Enzyme Ornithine Decarboxylase Research Articles

Identification and validation of a metabolism-related gene signature for predicting the prognosis of paediatric medulloblastoma

Medulloblastoma (MB) is a malignant brain tumour that is highly common in children and has a tendency to spread to the brain and spinal cord. MB is thought to be a metabolically driven brain tumour. Understanding tumour cell metabolic patterns and characteristics can provide a promising foundation for understanding MB pathogenesis and developing treatments. Here, by analysing RNA-seq data of MB samples from the Gene Expression Omnibus (GEO) database, 12 differentially expressed metabolic-related genes (DE-MRGs) were chosen for the construction of a predictive risk score model for MB. This model demonstrated outstanding accuracy in predicting the outcomes of MB patients and served as a standalone predictor. An evaluation of functional enrichment revealed that the risk score showed enrichment in pathways related to cancer promotion and the immune response. In addition, a high risk score was an independent poor prognostic factor for MB in patients with different ages, sexes, metastasis stages and subgroups (SHH and Group 4). Consistently, the metabolic enzyme ornithine decarboxylase (ODC1) was upregulated in MB patients with poor survival time. Inhibition of ODC1 in primary and metastatic MB cell lines decreased cell proliferation, migration and invasion but increased immune infiltration. This study could aid in identifying metabolic targets for MB as well as optimizing risk stratification systems and individual treatment plans for MB patients via the use of a metabolism-related gene prognostic risk score signature.

Abstract 7154: Ivospemin/doxorubicin combination modulates polyamine metabolism to improve survival in murine ovarian cancer models

Abstract Polyamines are small polycationic alkylamines that are absolutely required for the continual growth and proliferation of cancer cells. As cancer cells maintain elevated polyamine pools through dysregulated polyamine metabolism, its pharmacological modulation is a promising avenue in cancer therapeutics. The polyamine analogue ivospemin has shown efficacy in slowing pancreatic and ovarian tumor progression in vitro and in vivo and has demonstrated encouraging results in pancreatic cancer clinical trials. Considering nearly 75% of late-stage ovarian cancer patients develop resistant to platinum-based chemotherapies, limiting treatment options, the aim of our study is to determine the potential efficacy of ivospemin in combination with doxorubicin, a common chemotherapeutic used in platinum-resistant ovarian tumors. We have previously shown that ivospemin exposure decreases polyamine content in a variety of cancer cell lines through downregulation of the polyamine biosynthetic enzyme ornithine decarboxylase (ODC) and induction of the polyamine catabolic enzyme spermidine/spermine-N1-acetyltransferase (SSAT). Here we examine the potential of combining ivospemin with doxorubicin. Ivospemin treatment reduces cell viability in ovarian adenocarcinoma cell lines and increases the toxicity of doxorubicin regardless of cisplatin sensitivity. Cells treated with the combination exhibit a greater decrease in polyamine levels than cells treated with either single agent. This increased polyamine depletion and decreased survival is accomplished through modulation of polyamine metabolism, predominately through an additive induction of SSAT activity. Using the syngeneic VDID8+ ovarian murine model, we further evaluated the ability of ivospemin to improve response to doxorubicin at clinical and sub-clinical dosing. Ascites fluid was used as a marker for tumor burden and evaluated for polyamine content. We found that the combination treatment increases median survival, delays tumor onset, and decreases overall tumor burden compared to either clinical or subclinical doxorubicin dosing schemes. Combination treatment also decreases overall polyamine content in the ascites by 75%. N1-acetylated spermidine is enriched in ascites from combination-treated mice, consistent with an upregulation of SSAT in response to treatment. Recognizing the non-representative mutational status of the VDID8+ model as a limitation, we are currently evaluating the combination of ivospemin and doxorubicin in genetically defined murine models that better recapitulate human high-grade serous ovarian carcinomas. Ongoing studies will determine influences on the tumor microenvironment and will mechanistically evaluate the cooperativity of ivospemin and doxorubicin on pathways outside of polyamine metabolism. Citation Format: Cassandra E. Holbert, Jackson R. Foley, Ashley C. Nwafor, Ting-Ann Liu, Elizabeth Bruckheimer, Jennifer K. Simpson, Tracy Murray Stewart, Robert A. Casero. Ivospemin/doxorubicin combination modulates polyamine metabolism to improve survival in murine ovarian cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7154.

Preventing Cancer by Inhibiting Ornithine Decarboxylase: A Comparative Perspective on Synthetic vs. Natural Drugs.

This perspective delves into the investigation of synthetic and naturally occurring inhibitors, their patterns of inhibition, and the effectiveness of newly utilized natural compounds as inhibitors targeting the Ornithine decarboxylase enzyme. This enzyme is known to target the MYC oncogene, thereby establishing a connection between polyamine metabolism and oncogenesis in both normal and cancerous cells. ODC activation and heightened polyamine activity are associated with tumor development in numerous cancers and fluctuations in ODC protein levels exert a profound influence on cellular activity for inhibition or suppressing tumor cells. This perspective outlines efforts to develop novel drugs, evaluate natural compounds, and identify promising inhibitors to address gaps in cancer prevention, highlighting the potential of newly designed synthetic moieties and natural flavonoids as alternatives. It also discusses natural compounds with potential as enhanced inhibitors.

Effects of Spermine Synthase Deficiency in Mesenchymal Stromal Cells Are Rescued by Upstream Inhibition of Ornithine Decarboxylase.

Despite the well-known relevance of polyamines to many forms of life, little is known about how polyamines regulate osteogenesis and skeletal homeostasis. Here, we report a series of in vitro studies conducted with human-bone-marrow-derived pluripotent stromal cells (MSCs). First, we show that during osteogenic differentiation, mRNA levels of most polyamine-associated enzymes are relatively constant, except for the catabolic enzyme spermidine/spermine N1-acetyltransferase 1 (SAT1), which is strongly increased at both mRNA and protein levels. As a result, the intracellular spermidine to spermine ratio is significantly reduced during the early stages of osteoblastogenesis. Supplementation of cells with exogenous spermidine or spermine decreases matrix mineralization in a dose-dependent manner. Employing N-cyclohexyl-1,3-propanediamine (CDAP) to chemically inhibit spermine synthase (SMS), the enzyme catalyzing conversion of spermidine into spermine, also suppresses mineralization. Intriguingly, this reduced mineralization is rescued with DFMO, an inhibitor of the upstream polyamine enzyme ornithine decarboxylase (ODC1). Similarly, high concentrations of CDAP cause cytoplasmic vacuolization and alter mitochondrial function, which are also reversible with the addition of DFMO. Altogether, these studies suggest that excess polyamines, especially spermidine, negatively affect hydroxyapatite synthesis of primary MSCs, whereas inhibition of polyamine synthesis with DFMO rescues most, but not all of these defects. These findings are relevant for patients with Snyder-Robinson syndrome (SRS), as the presenting skeletal defects-associated with SMS deficiency-could potentially be ameliorated by treatment with DFMO.

Identification and characterization of polyamine metabolism in citrus in response to 'Candidatus Liberibacter asiaticus' infection.

Citrus Huanglongbing, one of the most devastating citrus diseases, is caused by Candidatus Liberibacter asiaticus (CLas). Polyamines are aliphatic nitrogen-containing compounds that play important roles in disease resistance. However, the role of polyamine metabolism in the tolerance of citrus to infection with CLas have not been extensively studied. We used HPLC and UPLC-Q/TOF-MS to detect the contents of nine polyamine metabolism-related compounds (PMRCs) in six citrus cultivars with varying levels of tolerance to CLas. Moreover, we systematically detected the changes of PMRC and H2O2 contents, and compared the gene expression levels and activities of enzymes involved in the polyamine metabolic pathway among healthy, asymptomatic, and symptomatic leaves of Newhall navel oranges infected with CLas. The tolerant and moderately tolerant varieties showed higher PMRC levels than those of susceptible varieties. Compared with the healthy group, the symptomatic group showed significantly increased contents of arginine, ornithine, γ-aminobutyric acid, and putrescine by approximately 180, 19, 1.5, and 0.2 times, respectively, and up-regulated expression of biosynthetic genes. Arginase and ornithine decarboxylase enzyme activities were the highest in the symptomatic group, while arginine decarboxylase and agmatine deiminase enzyme activities were the highest in the asymptomatic group. The two polyamine biosynthetic pathways showed different trends with the increase of the titer of CLas, indicating that polyamines were mainly synthesized through the arginine decarboxylase pathway in the asymptomatic leaves, and were synthesized via the ornithine decarboxylase pathway in symptomatic leaves. These findings provide new insight into the changes of polyamine metabolism in citrus infected with CLas.

Long-term inhibition of ODC1 in APP/PS1 mice rescues amyloid pathology and switches astrocytes from a reactive to active state

Alzheimer’s disease (AD) is characterized by the loss of memory due to aggregation of misphosphorylated tau and amyloid beta (Aβ) plaques in the brain, elevated release of inhibitory neurotransmitter gamma-aminobutyric acid (GABA) and reactive oxygen species from astrocytes, and subsequent neurodegeneration. Recently, it was found that enzyme Ornithine Decarboxylase 1 (ODC1) acts as a bridge between the astrocytic urea cycle and the putrescine-to-GABA conversion pathway in the brain of AD mouse models as well as human patients. In this study, we show that the long-term knockdown of astrocytic Odc1 in APP/PS1 animals was sufficient to completely clear Aβ plaques in the hippocampus while simultaneously switching the astrocytes from a detrimental reactive state to a regenerative active state, characterized by proBDNF expression. Our experiments also reveal an effect of astrocytic ODC1 inhibition on the expression of genes involved in synapse pruning and organization, histone modification, apoptotic signaling and protein processing. These genes are previously known to be associated with astrocytic activation and together create a neuroregeneration-supportive environment in the brain. By inhibiting ODC1 for a long period of 3 months in AD mice, we demonstrate that the beneficial amyloid-clearing process of astrocytes can be completely segregated from the systemically harmful astrocytic response to insult. Our study reports an almost complete clearance of Aβ plaques by controlling an endogenous degradation process, which also modifies the astrocytic state to create a regeneration-supportive environment in the brain. These findings present the potential of modulating astrocytic clearance of Aβ as a powerful therapeutic strategy against AD.

The arginase 1/ornithine decarboxylase pathway suppresses HDAC3 to ameliorate the myeloid cell inflammatory response: implications for retinal ischemic injury

The enzyme arginase 1 (A1) hydrolyzes the amino acid arginine to form L-ornithine and urea. Ornithine is further converted to polyamines by the ornithine decarboxylase (ODC) enzyme. We previously reported that deletion of myeloid A1 in mice exacerbates retinal damage after ischemia/reperfusion (IR) injury. Furthermore, treatment with A1 protects against retinal IR injury in wild-type mice. PEG-A1 also mitigates the exaggerated inflammatory response of A1 knockout (KO) macrophages in vitro. Here, we sought to identify the anti-inflammatory pathway that confers macrophage A1-mediated protection against retinal IR injury. Acute elevation of intraocular pressure was used to induce retinal IR injury in mice. A multiplex cytokine assay revealed a marked increase in the inflammatory cytokines interleukin 1β (IL-1β) and tumor necrosis factor α (TNF-α) in the retina at day 5 after IR injury. In vitro, blocking the A1/ODC pathway augmented IL-1β and TNF-α production in stimulated macrophages. Furthermore, A1 treatment attenuated the stimulated macrophage metabolic switch to a pro-inflammatory glycolytic phenotype, whereas A1 deletion had the opposite effect. Screening for histone deacetylases (HDACs) which play a role in macrophage inflammatory response showed that A1 deletion or ODC inhibition increased the expression of HDAC3. We further showed the involvement of HDAC3 in the upregulation of TNF-α but not IL-1β in stimulated macrophages deficient in the A1/ODC pathway. Investigating HDAC3 KO macrophages showed a reduced inflammatory response and a less glycolytic phenotype upon stimulation. In vivo, HDAC3 co-localized with microglia/macrophages at day 2 after IR in WT retinas and was further increased in A1-deficient retinas. Collectively, our data provide initial evidence that A1 exerts its anti-inflammatory effect in macrophages via ODC-mediated suppression of HDAC3 and IL-1β. Collectively we propose that interventions that augment the A1/ODC pathway and inhibit HDAC3 may confer therapeutic benefits for the treatment of retinal ischemic diseases.

Free Energy Landscapes of the Tautomeric Interconversion of Pyridoxal 5'-Phosphate Aldimines at the Active Site of Ornithine Decarboxylase in Aqueous Media.

The pyridoxal 5'-phosphate (PLP) acts as a coenzyme for a large number of biochemical reactions. It exists in mainly two bound forms at the active site of the concerned enzyme: the internal aldimine, in which the PLP is bound with the ϵ-amino group of lysine at the active site, and the external aldimine, where the PLP is bound to the substrate amino acid. Both the internal and external aldimines have Schiff base linkage (N-H-O) and can exist in two tautomeric structures of ketoenamine and enolimine forms. In this work, we have investigated the free energy landscape for the tautomeric proton transfer in the internal and external aldimines at the active site of the ornithine decarboxylase enzyme in an aqueous medium. We performed hybrid quantum-classical metadynamics and force field-based molecular dynamics simulations, which revealed that the ketoenamine tautomer is more stable than the enolimine form. The QM/MM metadynamics calculations show that the free energy difference between the ketoenamine and enolimine forms for the internal aldimine is 3.9 kcal/mol, and it is found to be 5.8 kcal/mol for the external aldimine, with the ketoenamine form being more stable in both cases. The results are further supported by calculations of the binding free energies from classical simulations and static quantum chemical calculations in different environments. We have also analyzed the configurational structure of the microenvironment at the active site in order to have better insights into the interactions of the active site residues with the PLP in its two tautomeric forms.

Microbiota engage a HDAC3 rheostat to shape ILC3 metabolism and function in the gut

Abstract Group 3 innate lymphoid cells (ILC3s) respond to microbial colonization of the intestine to coordinate tissue health. This process involves interpretation of host- or microbial-derived signals that must be tightly regulated to preserve immunologic homeostasis. Here we examine a key epigenomic regulator that is responsive to microbial-derived factors, histone deacetylase 3 (HDAC3), and find that lineage-specific deletion results in reduced ILC3s and increased susceptibility to intestinal damage. In contrast, the lineage-specific deletion of an essential corepressor, NCOR1, results in elevated ILC3s and enhanced protection from intestinal damage. This indicates that HDAC3 mediates both a supportive deacetylase-independent role and a repressive deacetylase-dependent role in ILC3s. To explore genes promoted by HDAC3, we performed RNA sequencing and found that HDAC3 deficient ILC3s exhibit impaired transcription of genes involved in polyamine synthesis including the polyamine rate limiting enzyme ornithine decarboxylase 1 (ODC1) and spermine oxidase (SMOX). At steady state, ILC3s uniquely express high levels of ODC1 and SMOX compared to other lymphocytes, and transcription of these genes is influenced by IL-23 and the presence of the microbiota. Overall, this indicates that HDAC3 is a rheostat of ILC3 functionality in the intestine, with the hypothesis that HDAC3/NCOR1 limit tissue protective ILC3 responses, and NCOR1 independent HDAC3-mediated transcription of polyamine synthesis genes promotes ILC3 tissue protective responses. Supported by NIDDK National Research Service Award (NRSA) T32 Diversity Administrative Supplement (NOT-DK-21-016), R01AI143842, R01AI123368, R01AI145989, U01AI095608, R21CA249274, R01AI162936, R01CA274534 and the NIAID Mucosal Immunology Studies Team (MIST), an Investigators in the Pathogenesis of Infectious Disease Award from the Burroughs Wellcome Fund, Linda and Glenn Greenberg, the Dalton Family Foundation, and the Roberts Institute for Research in IBD. G.F.S. is a CRI Lloyd J. Old STAR.

Persistent Coxsackievirus B3 Infection in Pancreatic Ductal Cells In Vitro Downregulates Cellular Polyamine Metabolism.

Picornaviruses infect a wide variety of cell types in vitro, with rapid replication kinetics and pronounced cytopathic effect. Coxsackievirus B3 (CVB3) can also establish a persistent infection in vivo that can lead to pathology, including dilated cardiomyopathy and myocarditis. One model system to study persistent infection is the pancreatic ductal cell line PANC-1, which CVB3 infects and is maintained indefinitely. We have characterized this model for CVB3 infection to study persistent infection for over 6 months. We find that CVB3 rapidly replicates within PANC-1 cells without robust cytopathic effect, and after 1 month in culture, titers stabilize. We find that infection does not significantly affect cellular viability. Persistent virus reverts to lytic infection when transferred to Huh7 or Vero cells. We find that persistent CVB3 adapts to PANC-1 cells via mutation of its capsid proteins and, curiously, the viral polymerase (3Dpol) to generate a high-fidelity polymerase. Persistent infection is associated with reduced cleavage of eIF4G, reduced plaque size, and decreasing particle infectivity. We further find that polyamine metabolism is altered in persistently infected cells, with the rate-limiting enzyme ornithine decarboxylase (ODC1) reduced in translation. We further find that targeting polyamine synthesis reduces persistent infection without affecting the viability of the PANC-1 cells. Finally, we find that viral fidelity is essential to maintaining CVB3 infection, and targeting viral fidelity reduces persistent virus infection. Together, these data highlight a novel role for polyamines and fidelity in persistent CVB3 infection and suggest avenues for therapeutic development to target persistent infection. IMPORTANCE Enteroviruses are significant human pathogens that can cause severe disease, including cardiomyopathies. Viruses like coxsackievirus B3 (CVB3) can cause tissue damage by lytically infecting cells; however, CVB3 can also persistently infect, which has been associated with several pathologies. Studying persistent infection in vitro is challenging, as CVB3 lytically infects most cellular model systems. Here, we show that CVB3 establishes persistent infection in pancreatic ductal cells in vitro, similar to prior studies on other coxsackieviruses. We also show that this infection results in adaptation of the virus to these cells, as well as changes to cellular metabolism of polyamines.

Enzyme Cascade with Horseradish Peroxidase Readout for High-Throughput Screening and Engineering of Human Arginase-1

We report an enzyme cascade with horseradish peroxidase-based readout for screening human arginase-1 (hArg1) activity. We combined the four enzymes hArg1, ornithine decarboxylase, putrescine oxidase, and horseradish peroxidase in a reaction cascade that generated colorimetric or fluorescent signals in response to hArg1 activity and used this cascade to assay wild-type and variant hArg1 sequences as soluble enzymes and displayed on the surface of Escherichia coli. We screened a curated 13-member hArg1 library covering mutations that modified the electrostatic environment surrounding catalytic residues D128 and H141, and identified the R21E variant with a 13% enhanced catalytic turnover rate compared to wild type. Our scalable one-pot single-step arginase assay with continuous kinetic readout is amenable to high-throughput screening and directed evolution of arginase libraries and testing drug candidates for arginase inhibition.

Abstract 4944: Evaluating the efficacy of spermine analogue ivospemin (SBP-101) in combination with chemotherapy in ovarian cancer

Abstract Polyamines are small cationic alkylamines that play critical roles in essential cellular processes governing growth and proliferation. As such, cancers are fully reliant on increased polyamine pools maintained through dysregulation of polyamine metabolism. Pharmaceutical modulation of polyamine metabolism is a promising avenue in cancer therapeutics and has been attempted with enzyme inhibitors, including DFMO (difluoromethylornithine), and polyamine analogues. Ivospemin is a spermine analogue that has shown efficacy in slowing pancreatic and ovarian tumor progression both in vitro and in vivo and demonstrated encouraging results in pancreatic cancer clinical trials. We have shown that ivospemin decreases polyamine content through depression of the activity of the polyamine biosynthetic enzyme ornithine decarboxylase (ODC) in a variety of cancer cell lines. Treatment of the VDID8+ murine ovarian cancer model with ivospemin resulted in a marked increase in survival. Here we examine the potential of combining ivospemin and chemotherapeutic agents that are used to treat cisplatin-resistant ovarian cancer. Treatment with gemcitabine, topotecan, and doxorubicin increased the in vitro toxicity of ivospemin, while paclitaxel and docetaxel did not have any added benefit over ivospemin alone. Using the VDID8+ model, we further evaluated the efficacy of ivospemin in combination with gemcitabine, topotecan, and doxorubicin in vivo. Ascites fluid was used as a marker of tumor burden and evaluated for polyamine content. Addition of ivospemin improved the survival of mice treated with any of the three chemotherapeutics. The ivospemin and doxorubicin combination mice had the greatest median survival time; this combination is being further evaluated in mechanistic studies and additional murine studies. Ovarian cancers have extremely immunosuppressive tumor microenvironments (TME) and metabolic reprogramming of the TME to reduce immunosuppressive phenotypes is a promising approach for treatment. Sustained elevation of polyamine levels supports an immunosuppressive TME, and evidence suggests that pharmacologic depletion of polyamines may reduce immunosuppressive phenotypes. DFMO treatment in the immunosuppressive VDID8+ model influences the immune cells of the TME, and we therefore are investigating the combination of ivospemin and DFMO in ovarian cancer. In addition to the cooperativity of ivospemin and chemotherapeutic agents, we have observed a cooperative antiproliferative response in ovarian cancer cells following DFMO and ivospemin cotreatment. Together, these studies suggest the potential of polyamine modulation by ivospemin and DFMO in combination with standard of care chemotherapy. Future studies will determine influences on the immune microenvironment and will evaluate cooperativity between ivospemin, DFMO, and chemotherapy. Citation Format: Cassandra E. Holbert, Jackson R. Foley, Tracy Murray Stewart, Michael J. Walker, Elizabeth Bruckheimer, Jennifer K. Simpson, Robert A. Casero. Evaluating the efficacy of spermine analogue ivospemin (SBP-101) in combination with chemotherapy in ovarian cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4944.

The role of polyamines in the regenerative process of skin aerobic-purulent wounds

Aliphatic polyamines (putrescine, spermidine, spermine) are organic polycations that play an important role in wound healing by stimulating several cellular mechanisms. In a human skin wound sample, the activity of the enzyme ornithine decarboxylase, which regulates the rate of polyamine synthesis, rapidly increases along the wound edges and leads to the activation of the polyamine synthesis cascade. Under the influence of polyamines, some signaling systems are also activated in wounds, which are the main pathways for the release of cellular mechanisms, and thanks to them, the healing process begins in wounds. For example, spermine induces the synthesis of urokinase-type plasminogen activator, the binding of which to the corresponding receptor at the wound margins executes the urokinase-type plasminogen activator and its receptor signaling system, which is the main driver of keratinocyte migration. Eukaryotic cell proliferation depends on precise modification of the eukaryotic initiation factor 5A1, in which spermidine plays an indispensable role. However, in addition to the significant functions performed by polyamines in the human body, polyamines are also necessary for the normal growth and development of fungi and bacteria. Small amounts of some microorganisms have a positive effect on the healing of wounds, but their increase, on the contrary, leads to the impairment of the normal course of wound healing due to their enhanced synthesis of polyamines. On the other hand, many studies show that excess ornithine decarboxylase and polyamines increase the risk of skin cancer. Suppression of polyamine synthesis by pathogenic microflora during wound healing can contribute to both rapid healing and the prevention of skin cancer. In our study, we offer a way of inhibition of polyamine synthesis by wound microflora for rapid wound healing and prevention of subsequent cancer. The medicinal mixture “Armenicum/Eflornithine” is a mixture of the drug “Armenicum” and α-difluoromethylornithine.

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.

Hypusinated eIF5A Promotes Ribosomal Frameshifting during Decoding of ODC Antizyme mRNA in Saccharomyces cerevisiae

Polyamines are essential biogenic poly-cations with important roles in many cellular processes and diseases such as cancer. A rate-limiting step early in the biosynthesis of polyamines is the conversion of ornithine to putrescine by the homodimeric enzyme ornithine decarboxylase (ODC). In a conserved mechanism of posttranslational regulation, ODC antizyme (OAZ) binds to ODC monomers promoting their ubiquitin-independent degradation by the proteasome. Decoding of OAZ mRNA is unusual in that it involves polyamine-regulated bypassing of an internal translation termination (STOP) codon by a ribosomal frameshift (RFS) event. Using Saccharomyces cerevisiae, we earlier showed that high polyamine concentrations lead to increased efficiency of OAZ1 mRNA translation by binding to nascent Oaz1 polypeptide. The binding of polyamines prevents stalling of the ribosomes on OAZ1 mRNA caused by nascent Oaz1 polypeptide thereby promoting synthesis of full-length Oaz1. Polyamine depletion, however, also inhibits RFS during the decoding of constructs bearing the OAZ1 shift site lacking sequences encoding the Oaz1 parts implicated in polyamine binding. Polyamine depletion is known to impair hypusine modification of translation factor eIF5A. Using a novel set of conditional mutants impaired in the function of eIF5A/Hyp2 or its hypusination, we show here that hypusinated eIF5A is required for efficient translation across the OAZ1 RFS site. These findings identify eIF5A as a part of Oaz1 regulation, and thereby of polyamine synthesis. Additional experiments with DFMO, however, show that depletion of polyamines inhibits translation across the OAZ1 RFS site not only by reducing Hyp2 hypusination, but in addition, and even earlier, by affecting RFS more directly.

Ornithine decarboxylase supports ILC3 responses in infectious and autoimmune colitis through positive regulation of IL-22 transcription

Group 3 innate lymphoid cells (ILC3s) are RORγT+ lymphocytes that are predominately enriched in mucosal tissues and produce IL-22 and IL-17A. They are the innate counterparts of Th17 cells. While Th17 lymphocytes utilize unique metabolic pathways in their differentiation program, it is unknown whether ILC3s make similar metabolic adaptations. We employed single-cell RNA sequencing and metabolomic profiling of intestinal ILC subsets to identify an enrichment of polyamine biosynthesis in ILC3s, converging on the rate-limiting enzyme ornithine decarboxylase (ODC1). In vitro and invivo studies demonstrated that exogenous supplementation with the polyamine putrescine or its biosynthetic substrate, ornithine, enhanced ILC3 production of IL-22. Conditional deletion of ODC1 in ILC3s impaired mouse antibacterial defense against Citrobacter rodentium infection, which was associated with a decrease in anti-microbial peptide production by the intestinal epithelium. Furthermore, in a model of anti-CD40 colitis, deficiency of ODC1 in ILC3s markedly reduced the production of IL-22 and severity of inflammatory colitis. We conclude that ILC3-intrinsic polyamine biosynthesis facilitates efficient defense against enteric pathogens as well as exacerbates autoimmune colitis, thus representing an attractive target to modulate ILC3 function in intestinal disease.

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.

Modulation of Arginase-2 mRNA Levels by ω-3 PUFAs and Aspirin in Asthmatic Human Lung Fibroblasts.

Airway remodeling (AR) increases disease severity, and morbidity of asthmatic patients by contributing to irreversible airflow obstruction and progressive declines in lung function. Arginase isoenzymes and the downstream enzymes ornithine decarboxylase (ODC) and ornithine aminotransferase (OAT) have been implicated in the hyperplastic and fibrotic changes of AR, respectively. Omega-3 polyunsaturated fatty acids (ω-3 PUFAs) and resolvin metabolites have anti-AR effects, but whether they are mediated through the arginase pathway is unclear. Our study intended to determine the effects of the ω-3 PUFAs eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), resolvin D1 (RvD1), TH1 cytokines, acetylsalicylic acid (ASA), cAMP, and dexamethasone (DEX) on the expression of arginase isoenzymes arginase 1 (ARG1) and arginase 2 (ARG2), ODC, and OAT in human lung fibroblasts (HLF) from normal (NHLF) and diseased (DHLF) asthmatic donors using reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR). Our data showed that EPA and EPA+DHA downregulated ARG2 mRNA 2-fold in both types of HLF. DHA, RvD1, and DEX did not alter mRNA levels for any of the genes studied. EPA lowered the ARG2 protein levels in DHLF, but did not affect those levels in NHLF. ASA upregulated ARG2 mRNA 5-fold and 7-fold in NHLF and DHLF, respectively, TH1 cytokines downregulated ARG2, ODC, and OAT mRNA in DHLF 10-fold, 2-fold, and 2.5-fold, respectively, and cAMP downregulated ARG2 mRNA 2-fold in DHLF. These results are the first to show a direct effect of ω-3 PUFAs on ARG2 mRNA levels and provide further evidence for a role of ω-3 PUFAs in AR.

Cytotoxic Effects of Rheum khorasanicum Extract on MCF-7 Human Breast Cancer Cell Line and ODC1 Gene Expression

Background: Polyamine metabolism has a critical role in cancer initiation and progression. The abnormal overexpression of the ornithine decarboxylase (ODC1) enzyme gene could be a characteristic of cancer cells. Objectives: The current study evaluated the cytotoxic effects of Rheum khorasanicum extract in three different solvents, including distilled water, ethanol, and n-hexane, on the MCF-7 breast cancer cell line and assessed the expression level of the ODC1 gene upon the abovementioned treatments. Methods: We cultured MCF-7 cancer cells in complete cell culture media and subsequently subjected them to increasing concentrations (0 - 200 μg/mL) of the extract. The cytotoxic activity of the extract was investigated using an MTT assay. The quantitative analysis of ODC1 gene expression was performed using real-time PCR. Results: The cell viability was significantly decreased in concentration- and time-dependent manners. The mRNA expression of the ODC1 gene declined gradually with the increasing concentrations of extracts. Conclusions: Our findings indicated that R. khorasanicum extract could potentially influence ODC1 gene expression in breast cancer cells and exert inhibitory effects on cancer cell proliferation.

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.