Ornithine Decarboxylase Activity Research Articles

Antizyme Inhibitor 2-Deficient Mice Exhibit Altered Brain Polyamine Levels and Reduced Locomotor Activity.

Alterations in the neural polyamine system are known to be associated with different brain pathological conditions. In addition, the regulation of enzymes involved in polyamine metabolism such as ornithine decarboxylase (ODC), antizymes (AZs), and antizyme inhibitors (AZINs) is critical during brain development. However, while most studies focus on ODC and AZs, less is known about AZIN expression and function in the brain. Thus, our aim was to analyze the expression pattern of AZIN2 during postnatal development, its brain distribution, and its possible implication in phenotypical alterations. The expression pattern of Azin2 and other genes related to polyamine metabolism was analyzed by RT-qPCR. β-D-galactosidase staining was used to determine the anatomical distribution of AZIN2 in a Azin2 knockout model containing the βGeo marker. Brain polyamine content was determined by HPLC. The Rota-Rod and Pole functional tests were used to evaluate motor skills in Azin2-lacking mice. Our results showed that expression of genes codifying for AZs and AZINs showed a similar increasing pattern over time that coincided with a decrease in ODC activity and putrescine levels. The analysis of AZIN2 distribution demonstrated that it is strongly expressed in the cerebellum and distributed along the neuron body and dendrites. The ablation of Azin2 showed a decrease in putrescine levels and is related to reduced motor skills. Our study revealed that AZIN2 expression in the brain is particularly limited to the cerebellum. In addition, the ablation of Azin2 leads to a reduction in putrescine that relates to alterations in motor function, suggesting the role of AZIN2 in the functioning of dopaminergic neurons.

Heterologous expression and activity verification of ornithine decarboxylase from a wild strain of Shewanella xiamenensis.

Shewanella xiamenensis is widely found in spoilage fish, shrimp and other seafoods. Under suitable conditions, ornithine can be synthesized into putrescine, which may spoil food or endanger health. Our research used a wild strain of Shewanella xiamenensis isolated from "Yi Lu Xian" salted fish (a salting method for sea bass) as a research object. According to the database of National Center of Biotechnology Information (NCBI), the target ornithine decarboxylase (ODC) gene SpeF was successfully amplified using the wild strain of Shewanella xiamenensis as the template. Sequencing alignment showed that the SpeF of the wild strain had more than 98% homology compared with the standard strain. The amino acid substitution occurred in the residues of 343, 618, 705, and 708 in the wild strain. After optimizing the expression conditions, a heterologous expression system of ODC was constructed to achieve a high yield of expression. The amount of 253.38 mg of ODC per liter of LB broth was finally expressed. High performance liquid chromatography (HPLC) and subsequent ODC activity verification experiments showed that hetero-expressed ODC showed a certain enzyme activity for about 11.91 ± 0.38 U/mg. Our study gives a new way to the development of a low-cost and high-yield strategy to produce ODC, providing experimental materials for further research and elimination of putrescine in food hazards.

Salmonella enterica serovar Cerro displays a phylogenetic structure and genomic features consistent with virulence attenuation and adaptation to cattle.

Salmonella enterica subsp. enterica (S.) serovar Cerro is rarely isolated from human clinical cases of salmonellosis but represents the most common serovar isolated from cattle without clinical signs of illness in the United States. In this study, using a large, diverse set of 316 isolates, we utilized genomic methods to further elucidate the evolutionary history of S. Cerro and to identify genomic features associated with its apparent virulence attenuation in humans. Phylogenetic analyses showed that within this polyphyletic serovar, 98.4% of isolates (311/316) represent a monophyletic clade within section Typhi and the remaining 1.6% of isolates (5/316) form a monophyletic clade within subspecies enterica Clade A1. Of the section Typhi S. Cerro isolates, 93.2% of isolates (290/311) clustered into a large clonal clade comprised of predominantly sequence type (ST) 367 cattle and environmental isolates, while the remaining 6.8% of isolates (21/311), primarily from human clinical sources, clustered outside of this clonal clade. A tip-dated phylogeny of S. Cerro ST367 identified two major clades (I and II), one of which overwhelmingly consisted of cattle isolates that share a most recent common ancestor that existed circa 1975. Gene presence/absence and rarefaction curve analyses suggested that the pangenome of section Typhi S. Cerro is open, potentially reflecting the gain/loss of prophage; human isolates contained the most open pangenome, while cattle isolates had the least open pangenome. Hypothetically disrupted coding sequences (HDCs) displayed clade-specific losses of intact speC and sopA virulence genes within the large clonal S. Cerro clade, while loss of intact vgrG, araH, and vapC occurred in all section Typhi S. Cerro isolates. Further phenotypic analysis suggested that the presence of a premature stop codon in speC does not abolish ornithine decarboxylase activity in S. Cerro, likely due to the activity of the second ornithine decarboxylase encoded by speF, which remained intact in all isolates. Overall, our study identifies specific genomic features associated with S. Cerro's infrequent isolation from humans and its apparent adaptation to cattle, which has broader implications for informing our understanding of the evolutionary events facilitating host adaptation in Salmonella.

The inhibition of putrescine synthesis affects the in vitro shoot development of Cedrela fissilis Vell. (Meliaceae) by altering endogenous polyamine metabolism and the proteomic profile

Polyamines —(PAs) and proteins have been demonstrated to be fundamental for in vitro shoot development of Cedrela fissilis. We evaluated the influence of 6-benzyladenine (BA) and putrescine (Put) on the growth of shoots, PA metabolism and proteomic profiles of C. fissilis. The longest shoots were obtained under 2.5 µM BA + 2.5 mM Put treatment. The inhibition of Put synthesis by d-arginine (d-arg) reduced the activities of the Put biosynthesis enzymes arginine decarboxylase (ADC) and ornithine decarboxylase (ODC) and the endogenous contents of free Put, resulting in reduced shoot growth. The ODC activity was higher than that of ADC, being ODC the main enzyme in the synthesis of Put in C. fissilis. Inhibition of Put synthesis affected the proteomic profile, reducing the accumulation of the ubiquitin receptor RAD23c, peroxidase 15, ADP-ribosylation factor 1, ADP-ribosylation factor-like protein 8a, profilin-4, profilin-2, glucan endo-1,3-beta-glucosidase, and expansin-like B1 and increasing the accumulation of V-type proton ATPase catalytic subunit A and methionine gamma-lyase, highlighting the relevance of these proteins in promoting the length of shoots. Moreover, the transport protein SEC13 homolog B and the basic isoform glucan endo-1,3-beta-glucosidase, unique proteins in shoots treated with BA + Put, were related to the promotion of shoot growth. Our results show that the modulation of endogenous PAs and proteomic profiles is necessary to regulate in vitro morphogenesis in C. fissilis. Moreover, the ODC enzyme is highly involved in the synthesis of Put during in vitro shoot development and is described for the first time in this species.

Analytical assays to evaluate enzymatic activity and screening of inhibitors for ornithine decarboxylase

Ornithine decarboxylase (ODC) catalyzes the decarboxylation of ornithine to produce putrescine, the first step in the metabolism of polyamines (putrescine, spermidine, and spermine), which are essential growth factors in eukaryotic cells. ODC is active as a homodimer and depends on pyridoxal 5′-phosphate (PLP) as a cofactor. An increase in the concentration of polyamines has been associated with carcinogenesis. Therefore, there is much interest in identifying inhibitors of this pathway as potential chemotherapeutic and chemopreventive agents. The best-known inhibitor of mammalian ODC is α-difluoromethylornithine (DFMO), a highly selective compound that alkylates Cys-360 (a residue of the ODC active site). Although DFMO was initially developed for the treatment of cancer, the World Health Organization recommends its use in combination with nifurtimox for the treatment of human African trypanosomiasis. Considering the importance of ODC as a promising target for the treatment of various types of cancer and other infectious diseases, choosing the right method for screening potential inhibitors can help to accelerate the discovery of new drugs. Several methods for the determination of ODC activity are found in the literature. Among these, we can mention analysis with radioactive markers, colorimetric assays using auxiliary enzymes to detect CO2 or H2O2 release, chromatographic separations with putrescine derivatization, mass spectrometry, and spectroscopic techniques. In this review, the main analysis methods used will be described, highlighting their advantages and disadvantages, as well as identifying the most promising methods for high-throughput screening.

Structure and Enzymatic Activity of an Intellectual Disability-Associated Ornithine Decarboxylase Variant, G84R.

Ornithine decarboxylase (ODC) is a rate-limiting enzyme for the synthesis of polyamines (PAs). PAs are required for proliferation, and increased ODC activity is associated with cancer and neural over-proliferation. ODC levels and activity are therefore tightly regulated, including through the ODC-specific inhibitor, antizyme AZ1. Recently, ODC G84R has been reported as a partial loss-of-function variant that is associated with intellectual disability and seizures. However, G84 is distant from both the catalytic center and the ODC homodimerization interface. To understand how G84R modulates ODC activity, we have determined the crystal structure of ODC G84R in both the presence and the absence of the cofactor pyridoxal 5-phosphate. The structures show that the replacement of G84 by arginine leads to hydrogen bond formation of R84 with F420, the last residue of the ODC C-terminal helix, a structural element that is involved in the AZ1-mediated proteasomal degradation of ODC. In contrast, the catalytic center is essentially indistinguishable from that of wildtype ODC. We therefore reanalyzed the catalytic activity of ODC G84R and found that it is rescued when the protein is purified in the presence of a reducing agent to mimic the reducing environment of the cytoplasm. This suggests that R84 may exert its neurological effects not through reducing ODC catalytic activity but through misregulation of its AZ1-mediated proteasomal degradation.

Exogenous Hemin alleviated cadmium stress in maize (Zea mays L.) by enhancing leaf photosynthesis, AsA-GSH cycle and polyamine metabolism.

Cadmium (Cd) stress is one of the principal abiotic stresses that inhibit maize growth. The research was to explore (hemin chloride) Hemin (100 μmol L−1) on photosynthesis, ascorbic acid (AsA)-glutathione (GSH) cycle system, and polyamine metabolism of maize under Cd stress (85 mg L−1) using nutrient solution hydroponics, with Tiannong 9 (Cd tolerant) and Fenghe 6 (Cd sensitive) as experimental materials. The results showed that Hemin can increase leaf photosynthetic pigment content and ameliorate the ratio of Chlorophyll a/chlorophyll b (Chla/Chlb) under Cd stress. The values of ribose 1, 5-diphosphate carboxylase/oxygenase (RuBPcase) and phosphoenolpyruvate carboxylase (PEPCase), and total xanthophyll cycle pool [(violoxanthin (V), antiflavin (A) and zeaxanthin (Z)] increased, which enhancing xanthophyll cycle (DEPS) de-epoxidation, and alleviating stomatal and non-stomatal limitation of leaf photosynthesis. Hemin significantly increased net photosynthetic rate (Pn), stomatal conductance (gs), transpiration rate (Tr), photochemical quenching coefficient (qP), PSII maximum photochemical efficiency (Fv/Fm), and electron transfer rate (ETR), which contributed to the improvement of the PSII photosynthetic system. Compared with Cd stress, Hemin can reduce thiobartolic acid reactant (TBARS) content, superoxide anion radical (O2−) production rate, hydrogen peroxide (H2O2) accumulation, and the extent of electrolyte leakage (EL); decreased the level of malondialdehyde (MDA) content and increased the activities of superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT); slowed the decrease in dehydroascorbic acid reductase (DHAR) and monodehydroascorbate reductase (MDHAR) activity and the increase in glutathione reductase (GR) and ascorbate peroxidase (APX) activity in leaves; promoted the increase in AsA and GSH content, decreased dehydroascorbic acid (DHA) and oxidized glutathione (GSSG), and increased AsA/DHA and GSH/GSSG ratios under Cd stress. Hemin promoted the increase of conjugated and bound polyamine content, and the conversion process speed of free putrescine (Put) to free spermine (Spm) and spermidine (Spd) in maize; decreased polyamine oxidase (PAO) activity and increased diamine oxidase (DAO), arginine decarboxylase (ADC), ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (SAMDC) enzyme activities in leaves under Cd stress.

Brassinosteroids enhance resistance to manganese toxicity in Malus robusta Rehd. via modulating polyamines profile

Manganese (Mn) toxicity in soil is a widely observed phenomenon, which seriously restricts growth, quality, and yield of various crops and fruits including apples. However, mechanisms underlying the regulation of polyamines (PAs) by brassinosteroids (BRs) to improve tolerance to Mn stress are still unclear. In this study, we investigated the effects of 2,4-epibrassinolide (EBL; a BR) on the expression of genes involved in BR signaling pathway, Mn accumulation, PAs-mediated responses (PA precursor levels, metabolic enzymes, and genes), and growth parameters in Mn-stressed Malus robusta Rehd. EBL application significantly modulated the expressions of genes related to BR signaling (MdBRI, MdBSK, etc.) and reduced Mn accumulation, along with improving the rate of increase in root length and plant height, relative water content, chlorophyll content, maximum photochemical efficiency of PSII (Fv/Fm), and actual photochemical efficiency (ΦPSII) and decreasing electrical conductivity. Furthermore, EBL application significantly reduced putrescine (Put) accumulation and increased spermine (Spm) content and (Spd + Spm)/Put ratio. EBL weakened ornithine (Orn) pathway, decreased ornithine decarboxylase (ODC) activity, and increased biosynthesis of Spm from Put via elevating the PA oxidase (PAO) activity and expression of MdSPDS, MdSPMS, and MdPAO. The trends for free, PS-conjugated, and PIS-bound PAs were similar to that of total PAs, except that no significant change was observed in free Spm, PS-conjugated Spd, and Spm, as well as PIS-bound Spd. This study revealed that BR-regulated PAs help in mitigating Mn toxicity and clarified the mechanisms of regulation of PAs by BRs in apple trees.

Ornithine Decarboxylase in Gastric Epithelial Cells Promotes the Immunopathogenesis of Helicobacter pylori Infection.

Colonization by Helicobacter pylori is associated with gastric diseases, ranging from superficial gastritis to more severe pathologies, including intestinal metaplasia and adenocarcinoma. The interplay of the host response and the pathogen affect the outcome of disease. One major component of the mucosal response to H. pylori is the activation of a strong but inefficient immune response that fails to control the infection and frequently causes tissue damage. We have shown that polyamines can regulate H. pylori-induced inflammation. Chemical inhibition of ornithine decarboxylase (ODC), which generates the polyamine putrescine from l-ornithine, reduces gastritis in mice and adenocarcinoma incidence in gerbils infected with H. pylori However, we have also demonstrated that Odc deletion in myeloid cells enhances M1 macrophage activation and gastritis. Here we used a genetic approach to assess the specific role of gastric epithelial ODC during H. pylori infection. Specific deletion of the gene encoding for ODC in gastric epithelial cells reduces gastritis, attenuates epithelial proliferation, alters the metabolome, and downregulates the expression of immune mediators induced by H. pylori Inhibition of ODC activity or ODC knockdown in human gastric epithelial cells dampens H. pylori-induced NF-κB activation, CXCL8 mRNA expression, and IL-8 production. Chronic inflammation is a major risk factor for the progression to more severe pathologies associated with H. pylori infection, and we now show that epithelial ODC plays an important role in mediating this inflammatory response.

Is Glyceryl Trinitrate, a Nitric Oxide Donor Responsible for Ameliorating the Chemical-Induced Tissue Injury In Vivo?

Oxidative stress induced by well-known toxins including ferric nitrilotriacetate (Fe-NTA), carbon tetrachloride (CCl4) and thioacetamide (TAA) has been attributed to causing tissue injury in the liver and kidney. In this study, the effect of glyceryl trinitrate (GTN), a donor of nitric oxide and NG-nitroarginine methyl ester (l-NAME), a nitric oxide inhibitor on TAA-induced hepatic oxidative stress, GSH and GSH-dependent enzymes, serum transaminases and tumor promotion markers such as ornithine decarboxylase (ODC) activity and [3H]-thymidine incorporation in rats were examined. The animals were divided into seven groups consisting of six healthy rats per group. The six rats were injected intraperitoneally with TAA to evaluate its toxic effect, improvement in its toxic effect if any, or worsening in its toxic effect if any, when given in combination with GTN or l-NAME. The single necrogenic dose of TAA administration caused a significant change in the levels of both hepatic and serum enzymes such as glutathione S-transferase (GST), glutathione reductase (GR), glutathione peroxidase (GPx), γ-glutamyl transpeptidase (GGT), glucose 6-phosphate dehydrogenase (G6PD), alanine aminotransferase (AST) and aspartate aminotransferase (ALT). In addition, treatment with TAA also augmented malondialdehyde (MDA), ornithine decarboxylase (ODC) activity and [3H]-thymidine incorporation in rats liver. Concomitantly, TAA treatment depleted the levels of GSH. However, most of these changes were alleviated by the treatment of animals with GTN dose-dependently. The protective effect of GTN against TAA was also confirmed histopathologically. The present data confirmed our earlier findings with other oxidants including Fe-NTA and CCl4. The GTN showed no change whatsoever when administered alone, however when it was given along with TAA then it showed protection thereby contributing towards defending the role against oxidants-induced organ toxicity. Overall, GTN may contribute to protection against TAA-induced oxidative stress, toxicity, and proliferative response in the liver, according to our findings.

Abstract 5488: The potential of spermine analogue SBP-101 (diethyl dihydroxyhomospermine) as a polyamine metabolism modulator in ovarian cancer

Abstract The naturally occurring polyamines, putrescine, spermidine and spermine, are polycationic alkylamines that are essential for cellular growth and proliferation. As such, many cancers are reliant on elevated polyamine levels that are maintained through dysregulated polyamine metabolism. Polyamine metabolism is thus a promising target for cancer therapeutics, and modulation of polyamine metabolism has been attempted with numerous enzyme inhibitors and polyamine analogues. SBP-101 (diethyl dihydroxyhomospermine) is a novel spermine analogue that has shown efficacy in slowing pancreatic tumor progression both in vitro and in vivo. Here we determined the effect of SBP-101 treatment on polyamine metabolism in a variety of cancer cell types in vitro including lung, ovarian, prostate, pancreatic and breast. We evaluated the activity of four enzymes involved in the polyamine pathway following treatment with either SBP-101 or the well-characterized spermine analogue, BENSpm (N1,N11-bisethylnorspermine). Additionally, we determined by high performance liquid chromatography the effect of SBP-101 on intracellular polyamine pools and the accumulation of the analogue itself. The activity of the biosynthetic enzymes ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase and the catabolic enzymes spermidine/spermine-N-(1)-acetyltransferase and spermine oxidase were determined with and without treatment with the polyamine analogues. SBP-101 treatment resulted in a varying increase in the activity of polyamine catabolic enzymes in a subset of tested cell lines, while it downregulated the activity of the biosynthetic enzyme ODC across all cell types studied. These results indicate that SBP-101 likely exerts its effect predominately through decreased polyamine biosynthesis with minor upregulation of catabolism in contrast to the structurally similar BENSpm where the increase in polyamine catabolism is the predominant response. A sustained elevation of polyamine levels plays a role in the immunosuppressive environment of some cold tumors, and the pharmacologic and genetic modulation of polyamine metabolism has demonstrated success in reducing immunosuppressive phenotypes. Therefore, to extend our in vitro results, we evaluated the efficacy of SBP-101 in the immunosuppressive VDID8+ murine ovarian cancer model. SBP-101 caused a marked increase in median survival comparable to that of some promising combination therapies. Future studies will determine the synergistic effects, if any, of SBP-101 in combination with other polyamine metabolism modulators as well as with immune modulators. Citation Format: Cassandra E. Holbert, Tracy Murray Stewart, Jennifer K. Simpson, Michael J. Walker, Robert A. Casero. The potential of spermine analogue SBP-101 (diethyl dihydroxyhomospermine) as a polyamine metabolism modulator in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5488.

Novel Chemical Probes targeting the Polyamine Regulatory Circuit

Polyamines are vital for cellular life. Polyamines such as putrescine, spermidine, and spermine regulate the essential cellular process, including gene expression and cell proliferation. Given its cellular role, polyamine concentration in cells is tightly controlled. However, aberrant polyamine metabolism is associated with numerous diseases, including cancer. Growing evidence suggests that elevated polyamine levels are the absolute requirement for tumor growth and progression. The concentrations of cellular polyamines are controlled by the polyamine regulatory circuit comprising three different proteins: Ornithine Decarboxylase (ODC), Ornithine Decarboxylase Antizyme (OAZ), and Antizyme Inhibitor (AZIN). While ODC is directly involved in polyamine biosynthesis, OAZ and AZIN regulate the ODC activity via protein‐protein interactions. The dysregulation of ODC, OAZ, and AZIN leads to elevated polyamines in numerous pathologies, making them attractive targets to control polyamine levels. Besides regulating polyamine synthesis, OAZ is believed to modulate polyamine transport via multiple pathways. However, the precise mechanism of OAZ‐mediated polyamine regulation outside of the polyamine biosynthetic pathway remains elusive. We hypothesize that OAZ sequestration results in increased polyamine uptake. To provide evidence to our hypothesis, we generated covalent OAZ‐mimetic inactivators targeting both ODC and AZIN. Using biochemical experiments, we demonstrate that these OAZ‐mimetics inhibit the activity of ODC and prevent the sequestration of OAZ by AZIN. Using Mass spectrometric experiments, we determine the covalent binding of OAZ‐mimetics to both ODC and AZIN. We employ a novel gel assay to assess the rate of covalent modification of ODC and AZIN. Finally, using computational modeling, we determine the mode of binding of OAZ‐mimetics to both ODC and AZIN to provide insights into inhibition mechanisms.

Role of Polyamine-Induced Dimerization of Antizyme in Its Cellular Functions.

The polyamines, spermine (Spm) and spermidine (Spd), are important for cell growth and function. Their homeostasis is strictly controlled, and a key downregulator of the polyamine pool is the polyamine-inducible protein, antizyme 1 (OAZ1). OAZ1 inhibits polyamine uptake and targets ornithine decarboxylase (ODC), the rate-limiting enzyme of polyamine biosynthesis, for proteasomal degradation. Here we report, for the first time, that polyamines induce dimerization of mouse recombinant full-length OAZ1, forming an (OAZ1)2-Polyamine complex. Dimerization could be modulated by functionally active C-methylated spermidine mimetics (MeSpds) by changing the position of the methyl group along the Spd backbone—2-MeSpd was a poor inducer as opposed to 1-MeSpd, 3-MeSpd, and Spd, which were good inducers. Importantly, the ability of compounds to inhibit polyamine uptake correlated with the efficiency of the (OAZ1)2-Polyamine complex formation. Thus, the (OAZ1)2-Polyamine complex may be needed to inhibit polyamine uptake. The efficiency of polyamine-induced ribosomal +1 frameshifting of OAZ1 mRNA could also be differentially modulated by MeSpds—2-MeSpd was a poor inducer of OAZ1 biosynthesis and hence a poor downregulator of ODC activity unlike the other MeSpds. These findings offer new insight into the OAZ1-mediated regulation of polyamine homeostasis and provide the chemical tools to study it.

Carvacrol Arrests the Proliferation of Hypopharyngeal Carcinoma Cells by Suppressing Ornithine Decarboxylase and Hyaluronidase Activities.

Carvacrol, a monoterpene known for its pharmacological activities, is present in the essential oil of Origanum majorana, Origanum vulgare, Thymus vulgaris, and Lippia graveolens. It is used in food as a flavoring and preservative agent in cosmetics and medicines because of its useful bioactivities in clinical practice. However, carvacrol was not much explored for its anticancer potential. Targeting enzymes involved in carcinogenesis, such as ornithine decarboxylase (ODC), cyclooxygenase-2 (COX-2), lipoxygenase-5 (LOX-5), and hyaluronidase (HYAL) by monoterpenes are amongst the efficient approaches for cancer prevention and treatment. In this study, the efficacy of carvacrol was investigated against deregulated cancer biomarkers/targets in organ-specific human cancer cell lines (FaDu, K562, and A549) utilizing in vitro, in silico, and in vivo approaches. The efficacy of carvacrol was evaluated on human cancer cell lines using neutral red uptake (NRU), sulpho rhodamine B (SRB), and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assays. The mechanistic study was carried out in cell-based test systems. Further, the potency of carvacrol was confirmed by the quantitative real-time PCR analysis and molecular docking studies. The in vivo anti-tumor potential of carvacrol was performed on mice S-180 model, and the toxicity examination was accomplished through in silico approach. Carvacrol significantly impeded the growth of FaDu, K562, and A549 cell lines with IC50 values ranging from 9.61 ± 0.05 to 81.32 ± 11.83 μM. Further, the efficacy of carvacrol was explored against different cancer targets in FaDu, K562, and A549 cell lines. Carvacrol inhibits the ODC, COX-2, LOX-5, and HYAL activities in FaDu cell line and ODC, COX-2, and HYAL activities in K562 cell line. The results were validated by expression analysis revealing the downregulation of the targeted gene with a significant change in the transcript level of ODC and HYAL in FaDu cell line with a fold change of 1.56 and 1.61, respectively. A non-significant effect of carvacrol was observed on the downstream signaling pathway of PI3K and HIF-1α/vascular endothelial growth factor (VEGF) in FaDu cells. The cell cycle, reactive oxygen species (ROS), mitochondrial membrane potential (MMP), and Annexin V-fluorescein isothiocyanate (FITC) experiments demonstrate that carvacrol induces apoptosis of FaDu cells. Further, the potency of carvacrol was also evaluated in vivo on mice S-180 tumor model, wherein it inhibits tumor growth (72%) at 75 mg/kg body weight (bw). ADMET studies predicted carvacrol as a safe molecule. Overall, carvacrol delayed the growth of FaDu, K562, and A549 cell lines by targeting enzymes involved in the carcinogenesis process. The existence of one hydroxyl group at the para position of carvacrol could be responsible for the anti-proliferative activity. Thus, carvacrol could be used as a pharmacophore to develop a safe and effective multi-targeted anti-cancer medicament.

Co-functioning of 2AP precursor amino acids enhances 2-acetyl-1-pyrroline under salt stress in aromatic rice (Oryza sativa L.) cultivars

Aromatic rice (Oryza sativa) fetches a premium price due to the pleasant aroma. The major aroma compound 2-acetyl-1-pyrroline (2AP) has been found to be enhanced under stress. This condition can be considered to study the genes, precursors, enzymes, and metabolites involved in elevated levels of 2AP biosynthesis. In the present study, 100 mM salt treatment was given to two aromatic rice cultivars Ambemohar-157 (A-157) and Basmati-370 (B-370) at the vegetative stage (VS3). After salt treatment, in the leaves, 2AP contents were elevated by 2.2 and 1.8 fold in A-157 and B-370, respectively. Under these elevated 2AP conditions, the precursor amino acids (glutamate, putrescine, ornithine, and proline), their related genes, enzymes, and metabolites (methylglyoxal and γ-aminobutyric acid (GABA) related to 2AP biosynthesis were analyzed. In addition, agronomic characters were also studied. It was observed that the proline content was enhanced in both the cultivars by 29% (A-157) and 40% (B-370) as compared to control. The Δ1-pyrroline-5-carboxylate synthetase (P5CS) enzyme activity was increased in salt-treated plants leaf tissue by 31% (A-157) and 40% (B-370) compared to control. The P5CS gene expression was enhanced by A-157 (1.8 fold) and B-370 (2.2 fold) compared to control, putrescine content in A-157 and B-370 decreased by 2.5 and 2.7 fold respectively as compared to control. The ornithine decarboxylase (ODC) activity was enhanced in A-157 (12%) and B-370 (35%) over control. Further, ODC gene expression was enhanced in both the cultivars A-157 (1.5 fold) and B-370 (1.3 fold). The diamino oxidase (DAO) enzyme activity was increased by 28% (A-157) and 35% (B-370) respectively over control. The GABA content marginally increased over control in both the cultivars namely, A-157 (1.9%) and B-370 (9.5%). The methylglyoxal levels were enhanced by 1.4 fold in A-157 and 1.6 fold in B-370. Interestingly, the enhancement in 2AP in the vegetative stage also helped to accumulate it in mature grains (twofold in A-157 and 1.5 fold in B-370) without test weight penalty. The study indicated that the ornithine and proline together along with methylglyoxal contribute towards the enhancement of 2AP under salt stress.

Polyamines Metabolism Interacts with γ-Aminobutyric Acid, Proline and Nitrogen Metabolisms to Affect Drought Tolerance of Creeping Bentgrass.

Due to increased global warming and climate change, drought has become a serious threat to horticultural crop cultivation and management. The purpose of this study was to investigate the effect of spermine (Spm) pretreatment on metabolic alterations of polyamine (PAs), γ-aminobutyric acid (GABA), proline (Pro), and nitrogen associated with drought tolerance in creeping bentgrass (Agrostis stolonifera). The results showed that drought tolerance of creeping bentgrass could be significantly improved by the Spm pretreatment, as demonstrated by the maintenance of less chlorophyll loss and higher photosynthesis, gas exchange, water use efficiency, and cell membrane stability. The Spm pretreatment further increased drought-induced accumulation of endogenous PAs, putrescine, spermidine, and Spm, and also enhanced PAs metabolism through improving arginine decarboxylases, ornithine decarboxylase, S-adenosylmethionine decarboxylase, and polyamine oxidase activities during drought stress. In addition, the Spm application not only significantly improved endogenous GABA content, glutamate content, activities of glutamate decarboxylase and α-ketoglutarase, but also alleviated decline in nitrite nitrogen content, nitrate reductase, glutamine synthetase, glutamate synthetase, and GABA aminotransferase activities under drought stress. The Spm-pretreated creeping bentgrass exhibited significantly lower ammonia nitrogen content and nitrite reductase activity as well as higher glutamate dehydrogenase activity than non-pretreated plants in response to drought stress. These results indicated beneficial roles of the Spm on regulating GABA and nitrogen metabolism contributing towards better maintenance of Tricarboxylic acid (TCA) cycle in creeping bentgrass. Interestingly, the Spm-enhanced Pro metabolism rather than more Pro accumulation could be the key regulatory mechanism for drought tolerance in creeping bentgrass. Current findings provide a comprehensive understanding of PAs interaction with other metabolic pathways to regulate drought tolerance in grass species.

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.

Inhibitory Effect of Ursolic Acid on the Migration and Invasion of Doxorubicin-Resistant Breast Cancer.

The cause of death in most breast cancer patients is disease metastasis and the occurrence of multidrug resistance (MDR). Ornithine decarboxylase (ODC), which is involved into multiple pathways, is closely related to carcinogenesis and development. Ursolic acid (UA), a natural triterpenoid compound, has been shown to reverse the MDR characteristics of tumor cells. However, the effect of UA on the invasion and metastasis of tumor cells with MDR is not known. Therefore, we investigated the effects of UA on invasion and metastasis, ODC-related polyamine metabolism, and MAPK-Erk-VEGF/MMP-9 signaling pathways in a doxorubicin-resistant breast cancer cell (MCF-7/ADR) model. The obtained results showed that UA significantly inhibited the adhesion and migration of MCF-7/ADR cells, and had higher affinities with key active cavity residues of ODC compared to the known inhibitor di-fluoro-methyl-ornithine (DFMO). UA could downregulate ODC, phosphorylated Erk (P-Erk), VEGF, and matrix metalloproteinase-9 (MMP-9) activity. Meanwhile, UA significantly reduced the content of metabolites of the polyamine metabolism. Furthermore, UA increased the intracellular accumulation of Dox in MCF-7/ADR cells. Taken together, UA can inhibit against tumor progression during the treatment of breast cancer with Dox, and possibly modulate the Erk-VEGF/MMP-9 signaling pathways and polyamine metabolism by targeting ODC to exert these effects.

Suppression of cytokine storm and associated inflammatory mediators by salicylaldehyde derivative of pregabalin: An innovative perspective for alleviating airway inflammation and lung remodeling

ObjectivesPregsal is a novel salicylaldehyde derivative of pregabalin, a structural analogue of gamma-aminobutyric acid approved for treating epilepsy, neuropathic pain and diabetic peripheral neuropathy but not trialled to date for airway inflammatory diseases. The current study evaluated a preclinical model of airway inflammation and lung remodelling induced by ovalbumin (OVA) for the potential benefits of pregsal. MethodsWistar rats (N = 10 per group) were divided into four groups sensitized intraperitoneally (i.p.) and then challenged intranasally with OVA, with or without i.p. pregsal (100 mg/kg) or methylprednisolone (MP) (15 mg/kg). Airway inflammation was assessed through inflammatory cell infiltration in the lungs, delayed-type hypersensitivity (DTH), and nitric oxide (NO) level using bronchoalveolar lavage fluid (BALF) and lung tissues. The mRNA expression levels of a panel of inflammatory mediators (cytokines, chemokine and growth factors) in the lungs were measured by the reverse transcription-polymerase chain reaction (RT-PCR). Systemic inflammation was assessed using splenocyte proliferation and total and differential leucocyte count in blood and BALF. Lung remodelling was assessed by wet/dry lung weight ratio, epithelial thickness and goblet cell hyperplasia, hydroxyproline and osteopontin (OPN) levels, arginase activity in lungs, and ornithine decarboxylase (ODC) activity in lung mitochondria. ResultsPregsal significantly alleviated the total and differential leucocyte count in blood and BALF, NO production in BALF and recruitment of inflammatory cells in the lungs. It suppressed the T-cell response and attenuated the OVA-induced lung epithelial thickness, goblet cell hyperplasia, wet/dry lung weight ratio, hydroxyproline and OPN levels, arginase and ODC activity. Levels of inflammatory mediators were also downregulated in the lungs by pregsal. ConclusionsThe key findings of this study indicate that pregsal significantly reduces the development of airway inflammation and lung remodelling by suppression of cytokine storm and associated inflammatory mediators.

A Tear Metabolomic Profile Showing Increased Ornithine Decarboxylase Activity and Spermine Synthesis in Thyroid-Associated Orbitopathy.

About half of patients with Graves’ disease develop an orbitopathy related to an inflammatory expansion of the periorbital adipose tissue and muscles. We used a targeted metabolomic approach measuring 188 metabolites by mass spectrometry to compare the metabolic composition of tears in patients with active (n = 21) versus inactive (n = 24) thyroid-associated orbitopathy. Among the 44 metabolites accurately measured, 8 showed a significant alteration of their concentrations between the two groups. Two short-chain acylcarnitines, propionylcarnitine and butyrylcarnitine, and spermine showed increased concentrations in the tears of patients with active orbitopathy, whereas ornithine, glycine, serine, citrulline and histidine showed decreased concentrations in this group. In addition, the ratio putrescine/ornithine, representing the activity of ornithine decarboxylase, was significantly increased in patients with active compared to inactive orbitopathy (p = 0.0011, fold change 3.75). The specificity of this candidate biomarker was maintained when compared to a control group with unclassified dry eye disease. Our results suggest that the stimulation of ornithine decarboxylase by TSH receptor autoantibodies in orbital fibroblasts could lead to increased synthesis of spermine, through the increased activity of ornithine decarboxylase, that may contribute to periorbital expansion in Graves’ ophthalmopathy.