Spermidine/spermine Acetyltransferase Research Articles

PEDV infection affects the expression of polyamine-related genes inhibiting viral proliferation

Porcine epidemic diarrhea virus (PEDV) is an alpha-coronavirus that causes epidemic diarrhea in swines. The mortality of PEDV infection in one-week-old piglets is extremely high, which causes a huge significant economic loss to the global pig husbandry and blocks its healthy development. There was a lack of adequate studies to elucidate pathogenic mechanism associated with PEDV infection. In the present study, we detected the expression profiles of polyamine metabolism associated genes in Vero cells infected with PEDV by RT-qPCR. It is shown that PAOX(acetylpolyamine oxidase), SMOX(spermine oxidase), SAT1(spermidine-spermine acetyltransferase 1), ODC1(ornithine decarboxylase 1), DHPS(deoxyhypusine synthase) and EIF5A( eukaryotic initiation factor 5A) were significantly upregulated. Through intervening SAT1 level in PEDV-infected Vero cells, it is identified that overexpression of SAT1 inhibited PEDV replication by reducing polyamine levels. Furthermore, polyamine depletion and upregulation were found to regulate the proliferation of PEDV. PEDV infection in Vero cells did not result in a significant change in the protein level of eIF5A, and in addition, the activated eIF5A did not affect the proliferation of PEDV. Our results provided new insights into the influence of polyamine metabolism on the proliferation of PEDV.

Abstract B091: Small-molecule inhibitors targeting a specific metabolic pathway for precision therapy of advanced castrate-resistant prostate cancer

Abstract Introduction: The mechanism of prostate cancer (PCa) progression to the mostly lethal, metastatic, castrate-resistant stage (mCRPC) remains generally unknown. This prevents identifying patients likely to progress to mCRPC and designing new therapies for their treatment. A transcription factor JunD is overexpressed in PCa, but not in normal prostate epithelial cells. It complexes with androgen receptor (AR) to induce spermidine/spermine acetyl transferase (SSAT) [3]. SSAT initiates polyamine oxidation that generates copious amounts of reactive oxygen species (ROS) production in polyamine-rich PCa cells. ROS activate NF-κB. NF-κB can also induce SSAT and activate AR. This sets up a feed-forward loop for PCa growth at low androgen. Here, we present one mechanism of PCa progression to CRPC as well as its invasion and metastasis that may provide predictive biomarkers to identify patients with potentially lethal PCa at an early stage. Effects of an agent that can block both AR-JunD and AR-NF-κB interactions and prevent growth of PCa cells in vitro as well as in vivo xenografts will also be presented. Method: We have used a novel microscale device to separate invading from noninvading PCa cells ex vivo in a bone microenvironment. Immunocytochemistry (ICC), proteomic and metabolomic techniques have been employed to analyze the separated cells to understand the mechanism of invasion. Gaussia luciferase reconstitution assay has been used in a high-throughput screen (HTS) to identify inhibitors of AR-JunD and AR-NF-κB interactions. State-of-the-art in vitro cell growth assay along with in vivo pharmacokinetic (PK), maximum tolerated dose (MTD) and enzalutamide-resistant CRPC cell xenograft growth in nude mice are used to optimize the lead. Summary of Unpublished Results: Immunocytochemistry (ICC) data show that in cultured enzalutamide-resistant C4-2 and -sensitive LNCaP cells as well as in some patient PCa cells, more SSAT-positive cells are in the migratory than in the stationary section of the microscale device. Metabolomic analysis show a decrease in 3-phospho-glycerate levels in C4-2 cells, suggesting a decrease in GAPDH enzymatic activity that is related to an enhanced oxidation of GAPDH protein. Our published data of screening of 27,000+ compounds from two different chemical libraries detected a single compound that specifically inhibits both AR-JunD and AR-NF-κB2 (p52) interactions. We have synthesized several of its analogs. Lead analogs inhibit growth of AR-positive C4-2 and LNCaP cells, but have little effect on the growth of AR-negative PC-3 and SSAT-silent LNCaP siSSAT cells in culture. Co-immunoprecipitation (co-IP) assay shows its efficacy in inhibiting AR-JunD and AR-p52 interactions in LNCaP cells. It has been formulated in 25% ethanol:water and administered to nude mice orally. It is well tolerated at 50 mg/kg p.o. daily for more than 28 days and has a serum Cmax of ~ 3 μM within 30 minutes and a plasma t1/2 of ~1 h after a single oral dose of 100 mg/kg. Twenty-four hours after dosing it is found in the flank tumor (1.5 ng/mg tissue; ~450 nM) and in the prostate tissues (10 ng/mg tissue; ~3 μM) in addition to liver and kidney. Its effects on the growth of C4-2 xenografts in nude mice will be presented. Conclusion: A mechanism of PCa progression to CRPC and then to mCRPC provides a rational basis for targeted drug design that prevents PCa progression and metastasis. This should open up a new avenue of precision therapy of potentially lethal PCa at an early stage. Citation Format: Hirak Basu, Nathaniel Wilganowski, Jessica Lieblich, Grace Wu, Izabela Fokt, Sumankalai Ramachandran, Jiaquin Yu, Mark Titus, Waldemar Priebe, David J. Beebe, George Wilding. Small-molecule inhibitors targeting a specific metabolic pathway for precision therapy of advanced castrate-resistant prostate cancer [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 B091.

Abstract 3846: A metabolic pathway targeted inhibitor for precision therapy of castrate-resistant prostate cancer

Abstract Background: We have previously reported that an AP-1 factor JunD is overexpressed in PCa. It complexes with androgen receptor (AR) to induce spermidine/spermine acetyl transferase (SSAT). SSAT initiates polyamine oxidation that generates copious amounts of reactive oxygen species (ROS) production in polyamine-rich PCa cells. ROS activate NF-κB. NF-κB also induces SSAT and activates AR. This sets-up a feed-forward loop for PCa growth at low androgen. An IHC assay for SSAT can be utilized to identify PCa patients with this loop activated. AR-N-terminal targeted inhibitors that block AR interactions with JunD and NF-κB can be developed as a precision therapy for these patients. Method: We applied Immunocytochemistry (ICC), proteomic and metabolomic techniques to analyze stationary and invading cells that are separated in a novel micro-scale dvice to understand the mechanism of invasion. Gaussia luciferase reconstitution assay in a high throughput screen (HTS) to identify inhibitors of AR-JunD and AR-NF-κB interactions. In vitro cell growth assay along with in vivo pharmacokinetic (PK), maximum tolerated dose (MTD) and enzalutamide-resistant CRPC cell xenograft growth in nude mice are used to optimize the lead. Unpublished data: Immunocytochemistry (ICC) data show that in cultured enzalutamide-resistant C4-2 and -sensitive LNCaP cells as well as in some patient PCa cells, more SSAT positive cells are in the migratory than in the stationary section of the microscale device. Screening of 27,000+ compounds from 2 different chemical libraries detected a single compound that specifically inhibits both AR-JunD and AR-NF-κB2 (p52) interactions. We have synthesized several of its analogs. Lead analogs inhibit growth of AR-positive C4-2 and LNCaP cells at nanomolar concentration, but have little effect on the growth of AR-negative PC-3 and SSAT silent siSSAT cells in culture. Co-immunoprecipitation (co-IP) assay shows its efficacy in inhibiting AR-JunD and AR-p52 interactions in situ. It has been formulated in 25% ethanol:water and administered orally. PCa tumor bearing nude mice tolerated it at 50 mg/kg p.o. daily for more than 28 days and has a serum Cmax of ~ 3 μM within 30 minutes and a plasma t1/2 of ~1 h after a single oral dose of 100 mg/kg. Twenty four hours after dosing it is found in the flank tumor (1.5 ng/mg tissue; ~450 nM) and in the prostate tissues (10 ng/mg tissue; ~3 μM) in addition to liver and kidney and inhibited growth of C4-2 xenografts in nude mice. Citation Format: Hirak S. Basu, Nathaniel L. Wilgonowski, Jessica L. Lieblich, Grace T. Wu, Izabela Fokt, Sumankalai Ramachandran, Jiaquin Yu, Mark Titus, Waldemar Priebe, David J. Beebe, George Wilding. A metabolic pathway targeted inhibitor for precision therapy of castrate-resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3846.

Metabolomic study of urinary polyamines in rat exposed to 915 MHz radiofrequency identification signal

Metabolomic analysis of urinary polyamines (PAs) from rat exposed to 915 MHz radiofrequency identification (RFID) signal for 8 h/day for 2 weeks was performed by gas chromatography-mass spectrometry as N-ethoxycarbonyl/N-pentafluoropropionyl derivatives. Large alterations in nine PA levels including four aliphatic and five acetylated PAs were monitored in sham-exposed and RFID-exposed groups. Total PA and urinary levels of N (1)-acetylputrescine, N (1)-acetylcadaverine, putrescine, cadaverine, N (1)-acetylspermidine, N (8)-acetylspermidine, spermidine and spermine were reduced, whereas N (1)-acetylspermine was significantly increased after sham and RFID exposure compared with those before exposure. Their levels were normalized to the corresponding group means before exposure and then plotted into star symbol patterns. N (1)-Acetylspermine after RFID exposure was 54 % higher compared to the level before RFID exposure, while it was elevated by only 17 % in the sham group. The results suggest that 915 MHz RFID exposure may induce metabolic disturbance of PA. It may also elevate spermidine/spermine acetyltransferase (SSAT) activity. Thus, the present metabolic profiling combined with star pattern recognition method might be useful for understanding the complexity of biochemical events after exposure to RFID signal.

Exercise training preserves ischemic preconditioning in aged rat hearts by restoring the myocardial polyamine pool.

Background. Ischemic preconditioning (IPC) strongly protects against myocardial ischemia reperfusion (IR) injury. However, IPC protection is ineffective in aged hearts. Exercise training reduces the incidence of age-related cardiovascular disease and upregulates the ornithine decarboxylase (ODC)/polyamine pathway. The aim of this study was to investigate whether exercise can reestablish IPC protection in aged hearts and whether IPC protection is linked to restoration of the cardiac polyamine pool. Methods. Rats aging 3 or 18 months perform treadmill exercises with or without gradient respectively for 6 weeks. Isolated hearts and isolated cardiomyocytes were exposed to an IR and IPC protocol. Results. IPC induced an increase in myocardial polyamines by regulating ODC and spermidine/spermine acetyltransferase (SSAT) in young rat hearts, but IPC did not affect polyamine metabolism in aged hearts. Exercise training inhibited the loss of preconditioning protection and restored the polyamine pool by activating ODC and inhibiting SSAT in aged hearts. An ODC inhibitor, α-difluoromethylornithine, abolished the recovery of preconditioning protection mediated by exercise. Moreover, polyamines improved age-associated mitochondrial dysfunction in vitro. Conclusion. Exercise appears to restore preconditioning protection in aged rat hearts, possibly due to an increase in intracellular polyamines and an improvement in mitochondrial function in response to a preconditioning stimulus.

Lack of evidence for the association of ornithine decarboxylase (+316 G>A), spermidine/spermine acetyl transferase (−1415 T>C) gene polymorphisms with calcium oxalate stone disease

Urolithiasis is a complex and multifactorial disorder characterized by the presence of stones in the urinary tract. Urea cycle is an important process involved in disease progression. L-ornithine is a key amino acid in the urea cycle and is converted to putrescine by ornithine decarboxylase (ODC). Putrescine, spermidine and spermine are natural polyamines that are catabolized by a specific enzyme, spermidine/spermine acetyltransferase (SSAT). The single-nucleotide polymorphisms (SNPs) in the intron region of ODC (+316 G>A) and promoter region of SSAT (-1415 T>C) genes have been found to be associated with the polyamines expression levels. The aim of this study was to examine whether the ODC (+316 G>A) intron 1 region gene polymorphism and SAT-1 promoter region (-1415 T>C) gene polymorphisms are potential genetic markers for susceptibility to urolithiasis. A control group of 104 healthy subjects and a group of 65 patients with recurrent idiopathic calcium oxalate stone disease were enrolled into this study. Polymerase chain reaction (PCR)-based restriction analysis was performed for the ODC intron 1 (+316 G>A) region and SAT-1 (-1415 T>C) promoter gene polymorphisms by PstI and MspI restriction enzyme digestion, respectively. The genotype distribution of polymorphisms studied in the ODC intron 1 region (+316 G>A) and SAT-1 -1415 T>C promoter region did not reveal a significant difference between urolithiasis and the control groups (P=0.713 and 0.853), respectively. Furthermore, no significant difference was observed between the control and patient groups for ODC +316 G>A and SAT-1 -1415 T>C allele frequencies (P=0.877 and 0.644), respectively. In conclusion, results of the present study suggest that ODC + 316 G>A and SAT-1 -1415 T>C gene polymorphisms might not be a risk factor for urolithiasis.

Acetaldehyde-induced cytotoxicity involves induction of spermine oxidase at the transcriptional level

Ethanol consumption causes serious liver injury including cirrhosis and hepatocellular carcinoma. Ethanol is metabolized mainly in the liver to acetic acid through acetaldehyde. We investigated the effect of ethanol and acetaldehyde on polyamine metabolism since polyamines are essential factors for normal cellular functions. We found that acetaldehyde induced spermine oxidase (SMO) at the transcriptional level in HepG2 cells. The levels and activities of ornithine decarboxylase (ODC) and spermidine/spermine acetyltransferase (SSAT) were not affected by acetaldehyde. Spermidine content was increased and spermine content was decreased by acetaldehyde treatment. Knockdown of SMO expression using siRNA reduced acetaldehyde toxicity. Acetaldehyde exposure increased free acrolein levels. An increase of acrolein by acetaldehyde was SMO dependent. Our results indicate that cytotoxicity of acetaldehyde involves, at least in part, oxidation of spermine to spermidine by SMO, which is induced by acetaldehyde.

Abstract 1989: The effect of resveratrol combined with DFMO or DenSpm on MCF-7 breast cancer cells and MCF-10a non-cancerous cells

Abstract Resveratrol, (3,5,49-trihydroxy-trans-stilbene), was first reported to act as a chemo-preventative anticancer compound in mice by Jang et al. in 1997. Resveratrol has been shown to induce apoptosis and reduce both cell viability and mitotic index in many cancer cell lines, including MCF-7. The current work extends these studies to determine its effects on glucose utilization and enzymes involved in polyamine metabolism. Glucose uptake was found to be reduced in MCF-7 cells treated with resveratrol. Work in other labs has shown resveratrol to sensitize cancer cells to other chemotherapeutic compounds. In the present study, the effects of combinations of resveratrol with compounds that affect polyamine metabolism, DFMO (difluoromethylornithine) and DenSpm (N1, N11-diethylnorspermine), were tested. Both DFMO and DenSpm were found to reduce cell proliferation and mitotic index in MCF-7 cell lines and more importantly, a synergistic effect was observed when these compounds were used in combination with resveratrol. In this investigation resveratrol was also found to induce apoptosis and reduce cell viability, mitotic index, and glucose uptake in non-cancerous, transformed mammary epithelial cells (MCF-10a). Similar findings have been reported with other non-cancerous cells including intestinal smooth muscle cells (Garcia et al., 2011). The combination of resveratrol and DFMO also reduced glucose uptake, proliferation, and mitotic index in this non-cancerous cell line (MCF-10a). These compounds were found to affect the expression of ODC (ornithine decarboxylase) and SSAT (spermidine/spermine acetyl transferase), enzymes involved in polyamine metabolism, and other proteins involved in cell cycle regulation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1989. doi:1538-7445.AM2012-1989

Abstract 2165: Identification of a novel pathway of castrate resistant prostate cancer (CRPCa) growth

Abstract Development of an effective therapy to prevent PCa occurrence, recurrence and/or progression remains an unmet medical need. This is mainly due to a poor understanding of the mechanism of PCa occurrence and progression. There is accumulating evidence that ROS (Reactive Oxygen Species) such as hydrogen peroxide, superoxide, hydroxyl radical, etc. that are produced in high amounts in PCa cells may be a major factor in PCa occurrence and its progression to androgen-independence. One of the ROS (H2O2) plays a key role in triggering growth signals and expression of transcription factors to sustain growth of androgen-dependent PCa cells in the absence of androgen. One such factor is Nuclear Factor κB (NF-κB) that may sustain cell survival by preventing apoptosis and inducing cell proliferation. NF-κB activation kinetically correlates with androgen-induced ROS production in cultured LNCaP human PCa cells. This activation is also seen in resected PCa tissues from patients, who subsequently progressed to CRPCa. Our published data show that androgen induces an up-regulation of spermidine/spermine acetyl transferase (SSAT) mRNA as well as enzyme activity that initiates polyamine oxidation in LNCaP cells after 72 h exposure. Because of the unusually high polyamine levels in the PCa cells, polyamine oxidation causes a large increase in ROS production that is abrogated by silencing SSAT expression in PCa cells stably transfected with shRNA against SSAT (siSSAT). To determine whether SSAT is required for NF-κB activation following prolonged androgen stimulus, we transfected 3xκB-luciferase vector containing NF-κB consensus binding sites-luciferase reporter construct along with a β-gal expression vector (to control for the transfection efficiency) into both wild type LNCaP cells and its siSSAT clones. While 72 h androgen exposure causes an over 30-fold induction of luciferase activity in wild type LNCaP cells, no activation of NF-κB was observed in the siSSAT clones. These data clearly show a strong SSAT induction is required for a robust activation of NF-κB in androgen-treated PCa cells. Multiple published reports show that NF-κB can also induce SSAT in several human cancer cells. Thus, our data suggest an intriguing mechanism of CRPCa progression, where androgen induced SSAT expression and consequent upregulation of NF-κB can set up an autocrine feed forward loop of SSAT-ROS-NFκB-SSAT that can sustain PCa cell proliferation in the absence of androgen that can lead to CRPCa growth. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2165. doi:10.1158/1538-7445.AM2011-2165

Abstract 2532: Triethylenetetramine metabolism and its implication in cancer chemotherapy

Abstract Triethylenetetramine (TETA), a copper II chelator and a polyamine analog traditionally used for the treatment of Wilson's disease, is currently in Phase I clinical trials for cancer in combination with carboplatin. However, the metabolism of TETA has never been thoroughly investigated, which is crucial for its further development in cancer treatment1. The two major metabolites of TETA found in humans are N1-acetyl-TETA (MAT) and N1,N10-diacetyl-TETA (DAT)2. Traditionally, acetylation of drug is thought to be catalyzed by N-acetyltransferase (NAT2). FDA requires that pharmacological properties of any drug metabolized via acetylation route have to be investigated in population with different NAT2 phenotype. However, our recent clinical study showed that there was no significant difference in pharmacokinetic and metabolites profiles of healthy volunteers with fast or slow NAT2 acetylation phenotype3. We therefore hypothesize that the enzyme responsible to TETA acetylation is spermidine/spermine acetyltransferase (SSAT). We carried out in vitro drug metabolism assays to pin-point the enzyme responsible for TETA metabolism. TETA (as substrate) and acetyl-coenzyme A were incubated with human liver microsome and cytosol from 7 individuals in the presence or absence of pentamidine or acetaminophen separately. The formation of MAT from TETA was detected and measured using our LC-MS method published previously2. Both liver microsome and cytosol could catalyze the biotransformation of TETA. There is a huge inter-individual difference in the rate of metabolism in microsome and cytosol (Km range 181 – 12,457 μM, Vmax range 110 – 3,068 pg/min/mg protein). Pentamidine, a specific inhibitor for SSAT, could inhibit the reaction and increase Km by over 30%. While acetaminophen, a specific inhibitor for NAT2, did not show any inhibition effect. Same experiments had been performed in rat liver microsome and cytosol, and similar results were obtained apart from the Km and Vmax values. Two other analogs of polyamine, diethylnorspermine and diethylspermine, all of which are under investigation for the treatment of cancer, were assayed as well, and similar results were obtained. The identification of SSAT, which has never been before considered to be a xenobiotic metabolizing enzyme, as the enzyme to catalyze the actylation of polyamine analogs, have a significant importance in the cancer combination chemotherapy. Finding SSAT (instead of NAT2) as the metabolic enzyme of TETA suggests that TETA may be an ideal candidate for cancer combination chemotherapy, as cancer drugs are rarely metabolized via this route; thus competition for drug metabolizing enzyme is less likely to occur. However, it seems that individuals do have different SSAT phenotypes, and this needs to be taken into account when using TETA as a chemotherapeutic agent. 1. Lu, J. (2010) MCT 9: 2458-67. 2. Lu, J. et al. (2007) DMD 35: 221-7. 3. Lu, J. et al. (2010) JCP 50: 647-58. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2532. doi:10.1158/1538-7445.AM2011-2532

Abstract LB-287: Spermidine/spermine actyltransferase is responsible for the metabolism of triethylenetetramine and other polyamine analogs in humans

Abstract Triethylenetetramine (TETA), a copper II chelator traditionally used for the treatment of Wilson's disease, and a number of structural analogs are currently in clinical trials for cancer. However, the metabolism of TETA has never been thoroughly investigated, which is crucial for its further development in cancer treatment. The two major metabolites of TETA found in humans are N1-acetyl-TETA (MAT) and N1, N10-diacetyl-TETA (DAT). Traditionally, acetylation of drug is thought to be catalyzed by N-acetyltransferase (NAT2). FDA requires that pharmacological properties of any drug metabolized via acetylation route have to be investigated in population with different NAT2 phenotype. However, our recent clinical study showed that there was no significant difference in pharmacokinetic, pharmacodynamic and metabolites profiles of healthy volunteers with fast or slow NAT2 acetylation phenotype. We therefore hypothesize that the enzyme responsible to TETA acetylation is spermidine/spermine acetyltransferase (SSAT), which has never before been considered as a drug metabolizing enzyme, rather than the NAT2. We carried out in vitro drug metabolism assays to pin-point the enzyme responsible for TETA metabolism. TETA and MAT (as substrate) and acetyl-coenzyme A were incubated with human liver microsome and cytosol from 5 individuals in the presence or absence of pentamidine or acetaminophen separately. The formation of MAT from TETA and DAT from MAT was detected and measured using our LC-MS method published previously. Both liver microsome and cytosol could catalyze the biotransformation of TETA and MAT. The rate of metabolism in microsome and cytosol within each individual varies. The rates of metabolism in microsome and cytosol also varied between individuals (Km range 223 - 909 M, Vmax range 96 - 482 pg/min/mg protein). Pentamidine, a specific inhibitor for x SSAT, could inhibit the reaction and increase Km by over 30%. While acetaminophen, a specific inhibitor for NAT2, did not show any inhibition effect. Same experiments had been performed in rat liver microsome and cytosol, and similar results were obtained apart from the Km and Vmax values. TETA and two other analogs of polyamine, diethylnorspermine and diethylspermine, all of which are under investigation for the treatment of cancer, were incubated with pure whole-recombinant human SSAT protein, and formation of acetylated metabolites were found. The identification of SSAT, which has never been before considered to be a xenobiotic metabolizing enzyme, as the enzyme to catalyze the actylation of polyamine analogs, have a significant importance in the clinic, especially in clinical trial situations. Many polyamine analogs have been developed as potential cancer drugs and many of them, including TETA, are being investigated in clinical trials. Finding the right enzyme (i.e. SSAT instead of traditional NAT2) to study their biotransformation is crucial for understanding their pharmacology, which is essential for the overall drug study. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. 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 LB-287.

The α9β1 integrin enhances cell migration by polyamine-mediated modulation of an inward-rectifier potassium channel

The alpha9beta1 integrin accelerates cell migration through binding of spermidine/spermine acetyltransferase (SSAT) to the alpha9 cytoplasmic domain. We now show that SSAT enhances alpha9-mediated migration specifically through catabolism of spermidine and/or spermine. Because spermine and spermidine are effective blockers of K(+) ion efflux through inward-rectifier K(+) (Kir) channels, we examined the involvement of Kir channels in this pathway. The Kir channel inhibitor, barium, or knockdown of a single subunit, Kir4.2, specifically inhibited alpha9-dependent cell migration. alpha9beta1 and Kir4.2 colocalized in focal adhesions at the leading edge of migrating cells and inhibition or knockdown of Kir4.2 caused reduced persistence and an increased number of lamellipodial extensions in cells migrating on an alpha9beta1 ligand. These results identify a pathway through which the alpha9 integrin subunit stimulates cell migration by localized polyamine catabolism and modulation of Kir channel function.

Mechanistic and Structural Analysis of Human Spermidine/Spermine N1-Acetyltransferase,

The N1-acetylation of spermidine and spermine by spermidine/spermine acetyltransferase (SSAT) is a crucial step in the regulation of the cellular polyamine levels in eukaryotic cells. Altered polyamine levels are associated with a variety of cancers as well as other diseases, and key enzymes in the polyamine pathway, including SSAT, are being explored as potential therapeutic drug targets. We have expressed and purified human SSAT in Escherichia coli and characterized its kinetic and chemical mechanism. Initial velocity and inhibition studies support a random sequential mechanism for the enzyme. The bisubstrate analogue, N1-spermine-acetyl-coenzyme A, exhibited linear, competitive inhibition against both substrates with a true Ki of 6 nM. The pH-activity profile was bell-shaped, depending on the ionization state of two groups exhibiting apparent pKa values of 7.27 and 8.87. The three-dimensional crystal structure of SSAT with bound bisubstrate inhibitor was determined at 2.3 A resolution. The structure of the SSAT-spermine-acetyl-coenzyme A complex suggested that Tyr140 acts as general acid and Glu92, through one or more water molecules, acts as the general base during catalysis. On the basis of kinetic properties, pH dependence, and structural information, we propose an acid/base-assisted reaction catalyzed by SSAT, involving a ternary complex.

Analysis of the interactions of Nrf-2, PMF-1, and CSN-7 with the 5′-flanking sequence of the mouse 4E-BP1 gene

Nuclear factor erythroid 2-related factor 2 (Nrf-2) binds to a specific polyamine responsive element (PRE) in the promoter region of the spermidine–spermine acetyltransferase (SSAT) gene, a key component of the polyamine catabolic pathway. Regulation of SSAT gene transcription requires the additional interaction of Nrf-2 with polyamine modulated factor 1 (PMF-1). Likewise, transcription of the eukaryotic initiation factor 4E binding protein 1 (4E-BP1) gene is regulated in a polyamine-dependent manner, but the actual mechanism has not previously been determined. Analysis of the 5′-flanking sequence of the murine 4E-BP1 gene indicated the presence of several potential PRE sites, which might be involved in regulating its transcription. Our goal in this research was to determine potential interactions between Nrf-2, PMF-1, the human homologue of the Arabidopsis signalosome complex (CSN-7), and these potential PRE sites. Four PCR fragments containing regions with considerable homology (78%) to the human PRE were generated from the 5′-flanking sequence of the mouse 4E-BP1 gene and the fragments were used in electrophoretic gel mobility shift and supershift assays. Purified Nrf-2 interacted with all four of these fragments, and similar gel shifts were observed with both cytoplasmic and nuclear fractions of NIH-3T3 cells. However, polyamine depletion with difluoromethylornithine (DFMO) eliminated the gel shift. Supershift assays indicated that the shift was due to the binding of Nrf-2, and the binding was competitive with a known Nrf-2 binding sequence. Purified PMF-1 did not bind any of the PCR fragments alone, but when added with Nrf-2, decreased the magnitude of the gel shift for one of the fragments (PRE located at − 2060 relative to the transcription start site). CSN-7 did not interact with the sequences, nor did it inhibit protein/DNA interaction. These data indicate a possible mechanism by which polyamines enhance the binding of a Nrf-2/PMF-1 complex to the 5′-flanking region of the 4E-BP1 gene. Since polyamines increase expression of the 4E-BP1 gene, it seems likely that formation of this complex is involved in its transcriptional regulation.

Phosphorylation of Recombinant Human Spermidine/Spermine N1-Acetyltransferase by CK1 and Modulation of Its Binding to Mitochondria: A Comparison with CK2

Cytosolic spermidine/spermine acetyltransferase (SSAT) catalyzes the acetylation of the N1-propylamino groups of spermine and spermidine. The enzyme has a very short half-life and is rapidly induced by various stimuli. Once acetylated, these polyamines are subjected to the action of polyamine oxidase, which, besides initiating polyamine catabolism, may produce reactive oxygen species that in turn trigger modifications in subcellular compartments such as mitochondria. The present work evaluates the ability of the cAMP-independent Ser/Thr-protein kinase CK1 to phosphorylate SSAT. Results demonstrate that SSAT is phosphorylated by CK1, in sites distinct from those phosphorylated by CK2. Moreover, both phosphorylation processes are involved in the uptake of SSAT into rat liver mitochondria. Although CK2 is less effective than CK1 in phosphorylating SSAT, CK2 phosphorylation is much more powerful in preventing binding of SSAT to mitochondrial structures. These results suggest the involvement of CK1- and CK2-mediated SSAT phosphorylation in regulating the contents of polyamines and SSAT itself within subcellular compartments and implicate SSAT and polyamines as indirect modulators of progression through the cell cycle.

Pneumocystis carinii Polyamine Catabolism

DL-alpha-Difluoromethylornithine (DFMO) causes polyamines of the AIDS-associated opportunistic pathogen Pneumocystis carinii to diminish 15 times more rapidly than mammalian host cells. The proposed mechanism was that, unlike mammalian cells, P. carinii is unable to regulate polyamine catabolism when synthesis is blocked. To test this, the responses of the polyamine catabolic enzymes spermidine/spermine acetyltransferase (SSAT) and polyamine oxidase (PAO) were determined using a new high-performance liquid chromatography assay to measure the products of these enzymes. The specific activities in untreated Pneumocystis carinii were 1.78 +/- 0.5 pmol min(-1) mg protein(-1) for SSAT, similar to mammalian cells, and 6.42 +/- 0.8 pmol min(-1) mg protein(-1) for PAO, 19% of that of mammalian cells. DFMO treatment for 12 h caused reductions of only 11 and 4% in SSAT and PAO, respectively, despite polyamine reductions of 94, 96, and 90% for putrescine, spermidine, and spermine, respectively. The P. carinii SSAT K(m) value of 25 microM spermidine is 20% of that of mammalian cells, and the PAO K(m) value of 14 nM N(1)-acetylspermidine is 0.01% of that of mammalian cells. Acetylated polyamines continue to be lost from P. carinii even when exposed to DFMO. Collectively, these results support the hypothesis that P. carinii is unable to regulate polyamine catabolism.

Agmatine modulates polyamine content in hepatocytes by inducing spermidine/spermine acetyltransferase.

Agmatine has been proposed as the physiological ligand for the imidazoline receptors. It is not known whether it is also involved in the homoeostasis of intracellular polyamine content. To show whether this is the case, we have studied the effect of agmatine on rat liver cells, under both periportal and perivenous conditions. It is shown that agmatine modulates intracellular polyamine content through its effect on the synthesis of the limiting enzyme of the interconversion pathway, spermidine/spermine acetyltransferase (SSAT). Increased SSAT activity is accompanied by depletion of spermidine and spermine, and accumulation of putrescine and N1-acetylspermidine. Immunoblotting with a specific polyclonal antiserum confirms the induction. At the same time S-adenosylmethionine decarboxylase activity is significantly increased, while ornithine decarboxylase (ODC) activity and the rate of spermidine uptake are reduced. This is not due to an effect on ODC antizyme, which is not significantly changed. All these modifications are observed in HTC cells also, where they are accompanied by a decrease in proliferation rate. SSAT is also induced by low oxygen tension which mimics perivenous conditions. The effect is synergic with that promoted by agmatine.

Significant Increases in the Steady States of Putrescine and Spermidine/SpermineN1-Acetyltransferase mRNA in HeLa Cells Accompanied by Growth Arrest

Polyamines are intrinsic polycations which play critical roles in cell proliferation. Ornithine decarbolylase (ODC) catalyzes the first step of polyamine biosynthesis converting ornithine to putrescine. In addition to polyamine degradation, spermidine/spermine acetyltransferase (SSAT) regulates interconversion pathway of spermine and spermidine to putrescine. We quantified the polyamines and mRNAs of ODC and SSAT in HeLa S3 cells at various stages during exponential and plateau phases of culturing. Unexpectedly, putrescine and SSAT mRNA levels increased remarkably at the plateau phase, in contrast to the decrease of ODC mRNA level. It will be suggested that the putrescine has a novel function linked to the arrest of cell growth in which the SSAT-mediated pathway producing putrescine takes part.

Effects of the Trypanocidal Agents Berenil and Pentamidine on Growth, Enzyme-Activities, and Polyamine Concentrations in the Rice Blast Pathogen Pyricularia oryzae and on Powdery Mildew Infection of Barley Seedlings

The effects of the trypanocidal agents Berenil and pentamidine on growth and polyamine biosynthesis in the rice blast fungus Pyricularia oryzae were examined. Fungal growth was substantially reduced by as little as 0.02 m M pentamidine and 0.01 m M Berenil. These compounds also controlled infection of barley seedlings by the powdery mildew fungus Erysiphe graminis f.sp. hordei, with 1 m M Berenil or pentamidine reducing infection by 80 and 85%, respectively. Although both compounds reduced in vitro activity of S-adenosylmethionine decarboxylase (AdoMetDC), the effects of pentamidine were more pronounced. Neither inhibitor had any effect on the in vitro activities of omithine decarboxylase (ODC) and spermidine/spermine acetyltransferase (SSAT). In in vivo studies, neither Berenil nor pentamidine significantly altered the activities of AdoMetDC, ODC or SSAT. Curiously, however, exposure of P. oryzae to either compound resulted in greatly increased putrescine and cadaverine concentrations and a much reduced spermine concentration. The changes in polyamine concentrations may be due, in part, to increased activity of polyamine oxidase in fungal tissue following treatment with Berenil or pentamidine.

Inhibition of enzymes of polyamine back-conversion by pentamidine and berenil

Pentamidine and berenil, clinical antiparasitic amidines, have been found to be potent competitive inhibitors of human spermidine/spermine acetyltransferase (SSAT), K i values were found to be 2.4 and 2 μM, respectively, with spermidine as substrate. A second enzyme of polyamine back-conversion, murine polyamine oxidase (PAO), was found to be competitively inhibited by pentamidine, with a K i of 7.6 μM when N-acetylspermine was the substrate. Berenil, on the other hand, was an extremely weak inhibitor ( K i = 120 μM). The implication of the effect of inhibition of polyamine back-conversion on the growth of mammalian parasites is discussed.