Spermidine/spermine N1-acetyltransferase Activity Research Articles

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

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

Abstract B052: Leveraging the metabolic stress of polyamine biosynthesis in prostate cancer towards a novel therapeutic approach

Abstract Prostatic epithelial cells secrete high levels of acetylated polyamines into the prostatic lumen. This distinctive characteristic places added strain on connected pathways, forcing increased metabolite production from both one-carbon metabolism and the methionine cycle to maintain nucleotide and S-adenosylmethionine (SAM) pools, respectively. More importantly, this stress is increased in prostate cancer (CaP) due to increased polyamine biosynthesis, DNA synthesis, and proliferation. The metabolic flux is driven by the activity of spermidine/spermine N1-acetyltransferase (SSAT), which acetylates the polyamines leading to their secretion into the lumen, which drives demand for biosynthesis of polyamines. To overcome this stress, the methionine salvage pathway (MSP) recycles the one-carbon unit lost to polyamine biosynthesis back to the methionine cycle, allowing for replenishment of SAM pools. The rate-limiting enzyme involved in this process is methylthioadenosine phosphorylase (MTAP). We have found that CaP relies on the MSP to relieve the strain caused by high polyamine biosynthesis and that both genetic and pharmacologic inhibition of MTAP blocks CaP xenograft growth. We hypothesized that this dependence can be enhanced by increasing the activity of SSAT. Recent results from our lab have shown that pharmacologic inhibition of MTAP alongside SSAT upregulation is synergistic in multiple androgen-sensitive and castration-recurrent CaP cell lines. We are currently exploring the effects of our combination treatment in castration-recurrent xenografts in vivo as well as in human CaP treatment-naïve tumors from radical prostatectomies grown as ex vivo explants. Preliminary results reveal that our treatments are effective at slowing growth in a subset of xenografts in vivo as well as impacting our target enzymes, polyamine levels, and increasing apoptosis in our ex vivo system. We expect that simultaneous targeting of multiple, converging pathways that are exceptionally important for prostate will lead to significant delay or prevention of disease recurrence. Citation Format: Hayley C. Affronti, Anthony J. Pellerite, Aryn Rowsam, Spencer R. Rosario, Robert A. Casero, Mikhail A. Nikiforov, James Phillips, Dominic J. Smiraglia. Leveraging the metabolic stress of polyamine biosynthesis in prostate cancer towards a novel therapeutic approach [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr B052.

Abstract 3765: Induction of amineoxidases as a possible mechanism of action of tamoxifen metabolites

Abstract To elucidate the mechanism of action of endoxifen in breast cancer, we determined its effects on estradiol-mediated growth of ERalpha-positive MCF-7 cells. Endoxifen is the active metabolite of tamoxifen; the most widely used selective estrogen receptor modulator for anti-estrogen therapy for women with estrogen receptor alpha-positive breast cancer. Clinical trials recognized that tamoxifen was a prodrug and got converted to active metabolites in the human body. Earlier studies recognized 4-hydroxytamoxifen (4HT) as the active metabolite of tamoxifen, although recent studies showed that the concentration of endoxifen was up to 10-fold higher than that of 4HT in patients treated with tamoxifen. These metabolites are formed by the action of cytochrome P450 2D6 (CYP2D6). We used quantitative PCR analysis to examine the effects of endoxifen on estrogenic stimulation of early response genes, c-myc, c-fos and Tff1. Since the natural polyamines, putrescine, spermidine and spermine, play important roles in estrogenic function, we also measured the activities of enzymes in the polyamine biosynthetic/metabolic pathway: ornithine decarboxylase (ODC), S-adenosyl-L-methionine decarboxylase (AdoMetDC), spermidine/spermine N1-acetyltransferase (SSAT), spermine oxidase (SMO), and acetylpolyamine oxidase (APAO). Polyamine levels were quantified by high performance liquid chromatography. Our results showed that estradiol increased the proliferation of MCF-7 cells by 2- to 3-fold and endoxifen suppressed the effects of estradiol on cell growth. The expression of c-myc, c-fos and Tff1 genes was significantly increased by estradiol, while the addition of endoxifen suppressed estradiol-mediated increase in gene expression. Estradiol increased the activity of ODC and AdoMetDC, whereas endoxifen suppressed the activity of these enzymes. Endoxifen had no effect on SSAT activity, whereas it significantly increased activities of amineoxidases, SMO and APAO. Estradiol increased putrescine and spermidine levels and endoxifen inhibited this increase. There was no significant effect on spermine levels. These data suggest that endoxifen exerts its growth inhibitory effects on MCF-7 cells by suppressing the expression of estradiol-stimulated oncogenes and polyamine biosynthetic pathway. The antiestrogenic effect of endoxifen is also manifested through the stimulation of polyamine oxidase enzymes, SMO and APAO. These results indicate for the first time that the upregulation of SMO and APAO is important in the mechanism of action of endoxifen in breast cancer cells. Citation Format: T. J. Thomas, Tuomo A. Keinanen, Mervi T. Hyvonen. Induction of amineoxidases as a possible mechanism of action of tamoxifen metabolites. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3765.

Decreased sensitivity to aspirin is associated with altered polyamine metabolism in human prostate cancer cells

Aspirin is a well-known analgesic, anti-inflammatory and antipyretic drug and is recognised as a chemopreventative agent in cardiovascular disease and, more recently, in colorectal cancer. Although several studies indicate that aspirin is capable of reducing the risk of developing cancers, there is a lack of convincing evidence that aspirin can prevent prostate cancer in man. In this study, aspirin was shown to be an effective inhibitor of the growth of human prostate cancer cells. In order to investigate the link between polyamine catabolism and the effects of aspirin we used a “Tet off” system that induced the activity of spermidine/spermine N1-acetyltransferase (SSAT) in human prostate cancer cells (LNCap). Treatment with aspirin was found to decrease induced SSAT activity in these cells. A negative correlation was observed between increased polyamine catabolism via increased SSAT activity and the sensitivity to aspirin. In the presence of increased SSAT activity high amounts of N1-acetylspermidine and putrescine were observed. These cells were also found to grow more slowly than the non-induced cells. The results indicate that SSAT and its related polyamine metabolism may play a key role in sensitivity of cancer cells to aspirin and possibly other NSAIDs and this may have implications for the development of novel chemopreventative agents.

Overexpression of spermidine/spermine N 1-acetyltransferase impairs osteoblastogenesis and alters mouse bone phenotype

Spermidine/spermine N (1)-acetyltransferase (SSAT) is a catabolic regulator of polyamines, ubiquitous molecules essential for cell proliferation and differentiation. In pathological conditions, the increased polyamine catabolism has been shown to mediate its cellular functions not only by changed polyamine levels but also by the availability of metabolites shared with other metabolic pathways or by production of toxic compounds. Our previous results showed that mice overexpressing SSAT (SSAT mice) developed a myeloproliferative disease and the bone marrow microenvironment partly contributed to its development. In this study, the physiological role of SSAT and polyamines in bone remodeling was characterized. Skeletal development of the SSAT mice appeared outwardly similar to wild-type mice until maturity, after which the SSAT mice developed kyphosis. With aging, the SSAT overexpression elicited increased bone perimeter with strikingly thinned cortical bone, decreased trabecular thickness and increased trabecular number in mice. In vitro studies showed that the maturation of SSAT overexpressing osteoblasts was impaired and the expression of bone formation marker genes was dramatically decreased. The polyamine pattern in osteoblasts of SSAT mice was distorted in comparison with wild-type mice. However, treatment of osteoblasts with a SSAT-inducing functional polyamine analogue suggested that defective osteoblastogenesis resulted rather from other consequences of enhanced SSAT activity than lowered levels of the higher polyamines. In comparison to SSAT overexpressing mice, SSAT deficiency led to opposite changes in osteoblastogenesis and differences in bone phenotype in mice. In conclusion, the level of SSAT enzyme activity affected osteoblastogenesis and hence influenced bone remodeling and the bone phenotype in mice. Furthermore, our results suggest the contribution of the catabolic part of the polyamine cycle, other than polyamine depletion, in pathophysiological processes of bone remodeling.

Spermidine/spermine N1-acetyltransferase activity associates with white blood cell count in myeloid leukemias

The metabolism of polyamines, the cationic small molecules essential for cell proliferation and differentiation, is altered in cancer cells and can be exploited in cancer diagnosis and therapy. Spermidine/spermine N(1)-acetyltransferase (SSAT), which regulates intracellular levels of polyamines by catabolizing spermidine and spermine, has a controversial role in the development of cancers. In this study, the polyamine metabolism and function of SSAT were characterized in acute myeloid leukemia (AML), chronic myeloid leukemia (CML), and acute lymphoid leukemia patient samples. Also, mice overexpressing SSAT and having a myeloproliferative phenotype were analyzed for their response to decitabine and histone deacetylase inhibitor trichostatin A. The presence of epigenetic factors in the bone marrow cells of SSAT mice was analyzed. Elevated levels of spermidine and spermine, as well as increased activity of SSAT, were detected in AML, CML, and acute lymphoid leukemia patients compared with the controls. However, we found SSAT activity to be associated with white blood cell count only in AML and CML patients. Decitabine treatment brought the peripheral blood and bone marrow cell counts of SSAT mice to the level of wild-type mice. Spermidine/spermine N(1)-acetyltransferase mice had increased histone methylation and an increased level of histone deacetylase 1 in their bone marrow cells. The study suggests that SSAT influences the development of myeloid malignancies, and epigenetic factors partly contribute to the SSAT overexpression-induced myeloproliferative disease in mice.

Assay of Ornithine Decarboxylase and Spermidine/Spermine N1-acetyltransferase Activities

The polyamines, spermidine (Spd) and spermine, and their diamine precursor putrescine, are important regulators of various cellular functions, such as proliferation and differentiation. Polyamine homeostasis is tightly regulated on the level of uptake, excretion, biosynthesis, interconversion and terminal catabolism. The rate-controlling enzymes of polyamine biosynthesis and interconversion are ornithine decarboxylase (ODC) and spermidine/spermine N1-acetyltransferase (SSAT), respectively. Here, we describe a protocol to assay ODC (Jänne and Williams-Ashman, 1971) and SSAT (Libby, 1978) activities from cell or tissue samples.

Enhanced polyamine catabolism disturbs hematopoietic lineage commitment and leads to a myeloproliferative disease in mice overexpressing spermidine/spermine N 1-acetyltransferase

Spermidine/spermine N(1)-acetyltransferase (SSAT) regulates intracellular polyamine levels by catabolizing spermidine and spermine which are essential for cell proliferation and differentiation. Hematological characterization of SSAT overexpressing mice (SSAT mice) revealed enhanced myelopoiesis and thrombocytopoiesis leading to increased amounts of myeloid cells in bone marrow, peripheral blood, and spleen compared to wild-type animals. The level of SSAT activity in the bone marrow cells was associated with the bone marrow cellularity and spleen weight which both were significantly increased in SSAT mice. The result of bone marrow transplantations indicated that both the intrinsic SSAT overexpression of bone marrow cells and bone marrow microenvironment had an impact on the observed hematopoietic phenotype. The Lineage-negative Sca-1(+) c-Kit(+) hematopoietic stem cell (HSC) compartment in SSAT mice, showed enhanced proliferation, increased proportion of long-term HSCs and affected expression of transcription factors associated with lineage priming and myeloid differentiation. The proportions of common myeloid and megakaryocytic/erythroid progenitors were decreased and the proportion of granulocyte-macrophage progenitors was increased in SSAT bone marrow. The data suggest that SSAT overexpression and the concomitantly accelerated polyamine metabolism in hematopoietic cells and bone marrow microenvironment affect lineage commitment and lead to the development of a mouse myeloproliferative disease in SSAT mice.

Integrin α9β1 in airway smooth muscle suppresses exaggerated airway narrowing

Exaggerated contraction of airway smooth muscle is the major cause of symptoms in asthma, but the mechanisms that prevent exaggerated contraction are incompletely understood. Here, we showed that integrin α9β1 on airway smooth muscle localizes the polyamine catabolizing enzyme spermidine/spermine N1-acetyltransferase (SSAT) in close proximity to the lipid kinase PIP5K1γ. As PIP5K1γ is the major source of PIP2 in airway smooth muscle and its activity is regulated by higher-order polyamines, this interaction inhibited IP3-dependent airway smooth muscle contraction. Mice lacking integrin α9β1 in smooth muscle had increased airway responsiveness in vivo, and loss or inhibition of integrin α9β1 increased in vitro airway narrowing and airway smooth muscle contraction in murine and human airways. Contraction was enhanced in control airways by the higher-order polyamine spermine or by cell-permeable PIP2, but these interventions had no effect on airways lacking integrin α9β1 or treated with integrin α9β1-blocking antibodies. Enhancement of SSAT activity or knockdown of PIP5K1γ inhibited airway contraction, but only in the presence of functional integrin α9β1. Therefore, integrin α9β1 appears to serve as a brake on airway smooth muscle contraction by recruiting SSAT, which facilitates local catabolism of polyamines and thereby inhibits PIP5K1γ. Targeting key components of this pathway could thus lead to new treatment strategies for asthma.

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.

Tissue-specific alternative splicing of spermidine/spermine N 1-acetyltransferase

The polyamines, spermidine and spermine, are abundant organic cations participating in many important cellular processes. We have previously shown that the rate-limiting enzyme of polyamine catabolism, spermidine/spermine N(1)-acetyltransferase (SSAT), has an alternative mRNA splice variant (SSATX) which undergoes degradation via nonsense-mediated mRNA decay (NMD) pathway, and that the intracellular polyamine level regulates the ratio of the SSATX and SSAT splice variants. The aim of this study was to investigate the effect of SSATX level manipulation on SSAT activity in cell culture, and to examine the in vivo expression levels of SSATX and SSAT mRNA. Silencing SSATX expression with small interfering RNA led to increased SSAT activity. Furthermore, transfection of SSAT-deficient cells with mutated SSAT gene (which produced only trace amount of SSATX) yielded higher SSAT activity than transfection with natural SSAT gene (which produced both SSAT and SSATX). Blocking NMD in vivo by protein synthesis inhibitor cycloheximide resulted in accumulation of SSATX mRNA, and like in cell culture, the increase of SSATX mRNA was prevented by administration of polyamine analog N(1),N(11)-diethylnorspermine. Although SSATX/total SSAT mRNA ratio did not correlate with polyamine levels or SSAT activity between different tissues, increasing polyamine levels in a given tissue led to decreased SSATX/total SSAT mRNA ratio and vice versa. Taken together, the regulated unproductive splicing and translation of SSAT has a physiological relevance in modulating SSAT activity. However, in addition to polyamine level there seems to be additional factors regulating tissue-specific alternative splicing of SSAT.

Novel anti-apoptotic effect of the retinoblastoma protein: implications for polyamine analogue toxicity

The retinoblastoma protein (pRb) pathway is frequently altered in breast cancer cells. pRb is involved in the regulation of cell proliferation and cell death. The breast cancer cell line L56Br-C1 does not express pRb and is extremely sensitive to treatment with the polyamine analogue N(1),N(11)-diethylnorspermine (DENSPM) which causes apoptosis. Polyamines are essential for the regulation of cell proliferation, cell differentiation and cell death. DENSPM depletes cells of polyamines, e.g., by inducing the activity of the polyamine catabolic enzyme spermidine/spermine N(1)-acetyltransferase (SSAT). In this study, L56Br-C1 cells were transfected with human pRb-cDNA. Overexpression of pRb inhibited DENSPM-induced cell death and DENSPM-induced SSAT activity. This suggests that the pRb protein level is a promising marker for polyamine depletion sensitivity and that there is a connection between pRb and the regulation of SSAT activity. We also show that SSAT protein levels and SSAT activity do not always correlate, suggesting that there is an unknown regulation of SSAT.

Polyamine Catabolism in Relation to Trypsin Activation and Apoptosis in Experimental Acute Pancreatitis

Background: Overinduced polyamine catabolism (PC) in a transgenic rat model has been suggested to be a mediator of trypsin activation which is important in acinar cell necrosis. PC has also been observed in experimental taurodeoxycholate pancreatitis. We hypothesized that PC may be a mediator of trypsin activation in taurodeoxycholate pancreatitis. Methods: Pancreatitis was induced in wild-type rats by 2 or 6% taurodeoxycholate infusion or in transgenic rats by overexpressing spermidine/spermine N1-acetyltransferase (SSAT). The time courses of necrosis, caspase-3 immunostaining, SSAT, polyamine levels, and trypsinogen activation peptide (TAP) were monitored. The effect of the polyamine analogue bismethylspermine (Me2Spm) was investigated. Results: In a transgenic pancreatitis model, TAP and acinar necrosis increased simultaneously after the activation of SSAT, depletion of spermidine, and development of apoptosis. In taurodeoxycholate pancreatitis, necrosis developed along with the accumulation of TAP. SSAT was activated simultaneously or after TAP accumulation and less than in the transgenic model, with less depletion of spermidine than in the transgenic model. Supplementation with Me2Spm ameliorated the extent of acinar necrosis at 24 h, but contrary to previous findings in the transgenic model, in the taurodeoxycholate model it did not affect trypsin activation. Compared with the transgenic model, no extensive apoptosis was found in taurodeoxycholate pancreatitis. Conclusions: Contrary to transgenic SSAT-overinduced pancreatitis, PC may not be a mediator of trypsin activation in taurodeoxycholate pancreatitis. The beneficial effect of polyamine supplementation on necrosis in taurodeoxycholate pancreatitis may rather be mediated by other mechanisms than amelioration of trypsin activation.

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.

Characterization of Polyamine Homeostasis in l-Ornithine-Induced Acute Pancreatitis in Rats

l-Ornithine is a precursor of polyamine synthesis that is essential for cell survival. In contrast, intraperitoneal (IP) administration of a large dose of l-ornithine results in death of pancreatic acinar cells in rats. We investigated changes in pancreatic and extrapancreatic polyamine homeostasis after injection of l-ornithine and tested the effects of the stable polyamine analogue methylspermidine (MeSpd) on l-ornithine-induced pancreatitis. Male Wistar rats were injected IP with 3 g/kg l-ornithine and were untreated, pretreated, or treated with 50 mg/kg MeSpd IP. Rats were killed after 0 to 168 hours for determinations of polyamines and activities of ornithine decarboxylase and spermidine/spermine N(1)-acetyltransferase (SSAT). Pancreatitis severity was assessed by measuring standard laboratory and histological parameters. Injection of l-ornithine paradoxically induced pancreatic spermidine catabolism, possibly via activation of SSAT, after (>6 hours) appearance of the first histological signs of acute pancreatitis. Polyamine levels generally increased in the lung and liver with the exception of lung spermidine levels, which decreased. Methylspermidine did not influence polyamine levels and SSAT activity and did not ameliorate the severity of l-ornithine-induced pancreatitis. l-Ornithine-induced pancreatitis was associated with activation of pancreatic polyamine catabolism. However, administration of a metabolically stable polyamine analogue did not affect disease severity.

Abstract 4603: Role of endoxifen as an antiestrogen: Effects on oncogene expression and polyamine pathway

Abstract The nonsteroid antiestrogen tamoxifen [trans-1-(4-b-dimethylaminoethoxyphenyl)-1,2-diphenylbut-1-ene] is the most commonly used endocrine treatment for estrogen receptor α (ERα)-positive breast cancer in pre- and post-menopausal women, and it has helped to reduce breast cancer death rate by one third. Tamoxifen is extensively metabolized, and several metabolites have been detected in human serum. It is metabolized to 4-hydroxytamoxifen and N-desmethyltamoxifen by the action of CYP2D6 and CYP3A4/5 enzymes, respectively. N-desmethyltamoxifen and 4-hydroxy-tamoxifen are further converted to endoxifen by the action of these enzymes. Steady state levels of serum tamoxifen/metabolite are achieved within 4 weeks of continuous administration of the drug. The therapeutic efficacy of tamoxifen is determined by the distribution of the drug into tissues and the availability of the parent drug and its active metabolites in target tissues. Recent reports indicate that endoxifen might act as an antiestrogen. In order to examine the mechanism of action of endoxifen on an ERα-positive breast cancer cell line, MCF-7, we determined the effects of 100, 250, 500 and 1000 nM concentrations of this agent on cell growth, c-myc oncogene expression and activity of the polyamine biosynthetic enzymes. MCF-7 breast cancer cells were treated for 1, 3 or 5 days with endoxifen. Endoxifen had a growth inhibitory effect on MCF-7 cells by day 3 and 5 of treatment, as measured by cell number. Estradiol increased cell number by >2-fold compared to untreated control by day 5 of treatment, whereas endoxifen inhibited the estrogenic effect. c-myc gene expression was increased by a significant 2- to 3-fold by 4 nM estradiol in 2 and 4 hours of treatment, while 250 and 500 nM endoxifen suppressed the increase in c-myc gene expression, as measured by real-time qPCR experiment. In the absence of estradiol, endoxifen slightly (1.5 fold) increased c-myc gene expression. Endoxifen decreased ornithine decarboxylase (ODC) and S-adenosine methyl decarboxylase (AdoMetDC) activities in a concentration- and time-dependent manner; however, it had little effect on spermidine/spermine N1-acetyl transferase (SSAT) activity. The change in biosynthetic enzyme activities were reflected in polyamine pools, as decreased putrescine and spermidine levels. There were no changes in spermine level. As expected, treatment of the cells with 4 nM estradiol induced ODC and AdoMetDC activities, and slightly decreased SSAT activity. Addition of endoxifen in combination with 4 nM estradiol led to even more dramatic decrease in ODC and AdoMetDC activities and reduction of putrescine and spermidine levels. These results support our hypothesis that endoxifen can act as an antiestrogen and suppress cell growth stimulatory effects of estradiol in ERα-positive breast cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4603.

The role of spermidine/spermine N1-acetyltransferase in endotoxin-induced acute kidney injury

The expression of catabolic enzymes spermidine/spermine N(1)-acetyltransferase (SSAT) and spermine oxidase (SMO) increases after ischemic reperfusion injury. We hypothesized that polyamine catabolism is upregulated and that this increase in catabolic response contributes to tissue damage in endotoxin-induced acute kidney injury (AKI). SSAT mRNA expression peaked at threefold 24 h following LPS injection and returned to background levels by 48 h. The activity of SSAT correlated with its mRNA levels. The expression of SMO also increased in the kidney after LPS administration. Serum creatinine levels increased significantly at approximately 15 h, peaking by 24 h, and returned to background levels by 72 h. To test the role of SSAT in endotoxin-induced AKI, we injected wild-type (SSAT-wt) and SSAT-deficient (SSAT-ko) mice with LPS. Compared with SSAT-wt mice, the SSAT-ko mice subjected to endotoxic-AKI had less severe kidney damage as indicated by better preservation of kidney function. The role of polyamine oxidation in the mediation of kidney injury was examined by comparing the severity of renal damage in SSAT-wt mice treated with MDL72527, an inhibitor of both polyamine oxidase and SMO. Animals treated with MDL72527 showed significant protection against endotoxin-induced AKI. We conclude that increased polyamine catabolism through generation of by-products of polyamine oxidation contributes to kidney damage and that modulation of polyamine catabolism may be a viable approach for the treatment of endotoxin-induced AKI.

Lactobacillus rhamnosus GG Influences Polyamine Metabolism in HGC-27 Gastric Cancer Cell Line: A Strategy Toward Nutritional Approach to Chemoprevention of Gastric Cancer

Chemoprevention by dietary constituents has recently emerged as a novel approach to control gastric cancer incidence. Over the past years, functional foods and food supplements, especially probiotics, have received much attention as potential dietary cancer prevention agents. The precise mechanisms by which these lactic cultures exert their antitumorigenic activities are not fully elucidated, but there is some evidence of their influence on cell proliferation and growth. Ornithine decarboxylase (ODC) and spermidine/spermine N1-acetyltransferase (SSAT) are the key enzymes involved in polyamine biosynthesis and catabolism, respectively. These polycationic compounds are significantly associated with cancer risk and represent a specific markers for neoplastic proliferation. The aim of this study was to investigate the effects of increasing concentrations of Lactobacillus rhamnosus strain GG (ATCC 53103) (L. GG) homogenate on polyamine biosynthesis and polyamine degradation as well as on resulting polyamine levels in HGC-27 human gastric cancer cells. The influence of this probiotic on cell proliferation was also evaluated. Administration of probiotic homogenate significantly reduced both ODC mRNA and activity as well as polyamine content and neoplastic proliferation. Besides, an increase in both SSAT mRNA and activity occurred after LGG administration in HGC-27. These data suggest that a nutritional component such as the probiotic L. GG could be proposed in an alternative approach to prevention of gastric cancer. This strategy could overcome the limitations due to a prolonged use of drugs and/or the occurrence of their adverse effects, and it could reasonably also start at a young age.

Polyamine Catabolism Is Enhanced after Traumatic Brain Injury

Polyamines spermine and spermidine are highly regulated, ubiquitous aliphatic cations that maintain DNA structure and function as immunomodulators and as antioxidants. Polyamine homeostasis is disrupted after brain injuries, with concomitant generation of toxic metabolites that may contribute to secondary injuries. To test the hypothesis of increased brain polyamine catabolism after traumatic brain injury (TBI), we determined changes in catabolic enzymes and polyamine levels in the rat brain after lateral controlled cortical impact TBI. Spermine oxidase (SMO) catalyzes the degradation of spermine to spermidine, generating H2O2 and aminoaldehydes. Spermidine/spermine-N(1)-acetyltransferase (SSAT) catalyzes acetylation of these polyamines, and both are further oxidized in a reaction that generates putrescine, H2O2, and aminoaldehydes. In a rat cortical impact model of TBI, SSAT mRNA increased subacutely (6-24 h) after TBI in ipsilateral cortex and hippocampus. SMO mRNA levels were elevated late, from 3 to 7 days post-injury. Polyamine catabolism increased as well. Spermine levels were normal at 6 h and decreased slightly at 24 h, but were normal again by 72 h post-injury. Spermidine levels also decreased slightly (6-24 h), then increased by approximately 50% at 72 h post-injury. By contrast, normally low putrescine levels increased up to sixfold (6-72 h) after TBI. Moreover, N-acetylspermidine (but not N-acetylspermine) was detectable (24-72 h) near the site of injury, consistent with increased SSAT activity. None of these changes were seen in the contralateral hemisphere. Immunohistochemical confirmation indicated that SSAT and SMO were expressed throughout the brain. SSAT-immunoreactivity (SSAT-ir) increased in both neuronal and nonneuronal (likely glial) populations ipsilateral to injury. Interestingly, bilateral increases in cortical SSAT-ir neurons occurred at 72 h post-injury, whereas hippocampal changes occurred only ipsilaterally. Prolonged increases in brain polyamine catabolism are the likely cause of loss of homeostasis in this pathway. The potential for simple therapeutic interventions (e.g., polyamine supplementation or inhibition of polyamine oxidation) is an exciting implication of these studies.

Polyamine catabolism and disease.

In addition to polyamine homoeostasis, it has become increasingly clear that polyamine catabolism can play a dominant role in drug response, apoptosis and the response to stressful stimuli, and contribute to the aetiology of several pathological states, including cancer. The highly inducible enzymes SSAT (spermidine/spermine N1-acetyltransferase) and SMO (spermine oxidase) and the generally constitutively expressed APAO (N1-acetylpolyamine oxidase) appear to play critical roles in many normal and disease processes. The dysregulation of polyamine catabolism frequently accompanies several disease states and suggests that such dysregulation may both provide useful insight into disease mechanism and provide unique druggable targets that can be exploited for therapeutic benefit. Each of these enzymes has the potential to alter polyamine homoeostasis in response to multiple cell signals and the two oxidases produce the reactive oxygen species H2O2 and aldehydes, each with the potential to produce pathological states. The activity of SSAT provides substrates for APAO or substrates for the polyamine exporter, thus reducing the intracellular polyamine concentration, the net effect of which depends on the magnitude and rate of any increase in SSAT. SSAT may also influence cellular metabolism via interaction with other proteins and by perturbing the content of acetyl-CoA and ATP. The goal of the present review is to cover those aspects of polyamine catabolism that have an impact on disease aetiology or treatment and to provide a solid background in this ever more exciting aspect of polyamine biology.