ostrichs faeces (34 strains). High production of tyramine (TYM) was measured with an average amount of 958.16 ± 28.18 mg/ml. Among the enterococci from pheasants, the highest was production of TYM compared to phenylethylamine, spermidine, and spermine. Enterococci featured high BA production; however, they were sensitive to seven enterocins with inhibition activity ranging from 100 up to 25,600 AU/ml.

Chemical control of xylem differentiation by thermospermine, xylemin, and auxin.

The xylem conducts water and minerals from the root to the shoot and provides mechanical strength to the plant body. The vascular precursor cells of the procambium differentiate to form continuous vascular strands, from which xylem and phloem cells are generated in the proper spatiotemporal pattern. Procambium formation and xylem differentiation are directed by auxin. In angiosperms, thermospermine, a structural isomer of spermine, suppresses xylem differentiation by limiting auxin signalling. However, the process of auxin-inducible xylem differentiation has not been fully elucidated and remains difficult to manipulate. Here, we found that an antagonist of spermidine can act as an inhibitor of thermospermine biosynthesis and results in excessive xylem differentiation, which is a phenocopy of a thermospermine-deficient mutant acaulis5 in Arabidopsis thaliana. We named this compound xylemin owing to its xylem-inducing effect. Application of a combination of xylemin and thermospermine to wild-type seedlings negates the effect of xylemin, whereas co-treatment with xylemin and a synthetic proauxin, which undergoes hydrolysis to release active auxin, has a synergistic inductive effect on xylem differentiation. Thus, xylemin may serve as a useful transformative chemical tool not only for the study of thermospermine function in various plant species but also for the control of xylem induction and woody biomass production.

Cotton S-adenosylmethionine decarboxylase-mediated spermine biosynthesis is required for salicylic acid- and leucine-correlated signaling in the defense response to Verticillium dahliae.

MAIN CONCLUSION: Cotton S-adenosylmethionine decarboxylase-, rather than spermine synthase-, mediated spermine biosynthesis is required for salicylic acid- and leucine-correlated signaling in the defense response to Verticillium dahliae. Spermine (Spm) signaling is correlated with plant resistance to the fungal pathogen Verticillium dahliae. We identified genes for key rate-limiting enzymes in the biosynthesis of Spm, namely S-adenosylmethionine decarboxylase (GhSAMDC) and Spm synthase (GhSPMS). These were found by screening suppression subtractive hybridization and cDNA libraries of cotton (Gossypium) species tolerant to Verticillium wilt. Both were induced early and strongly by inoculation with V. dahliae and application of plant hormones. Silencing of GhSPMS or GhSAMDC in cotton leaves led to a significant accumulation of upstream substrates and, ultimately, enhanced plant susceptibility to Verticillium infection. Exogenous supplementation of Spm to the silenced cotton plants improved resistance. When compared with the wild type (WT), constitutive expression of GhSAMDC in Arabidopsis thaliana was associated with greater Verticillium wilt resistance and higher accumulations of Spm, salicylic acid, and leucine during the infection period. By contrast, transgenic Arabidopsis plants that over-expressed GhSPMS were unexpectedly more susceptible than the WT to V. dahliae and they also had impaired levels of putrescine (Put) and salicylic acid (SA). The susceptibility exhibited in GhSPMS-overexpressing Arabidopsis plants was partially reversed by the exogenous supply of Put or SA. In addition, the responsiveness of those two transgenic Arabidopsis lines to V. dahliae was associated with an alteration in transcripts of genes involved in plant resistance to epidermal penetrations and amino acid signaling. Together, these results suggest that GhSAMDC-, rather than GhSPMS-, mediated spermine biosynthesis contributes to plant resistance against V. dahliae through SA- and leucine-correlated signaling.

Targeting polyamine biosynthetic pathway through RNAi causes the abrogation of MCF 7 breast cancer cell line.

The diamine putrescine and polyamines, spermidine (triamine) and spermine (tetraamine) are small organic polycations that play an indispensable role in key cellular processes such as the regulation of growth, differentiation, and macromolecular functions. Elevated levels of polyamines (PAs) have been shown to be one of the major factors involved in carcinogenesis. In this study, specific silencing of the expression of three genes of PA biosynthesis pathway, ornithine decarboxylase (ODC), S-adenosylmethionine decarboxylase (SAMDC), and spermidine synthase (SPDSYN) was achieved using RNA interference in MCF 7 breast cancer cell line. For optimizing the effective small interfering nucleic acid (siNA), three variants of ODC siNA [siRNA, locked nucleic acid (LNA)-modified siRNA, and siHybrid (RNA and DNA hybrid)] were used and a dose- and time-dependent study was conducted. The PA biosynthetic genes were targeted individually and in combination. RNAi-mediated reduction in the expression of PA biosynthesis genes resulted in distorted cell morphology, reduced cancer cell viability, and migration characteristic. The most promising results were observed with the combined treatment of siSPDSYN and siODC with 83 % cell growth inhibition. On analyzing the messenger RNA (mRNA) expression profile of the cell cycle and apoptosis-related genes, it was observed that RNAi against PA biosynthetic genes downregulated the expression of CDK8, CCNE2, CCNH, CCNT1, CCNT2, CCNF, PCNA, CCND1, and CDK2, and upregulated the expression of E2F4, BAX, FAS, TP53, CDKN1A, BAK1, CDKN1B, ATM, GRANB, and ATR genes when compared with control-transfected cells. These results suggest that the targeting polyamine biosynthesis through RNAi approach could be a promising strategy for breast cancer therapy and might be extended for therapy of other cancers.

In vivo investigation of homocysteine metabolism to polyamines by high-resolution accurate mass spectrometry and stable isotope labeling.

Polyamines are essential polycations, playing important roles in mammalian physiology. Theoretically, the involvement of homocysteine in polyamine synthesis via S-adenosylmethionine is possible; however, to our knowledge, it has not been established experimentally. Here, we propose an original approach for investigation of homocysteine metabolites in an animal model. The method is based on the combination of isotope-labeled homocysteine supplementation and high-resolution accurate mass spectrometry analysis. Structural identity of the isotope-labeled metabolites was confirmed by accurate mass measurements of molecular and fragment ions and comparison of the retention times and tandem mass spectrometry fragmentation patterns. Isotope-labeled methionine, spermidine, and spermine were detected in all investigated plasma and tissue samples. The induction of moderate hyperhomocysteinemia leads to an alteration in polyamine levels in a different manner. The involvement of homocysteine in polyamine synthesis and modulation of polyamine levels could contribute to a better understanding of the mechanisms connected with homocysteine toxicity.

Arginine decarboxylase and agmatinase: an alternative pathway for de novo biosynthesis of polyamines for development of mammalian conceptuses.

Ornithine decarboxylase (ODC1) is considered the rate-controlling enzyme for the classical de novo biosynthesis of polyamines (putrescine, spermidine, and spermine) in mammals. However, metabolism of arginine to agmatine via arginine decarboxylase (ADC) and conversion of agmatine to polyamines via agmatinase (AGMAT) is an alternative pathway long recognized in lower organisms, but only recently suggested for neurons and liver cells of mammals. We now provide evidence for a functional ADC/AGMAT pathway for the synthesis of polyamines in mammalian reproductive tissue for embryonic survival and development. We first investigated cellular functions of polyamines by in vivo knockdown of translation of mRNA for ODC1 in ovine conceptus trophectoderm using morpholino antisense oligonucleotides (MAOs) and found that one-half of the conceptuses were morphologically and functionally either normal or abnormal. Furthermore, we found that increases in ADC/AGMAT mRNA levels and in the translation of AGMAT mRNA among conceptuses in MAO-ODC1 knockdown compensated for the loss of ODC1, supporting polyamine synthesis from arginine and accounting for the normal and abnormal phenotypes of conceptuses. We conclude that the majority of polyamine synthesis is by the conventional ODC1-dependent pathway (arginine-ornithine-putrescine) and that deficiencies in ODC1 result in increased activity of the rescue ADC/AGMAT-dependent pathway (arginine-agmatine-putrescine) for production of polyamines. The presence of an alternative ADC/AGMAT pathway for converting arginine into putrescine is functionally important for supporting survival and development of mammalian conceptuses.

Hydroxylamine derivatives for regulation of spermine and spermidine metabolism.

The biogenic polyamines spermine, spermidine, and their precursor putrescine are present in micro-to-millimolar concentrations in all cell types and are vitally important for their normal growth. High intracellular content of spermine and spermidine determines the multiplicity of the cellular functions of the polyamines. Many of these functions are not well characterized at the molecular level, ensuring the ongoing development of this field of biochemistry. Tumor cells have elevated polyamine level if compared with normal cells, and this greatly stimulates the search for new opportunities to deplete the intracellular pool of spermine and spermidine resulting in decrease in cell growth and even cell death. O-Substituted hydroxylamines occupy their own place among chemical regulators of the activity of the enzymes of polyamine metabolism. Varying the structure of the alkyl substituent made it possible to obtain within one class of chemical compounds highly effective inhibitors and regulators of the activity of all the enzymes of putrescine, spermine and spermidine metabolism (with the exception of FAD-dependent spermine oxidase and acetylpolyamine oxidase), effectors of the polyamine transport system, and even actively transported in cells "proinhibitor" of ornithine decarboxylase. Some principles for the design of specific inhibitors of these enzymes as well as the peculiarities of cellular effects of corresponding O-substituted hydroxylamines are discussed.

Role of ornithine decarboxylase in regulation of estrogen receptor alpha expression and growth in human breast cancer cells.

Our previous studies demonstrated that specific polyamine analogues, oligoamines, down-regulated the activity of a key polyamine biosynthesis enzyme, ornithine decarboxylase (ODC), and suppressed expression of estrogen receptor alpha (ERα) in human breast cancer cells. However, the mechanism underlying the potential regulation of ERα expression by polyamine metabolism has not been explored. Here, we demonstrated that RNAi-mediated knockdown of ODC (ODC KD) down-regulated the polyamine pool, and hindered growth in ERα-positive MCF7 and T47D and ERα-negative MDA-MB-231 breast cancer cells. ODC KD significantly induced the expression and activity of the key polyamine catabolism enzymes, spermine oxidase (SMO) and spermidine/spermine N (1)-acetyltransferase (SSAT). However, ODC KD-induced growth inhibition could not be reversed by exogenous spermidine or overexpression of antizyme inhibitor (AZI), suggesting that regulation of ODC on cell proliferation may involve the signaling pathways independent of polyamine metabolism. In MCF7 and T47D cells, ODC KD, but not DFMO treatment, diminished the mRNA and protein expression of ERα. Overexpression of antizyme (AZ), an ODC inhibitory protein, suppressed ERα expression, suggesting that ODC plays an important role in regulation of ERα expression. Decrease of ERα expression by ODC siRNA altered the mRNA expression of a subset of ERα response genes. Our previous analysis showed that oligoamines disrupt the binding of Sp1 family members to an ERα minimal promoter element containing GC/CA-rich boxes. By using DNA affinity precipitation and mass spectrometry analysis, we identified ZBTB7A, MeCP2, PARP-1, AP2, and MAZ as co-factors of Sp1 family members that are associated with the ERα minimal promoter element. Taken together, these data provide insight into a novel antiestrogenic mechanism for polyamine biosynthesis enzymes in breast cancer.