Alex R. Khomutov

Monitoring of the uptake and metabolism of aminooxy analogues of polyamines in cultured cells by high-performance liquid chromatography

A high-performance liquid chromatographic method for the determination of polyamines and their aminooxy analogues is described. Oxime derivatization with a ketone is used to protect the aminooxy group during post-column reaction with o-phthalaldehyde. The amount of the polyamines and of the oximes of their aminooxy analogues can be determined simultaneously in cultured cells and cell culture media. The limit of detection is 20-30 pmol, and the response of the fluorescence detection is linear up to 4 nmol. The separation of the aminooxy analogues from the naturally occurring polyamines can be varied by using different ketones for oxime formation. The method was used to measure the stability of aminooxy analogues of putrescine (1-aminooxy-3-aminopropane) and spermidine [N-(2-aminooxyethyl)-1,4-diaminobutane and 1-aminooxy-3-N-(3-aminopropyl)aminopropane] in cell culture media and the uptake into cultured baby hamster kidney (BHK21/C13) cells.

3-Aminooxy-1-aminopropane and derivatives have an antiproliferative effect on cultured Plasmodium falciparum by decreasing intracellular polyamine concentrations.

ABSTRACT The intraerythrocytic development of Plasmodium falciparum correlates with increasing levels of the polyamines putrescine, spermidine, and spermine in the infected red blood cells; and compartmental analyses revealed that the majority is associated with the parasite. Since depletion of cellular polyamines is a promising strategy for inhibition of parasite proliferation, new inhibitors of polyamine biosynthesis were tested for their antimalarial activities. The ornithine decarboxylase (ODC) inhibitor 3-aminooxy-1-aminopropane (APA) and its derivatives CGP 52622A and CGP 54169A as well as the S-adenosylmethionine decarboxlyase (AdoMetDC) inhibitors CGP 40215A and CGP 48664A potently affected the bifunctional P. falciparum ODC-AdoMetDC, with Ki values in the low nanomolar and low micromolar ranges, respectively. Furthermore, the agents were examined for their in vitro plasmodicidal activities in 48-h incubation assays. APA, CGP 52622A, CGP 54169A, and CGP 40215A were the most effective, with 50% inhibitory concentrations below 3 μM. While the effects of the ODC inhibitors were completely abolished by the addition of putrescine, growth inhibition by the AdoMetDC inhibitor CGP 40215A could not be antagonized by putrescine or spermidine. Moreover, CGP 40215A did not affect the cellular polyamine levels, indicating a mechanism of action against P. falciparum independent of polyamine synthesis. In contrast, the ODC inhibitors led to decreased cellular putrescine and spermidine levels in P. falciparum, supporting the fact that they exert their antimalarial activities by inhibition of the bifunctional ODC-AdoMetDC.

A Polyamine Analogue Prevents Acute Pancreatitis and Restores Early Liver Regeneration in Transgenic Rats with Activated Polyamine Catabolism

We recently generated a transgenic rat model for acute pancreatitis, which was apparently caused by a massive depletion of pancreatic polyamines spermidine and spermine due to inducible activation of their catabolism (Alhonen, L., Parkkinen, J. J., Keinänen, T., Sinervirta, R., Herzig, K. H., and Jänne, J. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 8290–8295). When subjected to partial hepatectomy, these animals showed striking activation of polyamine catabolism at 24 h postoperatively with a profound decrease in hepatic spermidine and spermine pools and failure to initiate liver regeneration. Here we show that pancreatitis in this model could be totally prevented, as judged by histopathology and plasma α-amylase activity, by administration of 1-methylspermidine, a metabolically stable analogue of spermidine. Similarly, the analogue, given prior to partial hepatectomy, restored early liver regeneration in the transgenic rats, as indicated by a dramatic increase in the number of proliferating cell nuclear antigen-positive hepatocytes from about 1% to more than 40% in response to the drug. The present results suggest that the extremely high concentration of spermidine in the pancreas, in fact the highest in the mammalian body, may have a critical role in maintaining organ integrity. The failure to initiate liver regeneration in the absence of sufficient hepatic polyamine pools similarly indicates that polyamines are required for proper commencement of the regenerative process.

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

We have earlier shown that α-methylated spermidine and spermine analogues rescue cells from polyamine depletion-induced growth inhibition and maintain pancreatic integrity under severe polyamine deprivation. However, because α-methylspermidine can serve as a precursor of hypusine, an integral part of functional eukaryotic translation initiation factor 5A required for cell proliferation, and because α, ω-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 α-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.

Metabolic Stability of α-Methylated Polyamine Derivatives and Their Use as Substitutes for the Natural Polyamines

Metabolically stable polyamine derivatives may serve as useful surrogates for the natural polyamines in studies aimed to elucidate the functions of individual polyamines. Here we studied the metabolic stability of α-methylspermidine, α-methylspermine, and bis-α-methylspermine, which all have been reported to fulfill many of the putative physiological functions of the natural polyamines. In vivo studies were performed with the transgenic rats overexpressing spermidine/spermine N1-acetyltransferase. α-Methylspermidine effectively accumulated in the liver and did not appear to undergo any further metabolism. On the other hand, α-methylspermine was readily converted to α-methylspermidine and spermidine; similarly, bis-α-methylspermine was converted to α-methylspermidine to some extent, both conversions being inhibited by the polyamine oxidase inhibitor N1, N2-bis(2,3-butadienyl)-1,4-butanediamine. Furthermore, we used recombinant polyamine oxidase, spermidine/spermine N1-acetyltransferase, and the recently discovered spermine oxidase in the kinetic studies. In vitro studies confirmed that methylation did not protect spermine analogs from degradation, whereas the spermidine analog was stable. Both α-methylspermidine and bis-α-methylspermine overcame the proliferative block of early liver regeneration in transgenic rats and reversed the cytostasis induced by an inhibition of ornithine decarboxylase in cultured fetal fibroblasts.

Spermidine is indispensable in differentiation of 3T3-L1 fibroblasts to adipocytes

Impaired adipogenesis has been shown to predispose to disturbed adipocyte function and development of metabolic abnormalities. Previous studies indicate that polyamines are essential in the adipogenesis in 3T3‐L1 fibroblasts. However, the specific roles of individual polyamines during adipogenesis have remained ambiguous as the natural polyamines are readily interconvertible inside the cells. Here, we have defined the roles of spermidine and spermine in adipogenesis of 3T3‐L1 cells by using (S’)‐ and (R’)‐ isomers of α‐methylspermidine and (S,S’)‐, (R,S)‐ and (R,R’)‐diastereomers of α,ω‐bismethylspermine. Polyamine depletion caused by α‐difluoromethylornithine (DFMO), an irreversible inhibitor of ornithine decarboxylase, prevented adipocyte differentiation by suppressing the expression of its key regulators, peroxisome proliferator‐activated receptor γ and CCAAT/enhancer binding protein α. Adipogenesis was restored by supplementation of methylspermidine isomers but not of bismethylspermine diastereomers. Although both spermidine analogues supported adipocyte differentiation only (S)‐methylspermidine was able to fully support cell growth after extended treatment with α‐DFMO. The distinction between the spermidine analogues in maintaining growth was found to be in their different capability to maintain functional hypusine synthesis. However, the differential ability of spermidine analogues to support hypusine synthesis did not correlate with their ability to support differentiation. Our results show that spermidine, but not spermine, is essential for adipogenesis and that the requirement of spermidine for adipogenesis is not strictly associated with hypusine modification. The involvement of polyamines in the regulation of adipogenesis may offer a potential application for the treatment of dysfunctional adipocytes in patients with obesity and metabolic syndrome.

Antileishmanial Effect of 3-Aminooxy-1-Aminopropane Is Due to Polyamine Depletion

ABSTRACT The polyamines putrescine, spermidine, and spermine are organic cations that are required for cell growth and differentiation. Ornithine decarboxylase (ODC), the first and rate-limiting enzyme in the polyamine biosynthetic pathway, catalyzes the conversion of ornithine to putrescine. As the polyamine biosynthetic pathway is essential for the growth and survival of Leishmania donovani, the causative agent of visceral leishmaniasis, inhibition of the pathway is an important leishmaniacidal strategy. In the present study, we examined for the first time the effects of 3-aminooxy-1-aminopropane (APA), an ODC inhibitor, on the growth of L. donovani. APA inhibited the growth of both promastigotes in vitro and amastigotes in the macrophage model, with the 50% inhibitory concentrations being 42 and 5 μM, respectively. However, concentrations of APA up to 200 μM did not affect the viability of macrophages. The effects of APA were completely abolished by the addition of putrescine or spermidine. APA induced a significant decrease in ODC activity and putrescine, spermidine, and trypanothione levels in L. donovani promastigotes. Parasites were transfected with an episomal ODC construct, and these ODC overexpressers exhibited significant resistance to APA and were concomitantly resistant to sodium antimony gluconate (Pentostam), indicating a role for ODC overexpression in antimonial drug resistance. Clinical isolates with sodium antimony gluconate resistance were also found to overexpress ODC and to have significant increases in putrescine and spermidine levels. However, no increase in trypanothione levels was observed. The ODC overexpression in these clinical isolates alleviated the antiproliferative effects of APA. Collectively, our results demonstrate that APA is a potent inhibitor of L. donovani growth and that its leishmaniacidal effect is due to inhibition of ODC.

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

Summary.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 (Me2Spm) 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 Me2Spm pretreatment. Glutathione level and catalase activity changed transiently, and the effect of Me2Spm was clear at 24 h. Serum inflammatory cytokine levels increased already at 4 h whereas NF-κB was distinctly activated only at 24 h. Me2Spm prevented the increase in TNF-α and IL-6 while it had no effect on NF-κB 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.

Synthesis of Hydroxylamine Analogues of Polyamines

Abstract Novel analogues of spermine and spermidine with terminal H2NCH2-group substituted by H2NO-group, were prepared starting the synthesis from EtO(Me)CNOH and subsequent extension of a polyamine backbone. To prepare their earlier unknown tritium labelled analoques, ω-[[(1′-ethoxyethylidene)amino]oxy]-poly-(iminomethylene) nitriles were reduced to amines by NaBT 4 CoCl 2 complex, which did not effect the N-O or CN bonds of ethoxyethylidene group, whereas aminooxy group deprotection was performed at the final step of synthesis by mild acidic hydrolysis. Novel monoacetylated (AcHN- or AcNHO-) analoques of spermidine were also synthesised.

Helianthus tuberosus and polyamine research: Past and recent applications of a classical growth model

The earliest studies concerning polyamines (PAs) in plants were performed by using in vitro cultured explants of Helianthus tuberosus dormant tuber. This parenchyma tissue was particularly useful due to its susceptibility to several growth substances, including PAs. During tuber dormancy, PA levels are too low to sustain cell division; thus Helianthus represents a natural PA-deficient model. When cultivated in vitro in the presence of auxins, Helianthus tuber dormant parenchyma cells at the G(0) stage start to divide synchronously acquiring meristematic characteristics. The requirement for auxins to induce cell division can be substituted by aliphatic PAs such as putrescine, spermidine or spermine. Cylinders or slices of explanted homogeneous tuber parenchyma were cultured in liquid medium for short-term studies on the cell cycle, or on solid agar medium for long-term experiments. Morphological and physiological modifications of synchronously dividing cells were studied during the different phases of the cell cycle in relation to PAs biosynthesis and oxidation. Long-term experiments led to the identification of the PAs as plant growth regulators, as the sole nitrogen source, as tuber storage substances and as essential factors for morphogenetic processes and cell homeostasis. More recently this system was used to study the effects on plant cell proliferation of platinum- or palladium-derived drugs (cisplatin and platinum or palladium bi-substituted spermine) that are used in human cancer cell lines as antiproliferative and cytotoxic agents. Cisplatin was the most active both in cell proliferation inhibition and on PA metabolism. Similar experiments were performed using three agmatine analogous. Different effects of these compounds were observed on cell proliferation, free PA levels and enzyme activities, leading to a hypothesis of a correlation between their chemical structure and the agmatine metabolism in plants.