Polyamine Catabolism Research Articles

Inhibition of Polyamine Catabolism Reduces Cellular Senescence.

The aging of the global population has necessitated the identification of effective anti-aging technologies based on scientific evidence. Polyamines (putrescine, spermidine, and spermine) are essential for cell growth and function. Age-related reductions in polyamine levels have been shown to be associated with reduced cognitive and physical functions. We have previously found that the expression of spermine oxidase (SMOX) increases with age; however, the relationship between SMOX expression and cellular senescence remains unclear. Therefore, we investigated the relationship between increased SMOX expression and cellular senescence using human-liver-derived HepG2 cells. Intracellular spermine levels decreased and spermidine levels increased with the serial passaging of cells (aged cells), and aged cells showed increased expression of SMOX. The levels of acrolein-conjugated protein, which is produced during spermine degradation, also increases. Senescence-associated β-gal activity was increased in aged cells, and the increase was suppressed by MDL72527, an inhibitor of acetylpolyamine oxidase (AcPAO) and SMOX, both of which are enzymes that catalyze polyamine degradation. DNA damage accumulated in aged cells and MDL72527 reduced DNA damage. These results suggest that the SMOX-mediated degradation of spermine plays an important role in cellular senescence. Our results demonstrate that cellular senescence can be controlled by inhibiting spermine degradation using a polyamine-catabolizing enzyme inhibitor.

L-Aminoguanidine Induces Imbalance of ROS/RNS Homeostasis and Polyamine Catabolism of Tomato Roots after Short-Term Salt Exposure.

Polyamine (PA) catabolism mediated by amine oxidases is an important process involved in fine-tuning PA homeostasis and related mechanisms during salt stress. The significance of these amine oxidases in short-term responses to salt stress is, however, not well understood. In the present study, the effects of L-aminoguanidine (AG) on tomato roots treated with short-term salt stress induced by NaCl were studied. AG is usually used as a copper amine oxidase (CuAO or DAO) inhibitor. In our study, other alterations of PA catabolism, such as reduced polyamine oxidase (PAO), were also observed in AG-treated plants. Salt stress led to an increase in the reactive oxygen and nitrogen species in tomato root apices, evidenced by in situ fluorescent staining and an increase in free PA levels. Such alterations were alleviated by AG treatment, showing the possible antioxidant effect of AG in tomato roots exposed to salt stress. PA catabolic enzyme activities decreased, while the imbalance of hydrogen peroxide (H2O2), nitric oxide (NO), and hydrogen sulfide (H2S) concentrations displayed a dependence on stress intensity. These changes suggest that AG-mediated inhibition could dramatically rearrange PA catabolism and related reactive species backgrounds, especially the NO-related mechanisms. More studies are, however, needed to decipher the precise mode of action of AG in plants exposed to stress treatments.

Contribution of Putrescine and Glutamic Acid on γ-Aminobutyric Acid Accumulation of Malus baccata Borkh. Roots under Suboptimal Low Root-Zone Temperature

GABA (γ-aminobutyric acid) is found in plants and accumulates rapidly under stresses. However, the contributions of glutamic acid and a (Glu)-derived pathway and polyamines (PAs) catabolism pathway on GABA accumulation and the regulatory effects of exogenous putrescine (Put) on a GABA shunt under suboptimal low root-zone temperatures remain unknown. Our results showed that suboptimal low root-zone temperatures (treatment L) significantly increased GABA contents and GABA transaminase (GABA-T) activities. The contribution rate of the PAs catabolism pathway increased from 20.60% to 43.31%. Treatment L induced oxidative stress in Malus baccata Borkh. roots. Exogenous Put increased the contents of endogenous Put, spermine (Spm), and spermidine (Spd), promoted the transformation of PAs, increased the activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT), and decreased the contents of hydrogen peroxide (H2O2), superoxide anion (O2∙−), and malondialdehyde (MDA). Meanwhile, contrasting results were observed after aminoguanidine (AG, an inhibitor of diamine oxidase) application. These findings revealed that the Glu-derived pathway is the main route of GABA synthesis. The contribution rate of the Pas catabolism pathway increased gradually with the extension of treatment time, and the treatment of exogenous Put significantly improved the tolerance of Malus baccata Borkh. Roots to suboptimal low temperature by regulating the transformation of Pas, GABA shunt, and the antioxidant system.

Polyamine, 1,3-diaminopropane, regulates defence responses on growth, gas exchange, PSII photochemistry and antioxidant system in wheat under arsenic toxicity

The metalloid arsenic (As) is extremely hazardous to all living organisms, including plants. Pollution with As is very detrimental to the photosynthetic machinery, cell division, energy generation, and redox status. In order to cope with stress, the use of growth regulators such as polyamines (PA), which strengthen the antioxidant system of plants, has become widespread in recent years. PAs can modulate the plant growth through basic mechanisms common to all living organisms, such as membrane stabilization, free radical scavenging, deoxyribonucleic acid (DNA), ribonucleic acid (RNA) and protein synthesis, enzyme activities and second messengers. However, the effect of 1,3- diaminopropane (Dap), which is a product of PA catabolism, is not clear enough in plants exposed to As toxicity. In the current study, the different concentrations of 1,3-diaminopropane (0.1, 0.5 and 1 mM Dap) were hydroponically treated to wheat (Triticum aestivum) under arsenic stress (100 μM As) and then relative growth rate (RGR), relative water content (RWC), proline content (Pro), gas exchange parameters, PSII photochemistry, chlorophyll fluorescence kinetics, antioxidant activity and lipid peroxidation were assessed. RGR, RWC, osmotic potential and Pro content decreased in As-applied plants. The inhibition of these parameters could be reversed by Dap treatments. Besides, Dap applications mitigated the As toxicity-induced suppression on chlorophyll fluorescence (Fv/Fm, Fv/Fo and Fo/Fm) and the performance of PSII photochemistry. As impaired the balance on antioxidant capacity by decreased activities of catalase (CAT), peroxidase (POX), glutathione peroxidase (GPX), ascorbate peroxidase (APX), monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), and the contents of ascorbate (AsA) and glutathione (GSH) and then lipid peroxidation (TBARS content) increased. In the presence of Dap under As stress, the plants exhibited an increase in superoxide dismutase (SOD), POX, and GPX. Dap treatments contributed to the maintenance of cellular redox state (AsA/DHA and GSH/GSSG) by regulating the activities/contents of enzyme/non-enzyme involved in the AsA-GSH cycle. After Dap applications against stress, ROS accumulation (H2O2 content) and lipid peroxidation (TBARS) were effectively reduced. The findings showed that by eliminating As-induced oxidative damage and protecting the biochemical processes of photosynthesis, Dap treatments have a substantial potential to give resistance to wheat.

Polyamine catabolism is concentrated in tumor-associated histiocytes in diffuse large B-cell lymphoma and classic Hodgkin lymphoma

Polyamines are cationic molecules necessary for cell survival, growth, and replication [1–5]. Polyamines come in a variety of structural forms and are principally regulated by two enzymes, spermine/spermidine acetyltransferase-1 (SAT1) and ornithine decarboxylase-1 (ODC1). SAT1 targets the polyamines spermidine and spermine for degradation via acetylation, while ODC1 is involved in converting the polyamine precursor molecule to more complex polyamines [6–8]. Polyamines and their regulatory enzymes have been implicated in tumor metastasis [9,10] and in crosstalk between oncogenes [11–13] in numerous types of cancer, but their role has never been evaluated in B-cell malignancies. In this study, we examine the expression of SAT1 in diffuse large B-cell lymphoma (DLBCL) and classic Hodgkin lymphoma (HL). We found that SAT1 is expressed in all examined cases of DLBCL (n = 15) and HL (n = 5), though the levels of expression across cases vary. We also note that SAT1 expression appears to be concentrated in tumor-associated histiocytes, rather than tumor cells in both DLBCL and HL. We propose that these findings indicate that the polyamine catabolic enzyme, SAT1, plays an unappreciated role in the pathogenesis of B-cell neoplasms.

PpeERF115 regulates peach fruit ripening by increasing polyamine turnover through up-regulation of genes involved in polyamine synthesis and catabolism

Polyamines (PAs) are anti-senescence agents that promote maintenance of fruit quality after harvesting. Many studies have reported increases in PA synthesis and catabolism and decreases in PA levels during fruit ripening. However, few reports have addressed the modulation mechanisms of PA metabolism during fruit maturation. In this report, we found that the peach (Prunus persica L.) transcription factor PpeERF115 directly bound to the PpeADC1 and PpePAO1 promoters, activating these genes. Silencing PpeERF115 enhanced spermidine (Spd) and spermine (Spm) accumulation, reduced putrescine (Put) content, decreased ethylene production, suppressed the respiratory rate, promoted maintenance of fruit firmness, and delayed peach ripening. Conversely, fruit over-expressing PpeERF115 showed significantly reduced Spd and Spm accumulation and clear increases in Put levels. RNA-sequencing analysis indicated that PpeADC1, PpeCuAO2, and PpePAO1 were dramatically down-regulated in PpeERF115-known out fruit, whereas PpeSAMDC1 was significantly up-regulated. This study is the first to show that PpeERF115 regulates peach ripening by increasing PA turnover through up-regulation of PA synthesis and catabolism. These results extend our comprehension of the coordination between ethylene and PAs during regulation of fruit maturation.

Polyamine catabolism via spermine oxidase in the pathogenesis and treatment of hyperglycemia and diabetes mellitus-A short discussion

Diabetes mellitus can be described as chronic, endocrine dysfunction of pathophysiological regulation related to glucose and insulin. Hyperglycemia is the most common and intensely related complication of this dysfunction and has countless methods of pathogenicity. Hyperglycemia has the ability to exacerbate a degenerative positive feedback loop within critical tissues. Considering the fact that pancreatic hormonal regulation has played an increasingly considerable role in serum glucose homeostasis, here we begin a discussion into the implication of polyamine catabolism as a pathway with a mechanism that would contribute considerably to the necrosis and dysfunction of islet cells in the pancreas. The pathogenicity of this mechanism focuses on how the sustained increase in free radical production by hyperglycemic induction extenuates several complications in diabetes mellitus. The oxidation of spermine to spermidine produces hydrogen peroxide, as well as 3-aminopropanol (which spontaneously converts to acrolein) and spermidine (the precursor for the substrate of this reaction). This increase in reactive oxygen species exhibits the ability to inhibit autophagy and apoptosis as well as stimulate fat necrosis within the cells of the pancreas. Considering the islet cells of the pancreas play an extremely important role in glucose homeostasis, small changes in concentrations of these ROS are able to eliminate large regulatory factors while going undetected in the analysis of disease processes and therapeutics.

The drought-tolerant rhizobacterium, Pseudomonas putida AKMP7, suppresses polyamine accumulation under well-watered conditions and diverts putrescine into GABA under water-stress, in Oryza sativa

Drought-tolerant plant growth promoting rhizobacteria (PGPR) have been reported to alleviate water-stress in plants that they associate with. Here, we report the impact of a free-living, drought-tolerant PGPR strain, Pseudomonas putida AKMP7 on (a) morpho-physiological responses and (b) metabolism of the three major polyamines-namely, putrescine (Put), spermidine (Spd) and spermine (Spm) in well-watered and water-stressed Oryza sativa (BPT5204). The bacterium significantly improved the plant growth and biomass, decreased membrane damage, electrolyte leakage and plant proline levels under water-stress, indicating drought alleviation. Dehydration did not affect the root colonization efficiency of the bacterium. We observed that the concentrations of all three polyamines were downregulated in rice leaves under well-watered conditions by AKMP7 inoculation. Under water-stress, however, AKMP7 caused a significant decrease only in the levels of foliar Spm, while Put and Spd levels did not majorly fluctuate. Transcript levels of most of the polyamine biosynthetic genes studied (AIH involved in Put biosynthesis; SPD/SPM2, SPD/SPM3 and SAMDC2, involved in Spd and Spm biosynthesis) positively correlated with the low polyamine levels in the inoculated plants, vs. the non-inoculated ones under well-watered conditions. While similar trends were observed in the water-stressed, inoculated plants vs. water-stressed, non-inoculated plants, the reduction in only Spm (and not Put and Spd) was a potential consequence of the interplay of both, its biosynthesis and back-conversion. In water-stressed plants, AKMP7 caused a small downregulation in AIH, while stronger downregulations in the expressions of SPD/SPM2 as well as SAMDC2 were seen. Furthermore, the Spd to Put back-conversion gene, PAO3 exhibited a downregulation on AKMP7 treatment under water-stressed conditions. There was also an increase in the expression of BADH2 gene, leading to a concomitant increase in foliar GABA levels in inoculated, water-stressed plants vs. their non-inoculated counterparts. The overall conclusion from this study is- P. putida AKMP7 tightly regulates polyamine homeostasis in rice plants, both under well-watered and water-stressed conditions by lowering polyamine levels, through (a) transcriptional control of polyamine biosynthetic genes under well-watered conditions and (b) a combination of biosynthesis, back-conversion and catabolism of polyamines under water-stress.

Manganese abated indomethacin-induced gastrohepatorenal toxicities in Rats via suppression of oxidative stress, polyamine catabolism, inflammation and activation of Caspase-3

Indomethacin (IND) is a non-steroidal anti-inflammatory drug with many therapeutic benefits and multi-organ toxicities. Manganese (Mn) is a trace metal essential in many biological processes. However, there is dearth of information concerning the effect of Mn on IND-induced gastrohepatorenal damage in rats. This study investigated the mitigating effect of manganese on IND-induced gastrohepatorenal damage in rats. Adult male rats were assigned into four groups of eight rats each and were treated once daily for 14 days. Group 1 served as control; group 2 received IND alone (5 mg/kg); group 3 received IND (5 mg/kg) and Mn alone (10 mg/kg) and group 4 received Mn alone Mn alone (10 mg/kg). Administration of IND significantly increased ulcer score, ulcer index, titrable acidity and peptic activity and decreased pH, mucus content, hexosamine and sialic acid levels. IND also increased hepatorenal markers, myeloperoxidase (MPO), nitric oxide (•NO), malondialdehyde (MDA), lipid hydroperoxide (LOOH), protein carbonyl (PCO), spermine oxidase (SpmOX), putrescine Oxidase (PutOX), tumor necrosis factor-α (TNF-α) interleukin-1β (IL-1β), caspase-3 and decreased the activities of superoxide dismutase (SOD), catalase, glutathione S-transferase (GST), glutathione peroxidase (GPx) and glutathione (GSH) level relative to control. Co-administration of IND and Mn significantly decreased ulcerogenic parameters, hepatorenal markers, MPO, •NO, MDA, LOOH, PCO, SpmOX, PutOX, TNF-α, IL-1β, caspase-3 and increased antioxidant status relative to IND alone group.Histological examination showed that injuries induced by IND were ameliorated by Mn rats.Mn suppressed IND-induced oxidative stress, inflammation, polyamine catabolism and caspase-3 activation in the liver and kidney of the rats.

Transcriptome Analysis of Redox Systems and Polyamine Metabolic Pathway in Hepatoma and Non-Tumor Hepatocyte-like Cells.

Reactive oxygen species (ROS) play a major role in the regulation of various processes in the cell. The increase in their production is a factor contributing to the development of numerous pathologies, including inflammation, fibrosis, and cancer. Accordingly, the study of ROS production and neutralization, as well as redox-dependent processes and the post-translational modifications of proteins, is warranted. Here, we present a transcriptomic analysis of the gene expression of various redox systems and related metabolic processes, such as polyamine and proline metabolism and the urea cycle in Huh7.5 hepatoma cells and the HepaRG liver progenitor cell line, that are widely used in hepatitis research. In addition, changes in response to the activation of polyamine catabolism that contribute to oxidative stress were studied. In particular, differences in the gene expression of various ROS-producing and ROS-neutralizing proteins, the enzymes of polyamine metabolisms and proline and urea cycles, as well as calcium ion transporters between cell lines, are shown. The data obtained are important for understanding the redox biology of viral hepatitis and elucidating the influence of the laboratory models used.

Abstract 306: Alternatively activated macrophages induce bystander effects via arginase 1

Abstract Colorectal cancer is a leading cause of cancer mortality and arises from genetic and epigenetic changes in colon epithelial cells. One of the mechanisms driving colon epithelial cell transformation is the bystander effect (BSE). BSE primarily arises from polarized macrophages (MØs) in stromal tissue. Herein, we report that alternatively activated (M2) MØs, as generated by interleukin (IL)-4, induce double-strand DNA breaks in target cells through BSE. Unlike M1 MØs that are activated by intestinal microbiota, BSE and DNA damage due to M2-like MØs occur through a distinct mechanism that differs from M1 MØs and does not involve pro-inflammatory mediators (e.g., cyclooxygenase-2 or tumor necrosis factor-α). Murine MØs (RAW264.7 cells) treated with IL-4 were polarized to an M2-like phenotype and found to strongly express arginase 1 (ARG1), a prototypical enzyme that converts L-arginine to L-ornithine and urea. L-Ornithine is a precursor in polyamine synthesis with H2O2 being a key oxidizing byproduct of polyamine catabolism. In a dual-chamber system using Young Adult Mouse Colon (YAMC) cells as targets for BSE generated by M2-like MØs, inhibition of ARG1 by N-hydroxy-nor-L-arginine abolished DNA damage (as measured by γH2AX). Direct treatment of YAMC cells with L-ornithine resulted in similar levels of DNA damage as M2-like MØs. YAMC cells exposed to M2-like MØs had increased intracellular H2O2. MDL-72527, an inhibitor polyamine oxidase and polyamine catabolism, reduced intracellular H2O2 to background levels and decreased DNA damage. YAMC cells sustaining DNA damage due to BSE from M2-like MØs showed specific phosphorylation of p53(ser15) and NF-κB(ser536) with no evidence for active caspase-3. These data along with an increased expression of cyclinD1 in YAMC cells indicated that DNA damage caused by M2-like MØs occurred via polyamine catabolism and was not due to cellular toxicity or apoptosis. Pathway analysis of RNAseq data for YAMC cells exposed to M2-like MØs identified multiple activated pathways including DNA repair, ubiquitination, Nrf2-mediated stress responses, endoplasmic reticulum overload responses, and lipid biosynthesis. These results provide additional evidence for global cellular responses to BSE generated by M2-like MØs. In conclusion, our findings show that M2-like MØs can generate BSE via a novel mechanism involving ARG1. Induction of BSE by M2-like MØs results in acute DNA damage in colon epithelial cells and activates multiple signaling pathways in target epithelial cells. These data imply a role for alternatively activated M2-like MØs in the malignant transformation of epithelial cells. Citation Format: Ram B. Undi, Hunter L. Porter, Jonathan D. Wren, Naushad Ali, Mark M. Huycke. Alternatively activated macrophages induce bystander effects via arginase 1 [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 306.

Abstract 1760: ADI-PEG20 and GC7 as a novel anti-metabolite strategy for the treatment of malignant pleural mesothelioma

Abstract Malignant pleural mesothelioma (MPM) is a highly aggressive cancer with less than 10% of patients surviving 5-years from diagnosis. Arginine deprivation using pegylated arginine deiminase (ADI-PEG20) is a novel treatment for mesothelioma deficient in the arginine biosynthetic enzyme ASS1. However, resistance to ADI-PEG20 in MPM is a significant problem and is characterized in part by reprogramming of the urea and polyamine metabolism pathway (Locke et al., 2016). We sought to identify a targeted metabolism-based therapy to overcome the emerging resistance and increase the efficiency of ADI-PEG20 treatment for patients with MPM. New models of ADI-PEG20 resistance were generated by constant exposure of MPM cell lines to arginine deprivation for 6 months. ADI-PEG20 resistant and sensitive MPM cell lines were treated with a panel of polyamine targeting molecules. Metabolome alterations were studied by targeted metabolomics and glucose flux analyses. Our results confirmed the up-regulation of ASS1 expression from (50 to 3000-fold) alongside upregulation of polyamine catabolism and upon generation of ADI-PEG20 resistance. Treatment of ADI-PEG20 resistant cell lines with a range of different polyamine inhibitors demonstrated that ADI-PEG20 resistant cell lines were significantly more sensitive to the spermidine-analogue GC7 than their respective ASS1-ve parental cell lines. Moreover, the combination of GC7 and ADI-PEG20 prevented the emergence of resistant cells in vitro. We observed a significant synergistic effect of GC7 and ADI-PEG20 in both ASS1-ve and ASS1+ve cell lines in 2D and 3D cell culture models as well as in our mice in vivo model. Metabolomics analysis revealed that GC7 treatment significantly impaired the TCA cycle. We hypothesize that GC7 is repurposing the fate of Acetyl-CoA to polyamine catabolism therefore shutting down the TCA cycle. Our findings provide novel insights into a polyamine targeted therapy that may prevent resistance to ADI-PEG20 treatment in MPM and related arginine-auxotrophic cancers. Citation Format: Joséphine Carpentier, Peter W. Szlosarek, Sarah Anne Martin. ADI-PEG20 and GC7 as a novel anti-metabolite strategy for the treatment of malignant pleural mesothelioma [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 1760.

Abstract 5905: Enterotoxigenic Bacteroides fragilis, a colon microbe, dysregulates polyamine catabolism to promote breast cancer progression

Abstract Background: With more than 2.3 million cases worldwide, breast cancer persists as a major health burden and warrants a greater understanding of the molecular triggers underlying this disease. Focusing on microbial dysbiosis, our lab discovered the pivotal role of the enterotoxigenic Bacteroides fragilis (ETBF) in promoting breast tumorigenesis through activation of key oncogenic pathways. A gap in knowledge exists regarding how microbes/microbial toxins modulate the oncogenic pathways in cancer cells. Polyamines are imperative for various physiological processes, including cell growth, survival, differentiation and apoptosis and are strongly associated with breast cancer. We questioned the involvement of polyamine pathway in mediating the oncogenic effects of microbial dysbiosis on breast cancer. Methods: Human Her2 expressing spontaneous breast cancer model, MMTV.f.HuHer2 mice, were infected with ETBF and tumor incidence and progression were monitored for 18 months. We used qRT-PCR, western blotting, immunocytochemistry and immunohistochemistry to examine expression of key nodes of polyamine pathway. Various growth, migration and invasion assays were performed to assess carcinogenic properties in breast cancer cells. We employed a colorimetric-based assay to analyze enzymatic activities of key polyamine metabolic members. ROS generation was examined via DCFA staining. Results: Our in vivo data showed that ETBF colonization in mice led to a considerable increase in the level of spermine oxidase (SMOX) in breast tumors, hinting at a correlation between ETBF and polyamine metabolism. Moreover, a marked rise in expression of γH2AX foci was observed in these tumors and in breast cancer cells, suggesting a critical role of ETBF in SMOX-related DNA damage. In agreement, we found SMOX upregulation in breast cancer cells. Our findings displayed a substantial increase in SMOX enzymatic activity in the presence of ETBF toxin, BFT, in breast tumor cells. BFT caused a marked rise in ROS generation of breast tumor cells, suggesting a plausible association of ETBF in SMOX-mediated oxidative stress. Interestingly, we noted a significant induction of ornithine decarboxylase (ODC) expression and activity in response to ETBF or BFT in breast cancer cells. Treatment of BFT-exposed breast cancer cells with SMOX inhibitors led to a sharp inhibition of neoplastic transformations, like cellular migration and γH2AX induction. Conclusion: Our collated evidences suggest the existence of an important cross-talk between key polyamine metabolic enzymes and ETBF that aids in exacerbating breast tumorigenesis. Of importance, therapeutic inhibition of SMOX dramatically impeded BFT-related breast tumorigenesis. Collectively, our results demonstrated the attractive potential of intervening the polyamine catabolic pathway for the management of breast cancer. Citation Format: Deeptashree Nandi, Sheetal Parida, Sowjanya Thatikonda, Jackson Foley, Tracy Stewart, Cynthia L. Sears, Robert A. Casero, Dipali Sharma. Enterotoxigenic Bacteroides fragilis, a colon microbe, dysregulates polyamine catabolism to promote breast cancer progression. [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 5905.

Synergistic Impact of Melatonin and Putrescine Interaction in Mitigating Salinity Stress in Snap Bean Seedlings: Reduction of Oxidative Damage and Inhibition of Polyamine Catabolism

While the individual influences of melatonin (MT) and polyamines (PAs) have been widely studied under various abiotic stresses, little is known about their interaction under salinity stress. In the present study, salt stress applied by 50 mM of sodium chloride (NaCl) on snap bean seedlings has been supplemented with 20 μM of MT and/or 100 μM of putrescine (Put) (individually and in combination). The results indicated that under salinity stress, the combination of MT + Put achieved the highest significant increase in shoot fresh and dry weight, chlorophyll (Chl a), Chl a + b, carotenoids, total soluble sugars, proline, K, Ca, and cell membrane stability index (CMSI), as well as catalase (CAT) and peroxidase (POX) activities. This improvement was associated with an obvious decrease in Na, Na/K ratio, and oxidative damage as indicated by reducing leaf contents of methylglyoxal (MG), hydrogen peroxide (H2O2), and the rate of lipid peroxidation (malondialdehyde; MDA). Moreover, the combination of MT + Put demonstrated a significant decrease in the activities of diamine oxidase (DAO) and polyamine oxidase (PAO) leading to the reduction of the rate of polyamine oxidation. Meanwhile, MT applied individually gave the highest significant increase in leaf relative water content (RWC), Chl b, superoxide dismutase (SOD), and ascorbate peroxidase (APX). Conclusively, the combination treatment of MT + Put could decrease the degradation of polyamines and enhance tolerance to salinity stress in snap bean seedlings.

Metabolic Reprogramming of Breast Tumor-Educated Macrophages Revealed by NMR Metabolomics

The metabolic crosstalk between tumor cells and tumor-associated macrophages (TAMs) has emerged as a critical contributor to tumor development and progression. In breast cancer (BC), the abundance of immune-suppressive TAMs positively correlates with poor prognosis. However, little is known about how TAMs reprogram their metabolism in the BC microenvironment. In this work, we have assessed the metabolic and phenotypic impact of incubating THP-1-derived macrophages in conditioned media (CM) from two BC cell lines cultured in normoxia/hypoxia: MDA-MB-231 cells (highly metastatic, triple-negative BC), and MCF-7 cells (less aggressive, luminal BC). The resulting tumor-educated macrophages (TEM) displayed prominent differences in their metabolic activity and composition, compared to control cells (M0), as assessed by exo- and endometabolomics. In particular, TEM turned to the utilization of extracellular pyruvate, alanine, and branched chain keto acids (BCKA), while exhibiting alterations in metabolites associated with several intracellular pathways, including polyamines catabolism (MDA-TEM), collagen degradation (mainly MCF-TEM), adenosine accumulation (mainly MDA-TEM) and lipid metabolism. Interestingly, following a second-stage incubation in fresh RPMI medium, TEM still displayed several metabolic differences compared to M0, indicating persistent reprogramming. Overall, this work provided new insights into the metabolic plasticity of TEM, revealing potentially important nutritional exchanges and immunoregulatory metabolites in the BC TME.

Plant Peroxisomal Polyamine Oxidase: A Ubiquitous Enzyme Involved in Abiotic Stress Tolerance.

Polyamines (PAs) are positively charged amines that are present in all organisms. In addition to their functions specific to growth and development, they are involved in responding to various biotic and abiotic stress tolerance functions. The appropriate concentration of PA in the cell is maintained by a delicate balance between the catabolism and anabolism of PAs, which is primarily driven by two enzymes, namely diamine oxidase and polyamine oxidase (PAO). PAOs have been found to be localized in multiple subcellular locations, including peroxisomes. This paper presents a holistic account of peroxisomal PAOs. PAOs are flavin adenine dinucleotide-dependent enzymes with varying degrees of substrate specificity. They are expressed differentially upon various abiotic stress conditions, namely heat, cold, salinity, and dehydration. It has also been observed that in a particular species, the various PAO isoforms are expressed differentially with a spatial and temporal distinction. PAOs are targeted to peroxisome via a peroxisomal targeting signal (PTS) type 1. We conducted an extensive bioinformatics analysis of PTS1s present in various peroxisomal PAOs and present a consensus peroxisome targeting signal present in PAOs. Furthermore, we also propose an evolutionary perspective of peroxisomal PAOs. PAOs localized in plant peroxisomes are of potential importance in abiotic stress tolerance since peroxisomes are one of the nodal centers of reactive oxygen species (ROS) homeostasis and an increase in ROS is a major indicator of the plant being in stress conditions; hence, in the future, PAO enzymes could be used as a key candidate for generating abiotic stress tolerant crops.

Spermine oxidase induces DNA damage and sensitizes fusion negative rhabdomyosarcoma cells to irradiation.

Rhabdomyosarcoma (RMS) is a pediatric myogenic soft tissue sarcoma that includes fusion-positive (FP) and fusion-negative (FN) molecular subtypes. FP-RMS expresses PAX3-FOXO1 fusion protein and often shows dismal prognosis. FN-RMS shows cytogenetic abnormalities and frequently harbors RAS pathway mutations. Despite the multimodal heavy chemo and radiation therapeutic regimens, high risk metastatic/recurrent FN-RMS shows a 5-year survival less than 30% due to poor sensitivity to chemo-radiotherapy. Therefore, the identification of novel targets is needed. Polyamines (PAs) such as putrescine (PUT), spermidine (SPD) and spermine (SPM) are low-molecular-mass highly charged molecules whose intracellular levels are strictly modulated by specific enzymes. Among the latter, spermine oxidase (SMOX) regulates polyamine catabolism oxidizing SPM to SPD, which impacts cellular processes such as apoptosis and DNA damage response. Here we report that low SMOX levels are associated with a worse outcome in FN-RMS, but not in FP-RMS, patients. Consistently, SMOX expression is downregulated in FN-RMS cell lines as compared to normal myoblasts. Moreover, SMOX transcript levels are reduced FN-RMS cells differentiation, being indirectly downregulated by the muscle transcription factor MYOD. Noteworthy, forced expression of SMOX in two cell lines derived from high-risk FN-RMS: 1) reduces SPM and upregulates SPD levels; 2) induces G0/G1 cell cycle arrest followed by apoptosis; 3) impairs anchorage-independent and tumor spheroids growth; 4) inhibits cell migration; 5) increases γH2AX levels and foci formation indicative of DNA damage. In addition, forced expression of SMOX and irradiation synergize at activating ATM and DNA-PKCs, and at inducing γH2AX expression and foci formation, which suggests an enhancement in DNA damage response. Irradiated SMOX-overexpressing FN-RMS cells also show significant decrease in both colony formation capacity and spheroids growth with respect to single approaches. Thus, our results unveil a role for SMOX as inhibitor of tumorigenicity of FN-RMS cells in vitro. In conclusion, our in vitro results suggest that SMOX induction could be a potential combinatorial approach to sensitize FN-RMS to ionizing radiation and deserve further in-depth studies.

Genome-Wide Identification and Characterization of the Polyamine Uptake Transporter (Put) Gene Family in Tomatoes and the Role of Put2 in Response to Salt Stress

The polyamine uptake transporter (Put), an important polyamines-related protein, is involved in plant cell growth, developmental processes, and abiotic stimuli, but no research on the Put family has been carried out in the tomato. Herein, eight tomato Put were identified and scattered across four chromosomes, which were classified into three primary groups by phylogenetic analysis. Protein domains and gene structural organization also showed a significant degree of similarity, and the Put genes were significantly induced by various hormones and polyamines. Tissue-specific expression analysis indicated that Put genes were expressed in all tissues of the tomato. The majority of Put genes were induced by different abiotic stresses. Furthermore, Put2 transcription was found to be responsive to salt stress, and overexpression of Put2 in yeast conferred salinity tolerance and polyamine uptake. Moreover, overexpression of Put2 in tomatoes promoted salinity tolerance accompanied by a decrease in the Na+/K+ ratio, restricting the generation of reactive oxygen and increasing polyamine metabolism and catabolism, antioxidant enzyme activity (SOD, CAT, APX, and POD), and nonenzymatic antioxidant activity (GSH/GSSG and ASA/DHA ratios, GABA, and flavonoid content); loss of function of put2 produced opposite effects. These findings highlight that Put2 plays a pivotal role in mediating polyamine synthesis and catabolism, and the antioxidant capacity in tomatoes, providing a valuable gene for salinity tolerance in plants.

The Improvement of Soybean Salt Tolerance by Overexpressed GmPAO1

Polyamines play an important regulatory role during plant growth and development and adversity stress, and polyamine oxidase (PAO) is involved in polyamine catabolism. In this study, an up-regulated polyamine oxidase gene GmPAO1 was obtained by transcriptome sequencing analysis and screening at soybean seedling stages. Also, its expression pattern and function were analyzed. The identification results of transgenic GmPAO1 soybean positive lines showed that the relative expression level of GmPAO1 in the overexpressed lines was increased under salt stress. With increasing stress concentration, the seed germination rate decreased. However, the seed germination rate of the overexpressed lines was significantly higher than that of the control lines, and the phenotypic character of the root systems was also better than that of the control lines. The measurement of superoxide dismutase (SOD) and peroxidase (POD) activities and malondialdehyde and hydrogen peroxide contents revealed that the overexpressed soybean lines significantly increased the SOD and POD activities, significantly reducing the malondialdehyde content. Although the hydrogen peroxide content in the transformed plants gradually increased, the hydrogen peroxide content in the overexpression lines was still lower than that in the gene editing lines. Based on this, it was preliminarily judged that GmPAO1 can improve soybean salt tolerance.

The hepatoprotective effect of aminoguanidine in acute liver injury caused by CCl4 in rats

In this study, the hepatoprotective effect of aminoguanidine in acute liver damage caused by carbon tetrachloride-CCl4 at a dose of 1 mL/kg, i.p. was investigated in experimental rats. Ten days of preventive treatment with aminoguanidine before exposure to toxic CCl4, at a dose of 150 mg/kg, i.p., led to significant reduction in biochemical markers of acute liver injury-AST(p < 0.001), ALT (p < 0.01), SDH (p < 0.05) and reduction in pro-oxidative markers-H2O2 (p < 0.05), TOS (p < 0.01), TBARS, and LOOH (p < 0.001) in relation to rats treated only CCl4. Treatment with aminoguanidine resulted in a significant reduction in the consumption of antioxidant-GR (p < 0.01), GST, GPx, GSH (p < 0.001), and a decrease in pro-inflammatory-TNF-α (p < 0.01), IL-1β, IL-6, NO and NGAL (p < 0.001) markers relative to animals exposed to CCl4 alone. Also, aminoguanidine pre-treatment leads to an increase in arginase activity (p < 0.001), and a decrease in citrulline concentration (p < 0.01), as well as polyamine catabolism enzyme activity-putrescin oxidase and spermine oxidase (p < 0.001) in comparison to the CCl4 group. Aminoguanidine led to a striking reduction of the necrotic field (p < 0.001), and a significant increase in the number of apoptotic hepatocytes (p < 0.001), as well as the proapoptotic markers-BAX and Caspase-3 (p < 0.05), compared to CCl4. The hepatoprotective mechanisms in CCl4 induce hepatotoxicity of aminoguanidine are based on the strong antioxidant effects, inhibition of pro-oxidative and pro-inflammatory mediators, as well as induction of damaged hepatocytes into apoptosis.