Effect Of Spermine Research Articles

Rice Response to Spermine Foliar Application and Its Association with Aerial Imagery Monitoring Under Water Stress Conditions

Rice is the most consumed food in the world, mainly in Asia and Africa. Malaysia is the second-largest rice importer in Southeast Asia after Indonesia. However, rice yield is limited by water stress. One alternative for a quicker strategy to mitigate water stress is through a combination of foliar spermine application and efficient rice management practices via image monitoring techniques using drone technology. The present study was aimed at evaluating the effects of spermine on rice physiological response and its association with aerial imagery and yield under reproductive during reproductive stage under water stress. The experiment was carried out under greenhouse conditions using a two-factorial randomized complete block design (RCBD), with foliar spermine treatment as the first factor and water stress as the second factor. Physiological parameters showed significantly higher tiller number per pot and photosynthesis rate by 29% and 31%, respectively. Correspondingly, the Normalised Difference Vegetation Index (NDVI) using aerial imagery monitoring showed an increased value in spermine treatments by 2% compared to control. Furthermore, NDVI readings and photosynthetic rate were positively correlated linearly with R2= 0.51. Interestingly, spermine treatments alleviated water stress effects by 40%, 17% and 12% in grain weight per pot, grain number per panicle and percentage filled grain. Biomass partitioning in roots improved by 44% in spermine treatments, even under water stress, due to an efficient translocation of assimilates. In conclusion, spermine foliar application significantly improved growth, grain filling and rice yield production, which was also supported by NDVI values using aerial imagery monitoring.

Impaired arginine/ornithine metabolism drives severe HFMD by promoting cytokine storm.

The Hand, Foot and Mouth Disease (HFMD), caused by enterovirus 71 infection, is a global public health emergency. Severe HFMD poses a significant threat to the life and well-being of children. Numerous studies have indicated that the occurrence of severe HFMD is associated with cytokine storm. However, the precise molecular mechanism underlying cytokine storm development remains elusive, and there are currently no safe and effective treatments available for severe HFMD in children. In this study, we established a mouse model of severe HFMD to investigate the molecular mechanisms driving cytokine storm. We specifically analyzed metabolic disturbances, focusing on arginine/ornithine metabolism, and assessed the potential therapeutic effects of spermine, an ornithine metabolite. Our results identified disturbances in arginine/ornithine metabolism as a pivotal factor driving cytokine storm onset in severe HFMD cases. Additionally, we discovered that spermine effectively mitigated the inflammatory injury phenotype observed in mice with severe HFMD. In conclusion, our findings provide novel insights into the molecular mechanisms underlying severe HFMD from a metabolic perspective while offering a promising new strategy for its safe and effective treatment.

Products of oxidative and non-oxidative metabolism of L-arginine as potential regulators of Ca2+ transport in mitochondria of uterine smooth muscle

Mitochondria play a crucial role in maintaining Ca2+ homeostasis in cells. Due to the critical regulatory role of the products of oxidative and non-oxidative metabolism of L-arginine, it is essential to clarify their effect on Ca2+ transport in smooth muscle mitochondria.Experiments were performed on the uterine myocytes of rats and isolated mitochondria. The possibility of NO synthesis by mitochondria was demonstrated by confocal microscopy and spectrofluorimetry methods using the NO-sensitive fluorescent probe DAF-FM and Mitotracker Orange CM-H2TMRos. It was shown that 50 μM L-arginine stimulates the energy-dependent accumulation of Ca2+ in mitochondria using the fluorescent probe Fluo-4 AM. A similar effect occurred when using nitric oxide donors 100 μM SNP, SNAP, and sodium nitrite (SN) directly. The stimulating effect was eliminated in the presence of the NO scavenger C-PTIO. Nitric oxide reduces the electrical potential in mitochondria without causing them to swell. The stimulatory effect of spermine on the accumulation of Ca2+ by mitochondria is attributed to the enhancement of NO synthesis, which was demonstrated with the use of C-PTIO, NO-synthase inhibitors (100 μM NA and L-NAME), as well as by direct monitoring of NO synthesis fluorescent probe DAF-FM.A conclusion was drawn about the potential regulatory effect of the product of the oxidative metabolism of L-arginine – NO on the transport of Ca2+ in the mitochondria of the myometrium, as well as the corresponding effect of the product of non-oxidative metabolism –spermine by increasing the synthesis of NO in these subcellular structures.

High-performance liquid chromatography determination of polyamine contents in poultry based on precolumn benzoyl chloride derivatization

Polyamines are widely present in animal cells and perform important physiological and biochemical functions. Moderate intake of polyamines contributes to good health. Poultry is one of the common sources of polyamines in daily life. However, different tissues from poultry contain different amounts of polyamine. In this study, an efficient and accurate method for the determination of polyamines was developed by high-performance liquid chromatography (HPLC) with precolumn benzoyl chloride derivatization. The linearity of the polyamines was good between 10–400 nmol/mL, with the limit of detection (LOD, S/N = 3) and limits of quantification (LOQ, S/N = 10) were 0.37 and 1.23 nmol/mL. The recoveries of the method ranged from 94.73 % to 106.58 % with high accuracy (RSD < 7 %) and no significant matrix effects or interferences. The RSD of intra-day, inter-day and inter-batch repeatability in the range of 0.07–1.57 %, 0.08–1.03 % and 1.46–4.57 %. The method was successfully used to detect polyamines in poultry tissue, with putrescine, spermidine and spermine levels ranging from 3.24 to 29.85 mg/kg, 0.46 to 164.11 mg/kg and 63.39 to 489.57 mg/kg, respectively. Polyamine levels were higher in the spleen, thymus and bursa of fabricius and lower in the heart and muscle. Furthermore, the method was employed to study the effect of exogenous spermine on polyamine deposition in different tissues of geese. In summary, the method is suitable for the detection of polyamine in animal tissues.

Effects of Putrescine and Spermine on Pollen Germination Levels of Some Olive Cultivars

In this study, it is aimed to determine the effects of putrescine and spermine on pollen germination levels of 8 olive cultivars. Arbequina, Gemlik, Halhalı, Karamani, Kozan yerli, Saurani, Sayfi and Şami olive cultivars were used as material. In the study, 5 spermine doses (0,25mM, 0,125 mM, 0,025 mM, 0,05 mM and 0,005 mM) and 5 putrescine doses (0,20 mM, 0,10 mM, 0,05 mM, 0,025 mM and 0,1 mM) added to basal germination medium (15% sucrose + 1% agar + 100 ppm Boric acid) were tested for determining the effects on pollen germination of olive cultivars. Basal media was used for Control treatment. In terms of pollen germination media, pollen germination levels were increased with lower putrescine and higher spermine levels. In Sayfi olive cultivar, while pollen viability rate was 42,74%, pollen germination was 0,00% in control treatments. However, pollen germination was increased for Sayfi olive cultivar with 0,20 mM and 0,10 mM putrescine (3,64% and 3,40%, respectively) and 0,25 mM, 0,125 mM and 0,05 mM Spermine (12,56%, 4,73% and 3,40%, respectively). In conclusion, polyamine treatments were increased the low germinated cultivars. So, polyamine treatments reveal the potential of the pollen especially in low-germinated pollens.

THE EFFECT OF PARGILINE, SPERMINE AND THEIR COMBINATIONS ON ANDROGEN-DEPENDENT AND ANDROGEN-INDEPENDENT HUMAN PROSTATE CANCER CELLS in vitro

Summary. A series of studies on enhancing the cytotoxic effect of spermine on human prostate cells by affecting the enzymes of its metabolism has been continued. Aim: to investigate effects of pargyline, spermine and their combinations the survival, ζ-potential and surface charge and cytomorphological properties of human prostate cancer cells in culture. Object and methods: studies were conducted on cell cultures: differentiated androgen-dependent LNCaP cell line and low-differentiated androgen-independent DU-145 cell line. Cell survival was determined in the trypan blue assay. The effect of the investigated compounds, both individually and in combination, on the viability and proliferative activity of cells was assessed by the colorimetric method – by staining living cells with a solution of crystal violet in methanol. Biophysical parameters (ζ-potential and surface electric charge density) were determined according to the method cellular microelectrophoresis. Cytomorphological characteristics were studied on preparations of cells grown on coverslips, fixed and stained with hematoxylin and eosin (magnification 1000×). Results: for LNCaP cells, the IC50 of pargyline was 2.2 mM, and the IC50 of spermine was 1.5 mM. For DU-145 cells, the IC50 of pargyline was 2.35 mM, the IC50 of spermine was 5.0 mM. According to biophysical indicators, spermine caused an inversion of the sign of the electric charge in cells of the LNCaP line. Pargyline did not exert such an effect, the charge remained negative, demonstrating an inverse dependence on its concentration. The combination of pargyline and spermine increased the cytotoxic effect of spermine on LNCaP cells, but did not affect the value of their ζ-potential and surface charge density, the positive sign of which, caused by the effect of spermine, was preserved. At the same time, pargyline completely eliminated spermine-induced inversion of the sign of the total surface charge of DU-145 cells, returning it to the normal negative values. Conclusions: it is shown that the differentiated androgen-dependent cells of the LNCaP line with the combined use of pargyline and spermine demonstrated an enhancement of the cytotoxic effect of spermine. At the same time, low-differentiated cells of the DU-145 line, which do not have androgen receptors, were more resistant to the effects of both pargyline and pargyline in combination with spermine. Pargyline also reversed spermine-induced inversion of the sign of the total surface charge of DU-145 cells, returning it to normal negative values. Cytomorphological changes that occurred under the influence of both pargyline alone and in its combination with spermine demonstrate mainly the apoptotic pathway of cell death in the androgen-independent DU-145 line, while in the androgen-dependent LNCaP line, a smaller proportion of cells were in a state of apoptosis.

Potential therapeutic role of spermine via Rac1 in osteoporosis: Insights from zebrafish and mice.

Osteoporosis is a prevalent metabolic bone disease. While drug therapy is essential to prevent bone loss in osteoporotic patients, current treatments are limited by side effects and high costs, necessitating the development of more effective and safer targeted therapies. Utilizing a zebrafish ( Danio rerio) larval model of osteoporosis, we explored the influence of the metabolite spermine on bone homeostasis. Results showed that spermine exhibited dual activity in osteoporotic zebrafish larvae by increasing bone formation and decreasing bone resorption. Spermine not only demonstrated excellent biosafety but also mitigated prednisolone-induced embryonic neurotoxicity and cardiotoxicity. Notably, spermine showcased protective attributes in the nervous systems of both zebrafish embryos and larvae. At the molecular level, Rac1 was identified as playing a pivotal role in mediating the anti-osteoporotic effects of spermine, with P53 potentially acting downstream of Rac1. These findings were confirmed using mouse ( Mus musculus) models, in which spermine not only ameliorated osteoporosis but also promoted bone formation and mineralization under healthy conditions, suggesting strong potential as a bone-strengthening agent. This study underscores the beneficial role of spermine in osteoporotic bone homeostasis and skeletal system development, highlighting pivotal molecular mediators. Given their efficacy and safety, human endogenous metabolites like spermine are promising candidates for new anti-osteoporotic drug development and daily bone-fortifying agents.

EFFECT OF SPERMININE AND ITS COMBINATION WITH SPERMINOXIDASE INHIBITORS ON THE PROFILE OF POLYAMINE AND SURVIVAL OF HUMAN PROSTATE CANCER CELLS

Summary. Aim: to investigate the effect of spermine (Spn) and spermine oxidase inhibitors (SMO) and their effect in combination on the polyamine profile in human PCa cell lines and to compare the nature of polyamine changes with tumor cell viability. Object and methods: the study was performed on cell cultures of hormone-sensitive (LNCaP) and hormone-resistant (DU-145) cell lines. The profile of polyamines in tumor cells and the activities of spermine oxidase and polyamine oxidase (PAO) were determined by high-performance liquid chromatography. Results: the addition of Spn to the culture medium causes a significant accumulation of this polyamine in cells, a decrease in putrescine (Put) and spermidine (Spd). The use of SMO inhibitors (chlorhexidine and MDL-72527) is accompanied by a decrease in Spd levels and accumulation of acetylated spermidine (AcSpd). The use of Spn leads to a decrease in the proliferation rate — the molar ratio of spermidine/spermine (Spd/Spn). Particularly low values of the Spd/Spn ratio were observed when spermine was used in combination with SMO inhibitors. When Spn was used in combination with SMO inhibitors, the lowest cell viability rates were also observed, indicating a more effective inhibition of cell growth with the combined use of these factors compared to their use separately, even in significantly higher concentrations. The use of chlorhexidine and chlorhexidine together with spermine led to a decrease in the activity of SMO and PAO. Conclusions: the use of spermine and spermine in combination with SMO inhibitors leads to changes in the polyamine profile in tumor cells characteristic of growth inhibition (decrease in Spd, Put and increase in Spn) and a decrease in the molar ratio Spd/Spn. These changes are accompanied by a decrease in the survival of cells of hormone-sensitive (LNCaP) and hormone-resistant (DU-145) human prostate cancer lines. The obtained results show the prospects for further study of spermine and its combination with SMO inhibitors as potential agents for the treatment of prostate cancer.

The effect of spermine on Tetranychus urticae-Cucumis sativus interaction

BackgroundTwo spotted spider mite, Tetranychus urticae (Acari: Tetranychidae) is one of the most important plant pests in the world. Due to increased resistance of mites to acaricides, it is necessary to use other methods such as inducing resistance in plants by natural compounds for pests' management. Polyamins such as spermine are effective in increasing plant resistance to biotic and abiotic stressors. In this research, the effect of spermine treatments in cucumber plants on life table parameters of T. urticae was investigated. Also, top-down effect of spermine and T. urticae on cucumber biochemical parameters was measured. In the experiments, 1, 2 and 3 mM spermine concentrations were used.ResultsAmongst the spermine treatments, those mites that fed on cucumbers which received 1 mM spermine showed the shortest protonymphal period and higher ovipositon period, fecundity, gross and net reproductive rates and life expectancy compare to control. Treatment with 2 mM spermine lead to the longest teleochrysalis period and shortest range of age-stage-specific fecundity period. In addition, 2 mM spermine lowered intrinsic and finite rate of population increase in T. urticae. The longest larval period of T. urticae was observed in 3 mM spermine. Feeding of T. urticae from cucumber plants increased hydrogen peroxide (H2O2), malondialdehyde (MDA) content, electrolyte leakage (EL) level and ascorbate peroxidase (APX) activity but inhibited catalase (CAT) activity in this plant. Infested cucumber plants treated with 2 mM spermine showed lower H2O2 and MDA content and highest activity of APX and CAT on day 1 and 3 compare to the others. The 3 mM spermine increased H2O2 content in infested plants during the whole experiment as well as non-infested plants in day 5 and 9 only. This treatment induced the highest MDA content and lowest catalase activity on day1, 3 and 5 of experiment in infested plants.ConclusionThis study showed that 2 mM spermine was the only effective concentration that reduce cucumber sensitivity to T. urticae. The trend of changes in biochemical parameters, especially H2O2, in 3 mM spermine was abnormal, and this concentration could be considered toxic.

Polyamines modulate mouse sperm motility

Polyamines are polycationic molecules which contains two or more amino groups (-NH3 +) highly charged at physiological pH, and among them we found spermine, spermidine, putrescine, and cadaverine. They interact with proteins, nucleic acids, modulate Ca2+, K+, and Na+ channels, and protect sperm from oxidative stress. In this work, we evaluate the effect of spermine, spermidine, and putrescine on the total, progressive and kinematic parameters of motility, capacitation, acrosome reaction, also in presence and absence of the dbcAMP, an analogue of the cAMP, and the IBMX, a phosphodiesterase inhibitor. In addition, we evaluated the intracellular concentrations of cAMP [cAMP]i, and performed an in silico analysis between polyamines and the sAC from mouse to predict the possible interaction among them. Our results showed that all polyamines decrease drastically the total, progressive and the kinetic parameters of sperm motility, decrease the capacitation, and only spermidine and putrescine impeded the acquisition of acrosome reaction. Moreover, the effect of polyamines was attenuated but not countered by the addition of db-cAMP and IBMX, suggesting a possible inhibition of the sAC. Also, the presence of polyamines induced a decrease of the [cAMP]i, and the in silico analysis predicted a strong interaction among polyamines and the sAC. Overall, the evidence suggests that probably the polyamines interact and inhibit the activity of the sAC.

Deciphering the synergistic effect of Jasmonic acid and Spermine in mitigating root-knot nematode stress in tomato plants through enhancing growth and activity of antioxidant enzymes

Root-Knot nematodes (RKNs) are one of the major plant pathogens that can substantially reduce plant growth and development. Management of RKNs is one of the major concerns as chemical pesticides pose a potential risk to humans and other life forms and also have toxic effects to environment. There is a need to find out some alternative eco-friendly strategies for management of these parasites. Jasmonic acid (JA) and Spermine (Spm) are those plant growth regulators (PGRs) that have positive effects on plant growth and alleviates toxic effects caused by various stresses including RKNs. Keeping this in mind,present study accessed the potential of these (PGRs), JA and Spm on morphological and biochemical attributes in 45 days old Solanum lycopersicum plants under nematode stress. It was observed that nematode infection resulted in reduced plant growth and photosynthetic pigments. Application of JA and Spm improved growth parameters and photosynthetic pigments. Moreover, tomato plants after JA and Spm supplementation lowered the activities of H2O2, DPPH and MDA thus decreasing nuclear and membrane damage caused by nematode stress. Additionally, enzymatic CAT (Catalase), SOD (Superoxide dismutase), GuPOX (Guaiacol peroxidase), APOX (Ascorbate peroxidase), PPO (Polyphenol oxidase), GST (Glutathione-S-transferase) and non-enzymatic (ascorbic acid, glutathione, and tocopherol) antioxidant levels increased markedly under nematode stress, but application of JA and Spm further improved these attributes. Taken together, the current study demonstrated that JA and Spm are suitable agents for managing nematode stress resistance in tomato plants.

The calcium-sensing receptor modulates the prostaglandin E2 pathway in intestinal inflammation.

Introduction: The prostaglandin E2 (PGE2) pathway is one of the main mediators of intestinal inflammation. As activation of the calcium-sensing receptor (CaSR) induces expression of inflammatory markers in the colon, we assessed the impact of the CaSR on the PGE2 pathway regulation in colon cancer cells and the colon in vitro and in vivo. Methods and Results: We treated CaSR-transfected HT29 and Caco-2 colon cancer cell lines with different orthosteric ligands or modulators of the CaSR and measured gene expression and PGE2 levels. In CaSR-transfected HT29CaSR-GFP and Caco-2CaSR-GFP cells, the orthosteric CaSR ligand spermine and the positive allosteric CaSR modulator NPS R-568 both induced an inflammatory state as measured by IL-8 gene expression and significantly increased the expression of the PGE2 pathway key enzymes cyclooxygenase (COX)-2 and/or prostaglandin E2 synthase 1 (PGES-1). Inhibition of the CaSR with the calcilytic NPS 2143 abolished the spermine- and NPS R-568-induced pro-inflammatory response. Interestingly, we observed cell-line specific responses as e.g. PGES-1 expression was affected only in HT29CaSR-GFP but not in Caco-2CaSR-GFP cells. Other genes involved in the PGE2 pathway (COX-1, or the PGE2 receptors) were not responsive to the treatment. None of the studied genes were affected by any CaSR agonist in GFP-only transfected HT29GFP and Caco-2GFP cells, indicating that the observed gene-inducing effects of spermine and R-568 were indeed mediated by the CaSR. In vivo, we had previously determined that treatment with the clinically approved calcimimetic cinacalcet worsened symptoms in a dextran sulfate sodium (DSS)-induced colitis mouse model. In the colons of these mice, cinacalcet significantly induced gene expression of PGES-2 and the EP3 receptor, but not COX-2; while NPS 2143 increased the expression of the PGE2-degrading enzyme 15-hydroxyprostaglandin dehydrogenase (15-PGDH). Importantly, neither treatment had any effect on the colons of non-DSS treated mice. Discussion: Overall, we show that activation of the CaSR induces the PGE2 pathway, albeit with differing effects in vitro and in vivo. This may be due to the different microenvironment in vivo compared to in vitro, specifically the presence of a CaSR-responsive immune system. Since calcilytics inhibit ligand-mediated CaSR signaling, they may be considered for novel therapies against inflammatory bowel disease.

Enhanced cell growth, production, and mAb quality produced in Chinese hamster ovary-K1 cells by supplementing polyamine in the media.

Polyamines such as putrescine (PUT), spermidine (SPD), and spermine (SPM) are amine group-containing biomolecules that regulate multiple intracellular functions such as proliferation, differentiation, and stress response in mammalian cells. Although these biomolecules can be generated intracellularly, lack of polyamine-synthesizing activity has occasionally been reported in a few mammalian cell lines such as Chinese hamster ovary (CHO)-K1; thus, polyamine supplementation in serum-free media is required to support cell growth and production. In the present study, the effects of biogenic polyamines PUT, SPD, and SPM in media on cell growth, production, metabolism, and antibody quality were explored in cultures of antibody-producing CHO-K1 cells. Polyamine withdrawal from media significantly suppressed cell growth and production. On the other hand, enhanced culture performance was achieved in polyamine-containing media conditions in a dose-dependent manner regardless of polyamine type. In addition, in polyamine-deprived medium, distinguishing metabolic features, such as enriched glycolysis and suppressed amino acid consumption, were observed and accompanied by higher heterogeneity of antibody quality compared with the optimal concentration of polyamines. Furthermore, an excessive concentration of polyamines negatively affected culture performance as well as antibody quality. Hence, the results suggest that polyamine-related metabolism needs to be further investigated and polyamines in cell growth media should be optimized as a controllable parameter in CHO cell culture bioprocessing. KEY POINTS: • Polyamine supplementation enhanced cell growth and production in a dose-dependent manner • Polyamine type and concentration in the media affected mAb quality • Optimizing polyamines in the media is suggested in CHO cell bioprocessing.

Effect of Exogenous Spermine on Biofilm Formation in Mycobacteria by Stimulating the Synthesis of Glycopeptidolipids

Biofilm formation is of great interest by its ability to increase bacterial tolerance to antibiotics that represent a serious problem for modern medicine. Among mycobacteria, which are also capable of forming biofilms, there are pathogens of socially dangerous infections, including tuberculosis. Basing on these data, the strains of Mycolicibacterium smegmatis mc2 155 were chosen as the objects of this study, including the parent strain without deletions and its mutants with one (ΔrelMsm) and double (ΔrelMsmΔrelZ) chromosomal deletions of the genes responsible for the synthesis of alarmone synthetase enzymes. Biofilms of mutant strains exhibited defects in biofilm formation. We have shown that the integrity, hydrophobicity, and the level of biomass of surface mycobacterial biofilms are dependent on the amount of glycopeptidolipids (GPL) in cells. The level of GPL depends on the activity of alarmone synthetases. The biogenic polyamine spermine is able to enhance the production of GPLs, restoring the integrity of biofilms of mutant strains. It is possible that this effect of spermine is caused by the influence on the activity of mycobacterial alarmone synthetases, which makes promising the further studying the molecular mechanisms of its action.

Improving postharvest life, quality and bioactive compounds of strawberry fruits using spermine and spermidine.

Small fruits such as strawberries, are a good source of natural antioxidants. In recent decades, many efforts have been made to increase the shelf life of strawberries and maintain its nutritional value in post-harvest conditions. In the present study, the effects of spermine (Spm) and spermidine (Spd) (0, 1.0 and 1.5 mM) on the post-harvest life and quality of strawberry fruits during the 3rd, 6th, and 12th days of storage, were investigated. Applications of Spm and Spd decreased the rate of weight loss, fruit decay, soluble solids content, fruit juice pH and taste index during the storage period in compared to the control. However, titratable acids and vitamin C contents, tissue stiffness, phenolic compounds and antioxidant activity increased in compared to the control. These growth regulators prevented the aging and loss of bioactive compounds of the fruit by increasing the antioxidant activity and preventing the destruction of the fruit tissue. Among the studied treatments, applications of 1.5 mM of Spm and Spd were the most effective treatments to enhance the storage life and quality characters of strawberry fruits.

THE INFLUENCE OF SPERMINE AND AMINOGUANIDINE ON THE MORPHOFUNCTIONAL CHARACTERISTICS OF HUMAN PROSTATE CANCER CELL LINE LNCaP

Summary. Aim: to investigate the inhibitory effect of spermine (Spn) and the modifying effect of aminoguanidine (AG) on the structural and functional characteristics of human prostate cancer cells of the LNCaP line, depending on the mode of their use. Object and methods: studies were conducted in vitro on human prostate cancer (PC) cells of the hormone-dependent LNCaP line. Cell survival was determined by the trypan blue exclusion. The concentration of Spn used in the study was 1.5 and 5.0 mM, and AG in combination with Spn 1.5 mM. Changes in the morphology of LNCaP cells were evaluated under microscopr on the fixed cytological specimens of cells grown on coverslips and stained with hematoxylin and eosin. Results: Spn at a concentration of 1.5 mM exhibites a pronounced inhibitory effect on the growth of LNCaP cells reducing their survival. Cells die at a Spn concentration of 5.0 mM. The presence of AG (1.5 mM) in the culture medium contributes to a significant recovery of cell survival. Conclusions: the increase in Spn concentration from 1.5 mM to 5.0 mM seems to result in apoptotic death of LNCaP cells. In sublethal concentrations, Spn reduces the synthetic and suppresses the mitotic activity of LNCaP cells, while AG shows a certain protective effect.

THE INFLUENCE OF SPERMINE AND CHLORHEXIDINE ON THE SURVIVAL, ELECTROKINETIC AND CYTOMORPHOLOGICAL CHARACTERISTICS OF HUMAN PROSTATE CANCER CELLS

Summary. Aim: to study the effects of spermine and the spermine oxidase inhibitor chlorhexidine, applied alone or in combination, on the viability, electrokinetic and structural-functional characteristics of human prostate cancer cells in vitro. Object and methods: Studies were conducted on cell cultures: differentiated androgen-dependent LNCaP cell line and low-differentiated androgen-independent DU-145 cell line. Cell survival was determined by the method of exclusion of the vital dye trypan blue by living cells. The electrokinetic parameters of the cells (ζ-potential and total surface charge density) were determined according to the Smoluchovsky and Quincke-Helmholtz equations. Morphological changes were assessed by light microscopy of fixed preparations of cells stained with hematoxylin and eosin. Results: The fact established by previous studies that spermine at a concentration of 1.5 mM exhibits a pronounced inhibitory effect on the growth of differentiated human prostate cancer cells of the LNCaP line, which consists in reducing their survival to 48.5 ± 1.5% compared to intact control, and when this factor is used in a concentration exceeding 5.0 mM – in their death. It was shown that poorly differentiated cells of the DU-145 line were significantly more resistant to its influence: their survival decreased to 56.0 ± 1.9% only under the influence of spermine at a concentration of 5.0 mM; a concentration of 10.0 mM caused complete cell death of both lines. The trend persisted in the case of chlorhexidine: under its influence at a concentration of 15.0 μM, the survival of cells of the hormone-dependent LNCaP line was 33.0 ± 1.5%, and the hormone-independent DU-145 cell line was 79.0 ± 2.5%. The combined use of spermine and chlorhexidine (0.6 mM and 3.2 µM, respectively) caused a strong cytotoxic effect in cells of the LNCaP line (survival 19.0 ± 1.2%), while cells of the DU-145 line maintained a fairly high rate (76.0 ± 2.1%) even with a significantly higher content of spermine (3.0 mM) and chlorhexidine (5.0 μM) in the culture medium. It was established that under the influence of chlorhexidine, the cells of both studied lines show an inversion of the sign of the surface electric charge, changing it from negative to positive under physiological conditions, just as it was shown in experiments with spermine on cells of the LNCaP line. Certain quantitative differences caused by the investigated compounds in the electrokinetic indicators of cells of hormone-dependent and hormone-independent lines were revealed. Cells of the DU-145 line under the influence of spermine, or simultaneously spermine and chlorhexidine, showed a typical apoptotic morphology: they rounded and separated from each other and the substrate, chromatin condensation, blurred contours of the surface membrane, nuclear fragmentation and the presence of apoptotic bodies were noted. Conclusions: Spermine in concentrations of 1.5-10.0 mM and chlorhexidine in concentrations of 10.0-30.0 μM, applied separately, have a cytotoxic effect on human prostate cancer cells of LNCaP and DU-145 lines in culture. Cytotoxic properties of spermine, chlorhexidine or their combination are significantly more pronounced when acting on the hormone-dependent LNCaP line cells than on the hormone-independent DU-145 line. Their combined use demonstrates a significantly higher cytotoxic effect on cells of the LNCaP line, which is not observed in the DU-145 cell line. Chlorhexidine, like spermine, causes an inversion of the surface charge in the studied cell lines, changing its sign from negative to positive. The revealed differences like changes in survival and electrokinetic parameters of LNCaP and DU-145 cells can be explained by the presence or absence of androgen binding receptors on their surface membrane. Cells of the DU-145 line under the influence of spermine, or at the same time spermine and chlorhexidine, go to the state of apoptosis, as well as cells of the LNCaP line when they are exposed to spermine.

Treatment with polyamines alleviates the effects of concomitantly applied aluminum in sunflower (Helianthus annuus L.) leaves

Contamination of agricultural soils with heavy metal is a significant risk for the environment. Many substances are reported to alleviate the toxic effects of heavy metals such as polyamines. The aim of this study is to examine whether the toxic effects of 0.1 mM aluminum, which is previously detected in sunflower leaves, might be alleviated with 0.1 mM putrescine, spermine or spermidine and to compare the effects of putrescine, spermine and spermidine in the ameliorating process. Chlorophyll a, carotenoid and anthocyanin content increased after putrescine, spermine and spermidine treatment under aluminum toxicity. However, chlorophyll b and total chlorophyll content only increased after spermine treatment. Intense accumulation of reactive oxygen species under aluminum toxicity decreased after putrescine, spermine and spermidine treatment while the spermine showed the maximum decrease. Superoxide dismutase enzyme activity and hydrogen peroxide content increased after putrescine, spermine and spermidine treatment while the spermine showed the maximum increase. Besides, catalase enzyme activity increased only after spermine treatment. Results showed that 0.1 mM putrescine, spermine and spermidine increased the 0.1 mM aluminum toxicity tolerance of sunflower leaves by modulating the reactive oxygen species detoxification metabolism. Spermine was the most effective polyamine in improving the aluminum tolerance.

Elevation of spermine remodels immunosuppressive microenvironment through driving the modification of PD-L1 in hepatocellular carcinoma

BackgroundSpermine is frequently elevated in tumor tissues and body fluids of cancer patients and is critical for cancer cell proliferation, migration and invasion. However, the immune functions of spermine in hepatocellular carcinoma progression remains unknown. In the present study, we aimed to elucidate immunosuppressive role of spermine in hepatocellular carcinoma and to explore the underlying mechanism.MethodsWhole-blood spermine concentration was measured using HPLC. Human primary HCC tissues were collected to examine the expression of CaSR, p-Akt, β-catenin, STT3A, PD-L1, and CD8. Mouse model of tumorigenesis and lung metastasis were established to evaluate the effects of spermine on hepatocellular carcinoma. Western blotting, immunofluorescence, real time PCR, digital Ca2+ imaging, and chromatin immunoprecipitation assay were used to investigate the underlying mechanisms by which spermine regulates PD-L1 expression and glycosylation in hepatocellular carcinoma cells.ResultsBlood spermine concentration in the HCC patient group was significantly higher than that in the normal population group. Spermine could facilitate tumor progression through inducing PD-L1 expression and decreasing the CD8+ T cell infiltration in HCC. Mechanistically, spermine activates calcium-sensing receptor (CaSR) to trigger Ca2+ entry and thereby promote Akt-dependent β-catenin stabilization and nuclear translocation. Nuclear β-catenin induced by spermine then activates transcriptional expression of PD-L1 and N-glycosyltransferase STT3A, while STT3A in turn increases the stability of PD-L1 through inducing PD-L1 protein N-glycosylation in HCC cells.ConclusionsThis study reveals the crucial function of spermine in establishing immune privilege by increasing the expression and N-glycosylation of PD-L1, providing a potential strategy for the treatment of hepatocellular carcinoma.7ng36xqHia8rCQn62itWxRVideo

Analysis of absorption-enhancing mechanisms for combinatorial use of spermine with sodium taurocholate in Caco-2 cells

Previously, we reported that the combined use of spermine (SPM) and sodium taurocholate (STC) (SPM–STC) significantly improves the oral absorption of rebamipide (BCS class IV) and pulmonary absorption of interferon-α without any harmful histopathological changes in the gastrointestinal tract and lungs, respectively. In the present study, we examined the effect of SPM–STC on the transport of fluorescein isothiocyanate-labeled dextrans (FDs) across Caco-2 cell monolayers and attempted to clarify the mechanisms underlying the transport enhancement caused by SPM–STC. SPM–STC were found to significantly enhance the transport of FDs, while the treatment with SPM–STC was not harmful, and the decrease in transepithelial electrical resistance was transient and reversible. The voltage-clamp study clearly indicated that the opening of the paracellular route could be mainly responsible for the enhanced transport of FD-4. As for the mechanisms, it was found that SPM–STC caused a significant increase in membrane fluidity, which would lead to the enhanced transport of small-molecule drugs such as rebamipide. Since SPM–STC increased intracellular Ca2+ via Ca2+ uptake through Ca2+ channels and Ca2+ release from the endoplasmic reticulum stimulated by the IP3 pathway, the subsequent possible activation of the MLCK signaling pathway would have led to the contraction of the actin–myosin ring. The rearrangement of tight junction-constituting proteins induced through the MAPK pathway has also been suggested as a possible mechanism for opening tight junctions. Claudin-4, a key protein constituting the tight junction, merged with F-actin along with the plasma membrane, was significantly decreased, which would be at least partial structural evidence for the tight-junction opening.