Activation Of Polyamine Catabolism Research Articles

The Polyamine Putrescine Promotes Human Epidermal Melanogenesis

Hyperpigmentary conditions can arise when melanogenesis in the epidermis is misregulated. Understanding the pathways underlying melanogenesis is essential for the development of effective treatments. Here, we report that a group of metabolites called polyamines are important in the control of melanogenesis in human skin. Polyamines are cationic molecules present in all cells and are essential for cellular function. We report that polyamine regulator ODC1 is upregulated in melanocytes from melasma lesional skin. We report that the polyamine putrescine can promote pigmentation in human skin explants and primary normal human epidermal melanocytes through induction of tyrosinase which is rate-limiting for the synthesis of melanin. Putrescine supplementation on normal human epidermal melanocytes results in the activation of polyamine catabolism, which results in increased intracellular H2O2. Polyamine catabolism is also increased in human skin explants that have been treated with putrescine. We further report that inhibition of polyamine catabolism prevents putrescine-induced promotion of tyrosinase levels and pigmentation in normal human epidermal melanocytes, showing that polyamine catabolism is responsible for the putrescine induction of melanogenesis. Our data showing that putrescine promotes pigmentation has important consequences for hyperpigmented and hypopigmented conditions. Further understanding of how polyamines control epidermal pigmentation could open the door for the development of new therapeutics.

Activation of Polyamine Catabolism by N¹,N11-Diethylnorspermine in Hepatic HepaRG Cells Induces Dedifferentiation and Mesenchymal-Like Phenotype.

Tumorigenesis is accompanied by the metabolic adaptation of cells to support enhanced proliferation rates and to optimize tumor persistence and amplification within the local microenvironment. In particular, cancer cells exhibit elevated levels of biogenic polyamines. Inhibitors of polyamine biosynthesis and inducers of their catabolism have been evaluated as antitumor drugs, however, their efficacy and safety remain controversial. Our goal was to investigate if drug-induced modulation of polyamine metabolism plays a role in dedifferentiation using differentiated human hepatocyte-like HepaRG cell cultures. N1,N11-diethylnorspermine (DENSpm), a potent inducer of polyamine catabolism, triggered an epithelial-mesenchymal transition (EMT)-like dedifferentiation in HepaRG cultures, as shown by down-regulation of mature hepatocytes markers and upregulation of classical EMT markers. Albeit the fact that polyamine catabolism produces H2O2, DENSpm-induced de-differentiation was not affected by antioxidants. Use of a metabolically stable spermidine analogue showed furthermore, that spermidine is a key regulator of hepatocyte differentiation. Comparative transcriptome analyses revealed, that the DENSpm-triggered dedifferentiation of HepaRG cells was accompanied by dramatic metabolic adaptations, exemplified by down-regulation of the genes of various metabolic pathways and up-regulation of the genes involved in signal transduction pathways. These results demonstrate that polyamine metabolism is tightly linked to EMT and differentiation of liver epithelial cells.

DENSpm overcame Bcl-2 mediated resistance against Paclitaxel treatment in MCF-7 breast cancer cells via activating polyamine catabolic machinery.

The Bcl-2 mediated resistance is one of the most critical obstacle in cancer therapy. Conventional chemotherapeutics such as Paclitaxel, a commonly used in the treatment of metastatic breast cancer, is not sufficient to overcome Bcl-2 mediated drug resistance mechanism. Thus, combinational drug regimes are favored by researchers to overcome resistance phenotype against drugs. N1,N11-diethylnorspermine (DENSpm), a polyamine analogue, which is a promising drug candidate induced-cell cycle arrest and apoptosis in various cancer cells such as prostate, melanoma, colon and breast cancer cells via activated polyamine catabolism and reactive oxygen generation. Recent studies indicated the potential therapeutic role of DENSpm in phase I and II trials in breast cancer cases. Although the molecular targets of Paclitaxel in apoptotic cell death mechanism is well documented, the therapeutic effect of DENSpm and Paclitaxel in breast cancer cells has not been investigated yet. In this study, our aim was to determine the time dependent effect of DENSpm and Paclitaxel on apoptotic cell death via determination of polyamine metabolism related targets in wt and Bcl-2 overexpressing MCF-7 breast cancer cells. In our experimental study, Paclitaxel decreased cell viability in dose-dependent manner within 24h. Co-treatment of Paclitaxel (30nM) with DENSpm (20μM) further increased the cytoxicity of Paclitaxel (30nM) compared to alone Paclitaxel (30nM) treatment in MCF-7 Bcl-2+ breast cancer cells. In addition, we determined that resistance against Paclitaxel-induced apoptotic cell death in Bcl-2 overexpressed MCF-7 cells was overcome due to activation of polyamine catabolic pathway, which caused depletion of polyamines. DENSpm combinational treatment might increase the effect of low cytotoxic paclitaxel in drug-resistant breast cancer cases.

CDK Inhibitors Induce Mitochondria-mediated Apoptosis Through the Activation of Polyamine Catabolic Pathway in LNCaP, DU145 and PC3 Prostate Cancer Cells

Androgen signaling is critical in prostate cancer development and progression. The co-existence of hormone responsive and irresponsive cells due to functional androgen receptor (AR) in prostate gland is the major obstacle in prostate cancer therapy models. Targeting aberrant cell cycle by novel cell cycle blocking agents is a promising strategy to treat various types of malignancies. Purvalanol and roscovitine are cyclin dependent kinase (CDK) inhibitors able to activate apoptotic cell death by inducing cell cycle arrest at G1/S and G2/M phases in cancer cells. Polyamines are unique cationic amine derivatives involved in the regulation of cell proliferation. Although the elevated intracellular level of polyamines (putrescine, spermidine and spermine) is typical for prostate gland, abnormal regulation of polyamine metabolism might result in rapid cell proliferation and, thus in prostate cancer progression. Therefore, treatment with drug-induced depletion of intracellular polyamine levels through the activated polyamine catabolism is critical to achieve successful strategies for prostate cancer. In this study we aimed to investigate the apoptotic efficiency of CDK inhibitors in three prostate cancer cell lines (LNCaP, DU145 and PC3), showing different AR expression profile. We found that both purvalanol and roscovitine were able to induce apoptosis at moderate cytotoxic concentrations by decreasing mitochondria membrane potential. The apoptotic effect of both CDK inhibitors was due to activation of caspases by modulating Bcl-2 family members. The efficiency of drugs was quite similar on the three prostate cell lines used in this study. However, DU145 cells were found the least sensitive against CDK inhibitors while purvalanol was more potent than roscovitine. Similarly to classical chemotherapeutic agents, both drugs could up-regulate polyamine catabolic enzymes (SSAT, SMO and PAO) in cell type dependent manner. Transient silencing of SSAT and/or inhibition of PAO/ SMO with MDL72527 prevented CDK inhibitors- induced apoptotic cell death in DU145 and PC3 cells. Although roscovitine was less effective in DU145 cells, pre-treatment with α-difluoromethylornithine (DFMO), an inhibitor of ODC, enhanced the roscovitine-induced apoptotic cell death through the cleavage of caspase-9 and caspase-3. Therefore, we conclude that polyamine catabolism might have essential role in the cellular responses against CDK inhibitors in different androgen-responsive or irresponsive prostate cancer cells.

Altered glucose-stimulated insulin secretion in a mouse line with activated polyamine catabolism

Ubiquitous activation of polyamine catabolism has been demonstrated to have protective effects in mice on fat accumulation and insulin sensitivity/glucose tolerance in, both, normal conditions and after a high fat diet. We have analyzed the endocrine pancreas functionality in four months-old male mice overexpressing the rate limiting enzyme in the polyamine catabolism, spermidine/spermine N¹-acetyltransferase (SSAT). The pancreatic SSAT activity was 37-fold elevated in the transgenic mice, which reduced the total pancreatic and islet pools of spermidine (71%) and spermine (69%), and increased putrescine and N¹-acetyl spermidine. Reduction in the islet ATP levels (65%) was accompanied with increased transcription of 5'-AMP-activated protein kinase (AMPK) (1.5-fold) and Foxa2 (2.7-fold), and reduced HNF4α (67%) and HNF1α (92%), insulin 1 (47%), insulin 2 (50%), and Glut2 (57%). Moreover, the SSAT transgenic mice also presented increased beta cell area, decreased insulin production, and altered glucose-stimulated insulin secretion. It has been hypothesized that the acute activation of the polyamine catabolism produces a futile cycle that greatly decreases the energy reserves of the cell. The lower energy status would activate the energy expenditure regulator, AMPK, which would consequently repress the PI3K/Akt pathway, and activate the transcription factor Foxa2.

The activation of hepatic and muscle polyamine catabolism improves glucose homeostasis

The mitochondrial biogenesis and energy expenditure regulator, PGC-1α, has been previously reported to be induced in the white adipose tissue (WAT) and liver of mice overexpressing spermidine/spermine N (1)-acetyltransferase (SSAT). The activation of PGC-1α in these mouse lines leads to increased number of mitochondria, improved glucose homeostasis, reduced WAT mass and elevated basal metabolic rate. The constant activation of polyamine catabolism produces a futile cycle that greatly reduces the ATP pools and induces 5'-AMP-activated protein kinase (AMPK), which in turn activates PGC-1α in WAT. In this study, we have investigated the effects of activated polyamine catabolism on the glucose and energy metabolisms when targeted to specific tissues. For that we used a mouse line overexpressing SSAT under the endogenous SSAT promoter, an inducible SSAT overexpressing mouse model using the metallothionein I promoter (MT-SSAT), and a mouse model with WAT-specific SSAT overexpression (aP2-SSAT). The results demonstrated that WAT-specific SSAT overexpression was sufficient to increase the number of mitochondria, reduce WAT mass and protect the mice from high-fat diet-induced obesity. However, the improvement in the glucose homeostasis is achieved only when polyamine catabolism is enhanced at the same time in the liver and skeletal muscle. Our results suggest that the tissue-specific targeting of activated polyamine catabolism may reveal new possibilities for the development of drugs boosting mitochondrial metabolism and eventually for treatment of obesity and type 2 diabetes.

Association between remote organ injury and tissue polyamine homeostasis in acute experimental pancreatitis – treatment with a polyamine analogue bismethylspermine

Experimental pancreatitis is associated with activation of polyamine catabolism. The polyamine analog bismethylspermine (Me2Spm) can ameliorate pancreatic injury. We investigated the roles of polyamine catabolism in remote organs during pancreatitis and explored the mechanism of polyamine catabolism by administering Me2Spm. Acute pancreatitis was induced by an infusion of 2 or 6% taurodeoxycholate before Me2Spm administration. Blood, urine and tissues were sampled at 24 and 72h to assess multi-organ injury and polyamine catabolism. The effect of Me2Spm on mortality in experimental pancreatitis was tested separately. Liver putrescine levels were elevated following liver injury. Me2Spm increased the activity of spermidine/spermine N1-acetyltransferase (SSAT) and depleted the spermidine, spermine or putrescine levels. Lung putrescine levels increased, and SSAT and spermine decreased following lung injury. Me2Spm enhanced the activity of SSAT and decreased the spermidine and spermine levels. Renal injury was manifested as an increase in creatinine or a decrease in urine output. Decreases in kidney SSAT, spermidine or spermine and an increase in putrescine were found during pancreatitis. In the 2% taurodeoxycholate model, Me2Spm decreased urine output and raised plasma creatinine levels. Me2Spm increased SSAT and decreased polyamines. Excessive Me2Spm accumulated in the kidney, and greater amounts were found in the 6% taurodeoxycholate model in which this mortality was not reduced by Me2Spm. In the 2% taurodeoxycholate model, Me2Spm dose-dependently induced mortality at 72h.Like pancreatic injury, remote organ injury in pancreatitis is associated with increased putrescine levels. However, Me2Spm could not ameliorate multi-organ injury. Me2Spm administration was associated with significant renal toxicity and induced mortality, suggesting that the current dose is too high and needs to be modified.

Continuous oxidative stress due to activation of polyamine catabolism accelerates aging and protects against hepatotoxic insults

Enhanced polyamine catabolism via polyamine acetylation-oxidation elevates the oxidative stress in an organism due to increased production of reactive oxygen species (ROS). We studied a transgenic mouse line overexpressing the rate limiting enzyme in the polyamine catabolism, spermidine/spermine N (1)-acetyltransferase (SSAT) that is characterized by increased putrescine and decreased spermidine and spermine pools. In order to protect the mice from the chronic oxidative stress produced by the activation of polyamine catabolism, the hepatic expression of the transcription factor p53 was found threefold elevated in the transgenic mice. In addition, the prolonged activation of p53 accelerated the aging of transgenic mice and reduced their lifespan (50%). Aging was associated with decreased antioxidant enzyme activities. In the transgenic mice the activities of catalase and Cu, Zn-superoxide dismutase (SOD) were 42 and 23% reduced respectively, while the expression of CYP450 2E1 was 60% decreased and oxidative stress measured as protein carbonyl content was tenfold elevated. In the transgenic mice, the age-related repression of the different antioxidant enzymes served as a protection against the hepatotoxic effects of carbon tetrachloride and thioacetamide.

Acute Pancreatitis Induced by Activated Polyamine Catabolism Is Associated with Coagulopathy: Effects of α-Methylated Polyamine Analogs on Hemostasis

Background/Aims: Polyamines are ubiquitous organic cations essential for cellular proliferation and tissue integrity. We have previously shown that pancreatic polyamine depletion in rats overexpressing the catabolic enzyme, spermidine/spermine N1-acetyltransferase (SSAT), results in the development of severe acute pancreatitis, and that therapeutic administration of metabolically stable α-methylated polyamine analogs protects the animals from pancreatitis-associated mortality. Our aim was to elucidate the therapeutic mechanism(s) of α-methylspermidine (MeSpd). Methods: The effect of MeSpd on hemostasis and the extent of organ failure were studied in SSAT transgenic rats with either induced pancreatitis or lipopolysaccharide (LPS)-induced coagulopathy. The effect of polyamines on fibrinolysis and coagulation was also studied in vitro. Results: Pancreatitis caused a rapid development of intravascular coagulopathy, as assessed by prolonged coagulation times, decreased plasma fibrinogen level and antithrombin activity, enhanced fibrinolysis, reduced platelet count and presence of schistocytes. Therapeutic administration of MeSpd restored these parameters to almost control levels within 24 h. In vitro, polyamines dose-dependently inhibited fibrinolysis and intrinsic coagulation pathway. In LPS-induced coagulopathy, SSAT transgenic rats were more sensitive to the drug than their syngeneic littermates, and MeSpd-ameliorated LPS-induced coagulation disorders. Conclusion: Pancreatitis-associated mortality in SSAT rats is due to coagulopathy that is alleviated by treatment with MeSpd.

Proteomic analysis of livers from a transgenic mouse line with activated polyamine catabolism

We have generated a transgenic mouse line that over expresses the rate-controlling enzyme of the polyamine catabolism, spermidine/spermine N (1)-acetyltransferase, under the control of a heavy metal inducible promoter. This line is characterized by a notable increase in SSAT activity in liver, pancreas and kidneys and a moderate increase in the rest of the tissues. SSAT induction results in an enhanced polyamine catabolism manifested as a depletion of spermidine and spermine and an overaccumulation of putrescine in all tissues. To study how the activation of polyamine catabolism affects other metabolic pathways, protein expression pattern of the livers of transgenic animals was analyzed by two-dimensional polyacrylamide gel electrophoresis and mass spectrometry. A total of 23 proteins were shown to be differentially expressed in the transgenic from the wild-type animals. Many of the identified proteins showed expression patterns associated with polyamine catabolism activation. However, the expression pattern of other proteins, such as repression of GST pi and selenium-binding protein 2 and 60 kDa heat-shock protein, could be explained by the overexpression of peroxisome proliferator-activated receptor gamma co-activator 1alpha in response to depleted ATP pools. The activation of the latter proteins is thought to lead to the improved insulin sensitivity seen in the MT-SSAT animals.

Activated polyamine catabolism leads to low cholesterol levels by enhancing bile acid synthesis

Transgenic mice with activated polyamine catabolism due to overexpression of spermidine/spermine N(1)-acetyltransferase (SSAT) have significantly reduced plasma total cholesterol levels. In our study, we show that low cholesterol levels were attributable to enhanced bile acid synthesis in combination with reduced cholesterol absorption. Hepatic cholesterol 7alpha-hydroxylase (CYP7A1), the rate-limiting enzyme catalyzing the conversion of cholesterol to bile acids, plays an important role in the removal of excess cholesterol from the body. We suggest that by reducing activity of Akt activated polyamine catabolism increased the stability and activity of peroxisome proliferator-activated receptor gamma co-activator 1alpha, the critical activator of CYP7A1. This is supported by our finding that the treatment with SSAT activator, N (1) ,N(11)-diethylnorspermine, reduced significantly the amount of phosphorylated (active) Akt in HepG2 cells. In summary, activated-polyamine catabolism is a novel mechanism to regulate bile acid synthesis. Therefore, polyamine catabolism could be a potential therapeutic target to control hepatic CYP7A1 expression.

α-Methylspermidine protects against carbon tetrachloride-induced hepatic and pancreatic damage

The role of polyamines in carbon tetrachloride (CCl(4))-induced organ injury was studied in syngenic rats and transgenic rats with activated polyamine catabolism. In syngenic rats, administration of CCl(4) resulted in the induction of hepatic spermidine/spermine N(1)-acetyltransferase (SSAT), accumulation of putrescine, reduction in spermine level and appearance of moderate hepatic injury within 24 h. Upon treatment with CCl(4), transgenic rats overexpressing SSAT displayed induction of both hepatic and pancreatic SSAT, with subsequent accumulation of putrescine and decrease of both spermidine and spermine pools. Administration of CCl(4) in SSAT transgenic rats induced not only massive hepatic injury, but also severe acute necrotizing pancreatitis. Pretreatment of the animals with catabolically stable functional polyamine mimetic, alpha-methylspermidine (MeSpd) prevented pancreatic and hepatic injury in SSAT rats and markedly reduced liver damage in syngenic animals. As assessed by immunostaining of proliferating cell nuclear antigen, MeSpd increased the amount of regenerating hepatocytes in both genotypes. These results show that CCl(4) induces hepatic and pancreatic polyamine catabolism, and the extent of organ damage correlates with the degree of polyamine depletion. Furthermore, MeSpd protects against CCl(4)-induced hepatic and pancreatic damage and promotes tissue regeneration.

Changes in blood polyamine levels in human acute pancreatitis

Objective. Experimental studies have shown that pancreatic activation of polyamine catabolism occurs during the early stage of acute pancreatitis. Changes in pancreatic polyamines are reflected in the red blood cell (RBC) polyamine contents, correlating with the extent of pancreatic necrosis. The aim of this human study was to examine the changes in polyamine levels in the RBCs of patients with acute pancreatitis. Material and methods. Twenty-four patients with acute pancreatitis (7 alcoholic, 10 gallstone and 7 of unknown etiology) were recruited in the study. Eighteen patients with non-pancreatic acute abdominal diseases were included as controls, and 6 volunteers were studied as references. Blood samples were collected on admission and during hospitalization to assess polyamine levels. After clinical recovery, the patients revisited the clinic, and RBC polyamine levels were measured again. For comparison, plasma interleukin-6 (IL-6), IL-10 and C-reactive protein (CRP) were measured. Results. In acute pancreatitis patients, there was no difference in RBC polyamine levels on admission compared with those in controls or in volunteers. Putrescine levels on admission were higher in patients with pancreatic necrosis than in patients without necrosis, but there was no difference in spermidine and spermine levels. Patients with pancreatitis of unknown etiology had significantly higher levels of polyamines on admission and throughout hospitalization, but they also had more necrosis, which explained the difference in multivariate analysis. Spermidine and spermine levels increased after clinical recovery. RBC putrescine correlated with IL-6 and IL-10, and spermine correlated with CRP. Conclusions. The results of this study suggest that RBC polyamines change in human acute pancreatitis in several respects, as has been previously observed in experimental pancreatitis.

A polyamine analog bismethylspermine ameliorates severe pancreatitis induced by intraductal infusion of taurodeoxycholate

Stable polyamine homeostasis is important for cell survival and regeneration. Our experimental studies have shown that catabolism of spermidine and spermine to putrescine is associated with the development of pancreatitis. We investigated the pathogenetic role of polyamine catabolism by studying the effect of a methylated polyamine analog on taurodeoxycholate-induced acute experimental pancreatitis. Acute pancreatitis was induced by infusion of sodium taurodeoxycholate (2%) into the pancreatic duct. Bismethylspermine (Me(2)Spm) was administered as a pretreatment before the induction of pancreatitis or as a treatment after the induction of pancreatitis. The sham operation included laparotomy only. Pancreas tissue and blood were sampled at 24 h and 72 h after the infusion of taurodeoxycholate and studied for pancreatitis severity (serum amylase activity, pancreatic water content, and histology) and polyamine catabolism, which includes spermidine/spermine N(1)-acetyltransferase (SSAT) activity as well as spermidine, spermine, and putrescine concentrations in the pancreas. Sodium taurodeoxycholate-induced acute pancreatitis manifests as increases in serum amylase and pancreatic water content, leukocytosis, and acinar cell necrosis in the pancreas. The activity of SSAT increased significantly together with an increase in the ratios of pancreatic putrescine/spermidine and putrescine/spermine at 24 h, which indicates SSAT-induced polyamine catabolism. Pancreatic water content and necrosis were reduced significantly by the treatment with Me(2)Spm at 24 h but not at 72 h when the polyamine homeostasis had recovered, and the pancreatitis had progressed. Taurodeoxycholate-induced acute pancreatitis was associated with activation of polyamine catabolism in the pancreas. The polyamine analog Me(2)Spm ameliorated the injury in the early stage, but it did not ameliorate the late progression of the pancreatic necrosis at 72 h. Thus, besides proteolytic enzyme activation and the cascades of inflammation, polyamine catabolism may be an important pathogenetic mediator of the early stages of acute pancreatitis.

Role of Hypusinated Eukaryotic Translation Initiation Factor 5A in Polyamine Depletion-induced Cytostasis

We have earlier shown that alpha-methylated spermidine and spermine analogues rescue cells from polyamine depletion-induced growth inhibition and maintain pancreatic integrity under severe polyamine deprivation. However, because alpha-methylspermidine can serve as a precursor of hypusine, an integral part of functional eukaryotic translation initiation factor 5A required for cell proliferation, and because alpha, omega-bismethylspermine can be converted to methylspermidine, it is not entirely clear whether the restoration of cell growth is actually attributable to hypusine formed from these polyamine analogues. Here, we have used optically active isomers of methylated spermidine and spermine and show that polyamine depletion-induced acute cytostasis in cultured cells could be reversed by all the isomers of the methylpolyamines irrespective of whether they served or not as precursors of hypusine. In transgenic rats with activated polyamine catabolism, all the isomers similarly restored liver regeneration and reduced plasma alpha-amylase activity associated with induced pancreatitis. Under the above experimental conditions, the (S, S)- but not the (R, R)-isomer of bismethylspermine was converted to methylspermidine apparently through the action of spermine oxidase strongly preferring the (S, S)-isomer. Of the analogues, however, only (S)-methylspermidine sustained cell growth during prolonged (more than 1 week) inhibition of polyamine biosynthesis. It was also the only isomer efficiently converted to hypusine, indicating that deoxyhypusine synthase likewise possesses hidden stereospecificity. Taken together, the results show that growth inhibition in response to polyamine depletion involves two phases, an acute and a late hypusine-dependent phase.

Activation of polyamine catabolism by N1, N11-diethylnorspermine alters the cellular localization of mTOR and downregulates mTOR protein level in glioblastoma cells

N1, N11-Diethylnorspermine (DENSPM) is a spermine analog and prototype anti-cancer drug that depletes cellular polyamine, increases cellular oxidative stress through the generation of H2O2, and induces the death of multiple types of cancer cells. However, the survival pathways perturbed by DENSPM are uncertain. To identify these pathways, we examined a series of proteins in the phosphoinositide 3-kinase /AKT/mammalian target of rapamycin (PI3K/AKT/mTOR) pathways in glioblastoma cell lines before and after treatment with DENSPM. We found that DENSPM did not change the protein levels of PI3K but did reduce the levels of AKT, phosphorylated AKT, mTOR, phosphorylated mTOR, p70S6K, phosphorylated p70S6K, 4E-BP1, phosphorylated 4E-BP1, and eIF-4B proteins. From this it appears that DENSPM directly targets the mTOR protein level in these glioblastoma cells by inhibiting mTOR-mediated protein synthesis. Immunofluorescence analysis of mTOR showed that DENSPM sequestered mTOR in the perinuclear region of the cells. We also detected a marked collapse of microtubules in U87 cells and a detachment of cells in a process resembling anoikis. We further showed that the levels of many proteins regulating cell growth and cell adhesion were down-regulated, suggesting a broad effect of DENSPM on mTOR-mediated protein synthesis. We conclude that the activation of polyamine catabolism alters the cellular location of mTOR, thus negatively affecting mTOR-mediated protein synthesis and leading to the death of glioblastoma cells.

Activation of polyamine catabolism by N1,N11-diethylnorspermine leads to cell death in glioblastoma

Glioblastoma multiforme (GBM) is one of the most therapeutically refractory human cancers. Elevated cellular polyamine levels are a common feature of cancer cells, including GBM cells, and the polyamine pathway has been explored as a potential therapeutic target to inhibit polyamine biosynthesis or activate polyamine catabolism. In this study, we investigated the effect of N1,N11-diethyl-norspermine (DENSPM), a spermine analog that activates polyamine catabolism, in GBM cells. The in vitro cell culture experiments showed that DENSPM increased the sub-G1 apoptotic cell population in GBM cell lines but caused minimal cytotoxicity in normal astrocytes. Prior to apoptosis induction, DENSPM caused the elevation of spermidine/spermine N1-acetyltransferase (SSAT) expression accompanied by a decrease in polyamine levels and an increase of acetylated polyamine levels, which temporally coincided with the onset of hydrogen peroxide (H2O2) induction in the cells. The cytotoxic effects of DENSPM in the GBM cells could be partially attenuated by either turning down SSAT mRNA with small interference RNA or inhibiting H2O2 production with N1-acetylpolymine oxidase (APAO)/spermine oxidase (SMO) inhibitor. Though mitochondrial damage was induced, neither activation of the caspase cascade nor cytochrome c redistribution between the mitochondria and cytoplasm was observed. Systemic DENSPM treatment of mice with intracerebral GBM led to longer survival. Taken together, our studies indicate that DENSPM kills GBM cells through induction of SSAT coupled with H2O2 production, which is a potential target for GBM therapy.

Enhanced Polyamine Catabolism Alters Homeostatic Control of White Adipose Tissue Mass, Energy Expenditure, and Glucose Metabolism

Peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1 alpha) is an attractive candidate gene for type 2 diabetes, as genes of the oxidative phosphorylation (OXPHOS) pathway are coordinatively downregulated by reduced expression of PGC-1 alpha in skeletal muscle and adipose tissue of patients with type 2 diabetes. Here we demonstrate that transgenic mice with activated polyamine catabolism due to overexpression of spermidine/spermine N(1)-acetyltransferase (SSAT) had reduced white adipose tissue (WAT) mass, high basal metabolic rate, improved glucose tolerance, high insulin sensitivity, and enhanced expression of the OXPHOS genes, coordinated by increased levels of PGC-1 alpha and 5'-AMP-activated protein kinase (AMPK) in WAT. As accelerated polyamine flux caused by SSAT overexpression depleted the ATP pool in adipocytes of SSAT mice and N(1),N(11)-diethylnorspermine-treated wild-type fetal fibroblasts, we propose that low ATP levels lead to the induction of AMPK, which in turn activates PGC-1 alpha in WAT of SSAT mice. Our hypothesis is supported by the finding that the phenotype of SSAT mice was reversed when the accelerated polyamine flux was reduced by the inhibition of polyamine biosynthesis in WAT. The involvement of polyamine catabolism in the regulation of energy and glucose metabolism may offer a novel target for drug development for obesity and type 2 diabetes.

Oxidative stress and inflammation in the pathogenesis of activated polyamine catabolism-induced acute pancreatitis

The markers of oxidative stress and inflammation were studied in acute pancreatitis in transgenic rats exhibiting activated polyamine catabolism. In addition, the effect of bismethylspermine (Me(2)Spm) pretreatment, preventing pancreatitis in this model, on these mediators was investigated. Lipid peroxidation was increased at 6 and 24 h after induction of pancreatitis. These changes as well as the markedly decreased superoxide dismutase activity at 24 h were abolished by Me(2)Spm pretreatment. Glutathione level and catalase activity changed transiently, and the effect of Me(2)Spm was clear at 24 h. Serum inflammatory cytokine levels increased already at 4 h whereas NF-kappaB was distinctly activated only at 24 h. Me(2)Spm prevented the increase in TNF-alpha and IL-6 while it had no effect on NF-kappaB activation. These results show that typical inflammatory and, to a lesser degree, some oxidative stress mediators are involved and beneficially affected by the disease-ameliorating polyamine analogue in our pancreatitis model.

Mechanisms of polyamine catabolism-induced acute pancreatitis

Acute pancreatitis is an autodigestive disease, in which the pancreatic tissue is damaged by the digestive enzymes produced by the acinar cells. Among the tissues in the mammalian body, pancreas has the highest concentration of the natural polyamine, spermidine. We have found that pancreas is very sensitive to acute decreases in the concentrations of the higher polyamines, spermidine and spermine. Activation of polyamine catabolism in transgenic rats overexpressing SSAT (spermidine/spermine-N(1)-acetyltransferase) in the pancreas leads to rapid depletion of these polyamines and to acute necrotizing pancreatitis. Replacement of the natural polyamines with methylated polyamine analogues before the induction of acute pancreatitis prevents the development of the disease. As premature trypsinogen activation is a common, early event leading to tissue injury in acute pancreatitis in human and in experimental animal models, we studied its role in polyamine catabolism-induced pancreatitis. Cathepsin B, a lysosomal hydrolase mediating trypsinogen activation, was activated just 2 h after induction of SSAT. Pre-treatment of the rats with bismethylspermine prevented pancreatic cathepsin B activation. Analysis of tissue ultrastructure by transmission electron microscopy revealed early dilatation of rough endoplasmic reticulum, probable disturbance of zymogen packaging, appearance of autophagosomes and later disruption of intracellular membranes and organelles. Based on these results, we suggest that rapid eradication of polyamines from cellular structures leads to premature zymogen activation and autodigestion of acinar cells.