How important is the oxidative degradation of spermine?: Minireview article

Summary. Spermine is a constituent of most eucaryotic cells, however, it is not of vital importance for the vertebrate organism, as is demonstrated by the existence of transgenic (Gy) mice that lack spermine and spermine synthase. In contrast its degradation appears to be of vital importance, since mice die after chronic administration of N¹,N⁴-bis(2,3-butadienyl)-1,4-butanediamine (MDL 72517). Under this condition spermine accumulates in red blood cells and blood plasma. Lethal toxicity can be avoided by intervals of MDL 72527-free periods. During these periods spermine appears to be directly degraded to spermidine without an intermediary acetylation step within the red blood cells. Since this reaction is of enormous physiological significance, it will be important to characterise the red blood cell spermine oxidase, and it will be particularly important to determine whether this oxidase is identical with the FAD-dependent polyamine oxidase that is considered to be involved in the polyamine interconversion sequence, or whether it is one of the recently characterised spermine oxidase isoenzymes.     

Cytotoxic effects of the polyamine oxidase inactivator MDL 72527 to two human colon carcinoma cell lines SW480 and SW620

N ¹,N ⁴-bis(2,3-butadienyl)-1,4-butanediamine (MDL 72527) was considered to be a selective inactivator of FAD-dependent tissue polyamine oxidase. Recently MDL 72527 was reported to induce apoptosis in transformed hematopoietic cells through lysosomotropic effects. Since it is the only useful inhibitor of polyamine oxidase available at present, the re-evaluation of its properties seemed important. Human colon carcinoma-derived SW480 cells and their lymph node metastatic derivatives (SW620) were chosen for our study because they differ in various aspects of polyamine metabolism but have similar polyamine oxidase activities. MDL 72527 inhibited cell growth in a concentration-dependent manner, depleted intracellular polyamine pools, and caused the accumulation of N ¹-acetyl derivatives of spermidine and spermine. SW620 cells were more sensitive to the drug than were SW480 cells. At 150 μmol/L MDL 72527, SW620 cells accumulated in S-phase of the cell cycle, showed decreased polyamine transport rate, and showed no increase of polyamine N ¹-acetyltransferase activity. In contrast, SW480 cells were not arrested in a particular phase of the cell cycle, showed enhanced polyamine uptake, and showed a mild induction of acetyltransferase. The results suggest that MDL 72527 retains its value as a selective tool in short-term experiments only at concentrations not exceeding those necessary for the inactivation of polyamine oxidase. At concentrations above 50 μmol/L and at exposure times longer than 24 h, it may derange cell functions nonspecifically, and thus blur the results of studies intended to elucidate polyamine oxidase functions. This revised version was published online in July 2006 with corrections to the Cover Date.     

Targeted disruption of spermidine/spermine N1-acetyltransferase gene in mouse embryonic stem cells. Effects on polyamine homeostasis and sensitivity to polyamine analogues

We have generated mouse embryonic stem cells with targeted disruption of spermidine/spermine N(1)-acetyltransferase (SSAT) gene. The targeted cells did not contain any inducible SSAT activity, and the SSAT protein was not present. The SSAT-deficient cells proliferated normally and appeared to maintain otherwise similar polyamine pools as did the wild-type cells, with the possible exception of constantly elevated (about 30%) cellular spermidine. As expected, the mutated cells were significantly more resistant toward the growth-inhibitory action of polyamine analogues, such as N(1),N(11)-diethylnorspermine. However, this resistance was not directly attributable to cellular depletion of the higher polyamines spermidine and spermine, as the analogue depleted the polyamine pools almost equally effectively in both wild-type and SSAT-deficient cells. Tracer experiments with [C(14)]-labeled spermidine revealed that SSAT activity is essential for the back-conversion of spermidine to putrescine as radioactive N(1)-acetylspermidine and putrescine were readily detectable in N(1),N(11)-diethylnorspermine-exposed wild-type cells but not in SSAT-deficient cells. Similar experiments with [C(14)]spermine indicated that the latter polyamine was converted to spermidine in both cell lines and, unexpectedly, more effectively in the targeted cells than in the parental cells. This back-conversion was only partly inhibited by MDL72527, an inhibitor of polyamine oxidase. These results indicated that SSAT does not play a major role in the maintenance of polyamine homeostasis, and the toxicity exerted by polyamine analogues is largely not based on SSAT-induced depletion of the natural polyamines. Moreover, embryonic stem cells appear to operate an SSAT-independent system for the back-conversion of spermine to spermidine.     

Polyamine metabolism is involved in adipogenesis of 3T3-L1 cells

Polyamines spermidine and spermine are known to be required for mammalian cell proliferation and for embryonic development. Alpha-difluoromethylornithine (DFMO), an inhibitor of ornithine decarboxylase (ODC) a limiting enzyme of polyamine biosynthesis, depleted the cellular polyamines and prevented triglyceride accumulation and differentiation in 3T3-L1 cells. In this study, to explore the function of polyamines in adipogenesis, we examined the effect of polyamine biosynthesis inhibitors on adipocyte differentiation and lipid accumulation of 3T3-L1 cells. The spermidine synthase inhibitor trans-4-methylcyclohexylamine (MCHA) increased spermine/spermidine ratios, whereas the spermine synthase inhibitor N-(3-aminopropyl)-cyclohexylamine (APCHA) decreased the ratios in the cells. MCHA was found to decrease lipid accumulation and GPDH activity during differentiation, while APCHA increased lipid accumulation and GPDH activity indicating the enhancement of differentiation. The polyamine-acetylating enzyme, spermidine/spermine N ¹-acetyltransferase (SSAT) activity was increased within a few hours after stimulus for differentiation, and was found to be elevated by APCHA. In mature adipocytes APCHA decreased lipid accumulation while MCHA had the opposite effect. An acetylpolyamine oxidase and spermine oxidase inhibitor MDL72527 or an antioxidant N-acetylcysteine prevented the promoting effect of APCHA on adipogenesis. These results suggest that not only spermine/spermidine ratios but also polyamine catabolic enzyme activity may contribute to adipogenesis.     

Effect of the polyamine oxidase inactivator MDL 72527 on N(1)-(n-octanesulfonyl)spermine toxicity

N(1)-(n-octanesulfonyl)spermine (N(1)OSSpm) is a potent calmodulin antagonist. In the present work, its toxicity to DHD/K12/TRb and CaCo-2 cells, two colon carcinoma-derived cell lines, was studied with the aim to identify those properties of the cells, which determine their sensitivity to N(1)OSSpm and related structures. Exposure of the cells to MDL 72527, a compound considered to be a selective inactivator of polyamine oxidase (PAO) increased the cytotoxicity of N(1)OSSpm to both cell lines. In contrast, toxicity of trifluoperazine, a calmodulin antagonist with a polyamine-unrelated structure, was not enhanced by MDL 72527. Combined exposure of cells to 2-(difluoromethyl)ornithine (DFMO) (a selective inactivator of ornithine decarboxylase), MDL 72527 and N(1)OSSpm produced a synergistic cytotoxic effect. Neither the intrinsic PAO activity of the cells (as determined with N(1), N(12)-diacetylspermine as substrate), nor their ability to accumulate the drug was a determinant of the cytotoxic effect of N(1)OSSpm. These data suggest that MDL 72527 has a target unrelated to PAO, which is responsible for the enhancement of N(1)OSSpm (and spermine) toxicity. Identification of this target may be of use if the therapeutic potentials of MDL 72527 are to be exploited.     

Induction of polyamine oxidase 1 by Helicobacter pylori causes macrophage apoptosis by hydrogen peroxide release and mitochondrial membrane depolarization

Helicobacter pylori infects the human stomach by escaping the host immune response. One mechanism of bacterial survival and mucosal damage is induction of macrophage apoptosis, which we have reported to be dependent on polyamine synthesis by arginase and ornithine decarboxylase. During metabolic back-conversion, polyamines are oxidized and release H(2)O(2), which can cause apoptosis by mitochondrial membrane depolarization. We hypothesized that this mechanism is induced by H. pylori in macrophages. Polyamine oxidation can occur by acetylation of spermine or spermidine by spermidine/spermine N(1)-acetyltransferase prior to back-conversion by acetylpolyamine oxidase, but recently direct conversion of spermine to spermidine by the human polyamine oxidase h1, also called spermine oxidase, has been demonstrated. H. pylori induced expression and activity of the mouse homologue of this enzyme (polyamine oxidase 1 (PAO1)) by 6 h in parallel with ornithine decarboxylase, consistent with the onset of apoptosis, while spermidine/spermine N(1)-acetyltransferase activity was delayed until 18 h when late stage apoptosis had already peaked. Inhibition of PAO1 by MDL 72527 or by PAO1 small interfering RNA significantly attenuated H. pylori-induced apoptosis. Inhibition of PAO1 also significantly reduced H(2)O(2) generation, mitochondrial membrane depolarization, cytochrome c release, and caspase-3 activation. Overexpression of PAO1 by transient transfection induced macrophage apoptosis. The importance of H(2)O(2) was confirmed by inhibition of apoptosis with catalase. These studies demonstrate a new mechanism for pathogen-induced oxidative stress in macrophages in which activation of PAO1 leads to H(2)O(2) release and apoptosis by a mitochondrial-dependent cell death pathway, contributing to deficiencies in host defense in diseases such as H. pylori infection.     

Polyamine reutilization and turnover in brain

N¹, N²-bis-(2, 3-butadienyl)-1, 4-butanediamine (MDL 72527) is an irreversible, specific inhibitor of polyamine oxidase, which allows one to completely inactivate this enzyme in all organs of an experimental animal. As a result one observes a linear increase of N¹-acetylsperimidine and N¹-acetylspermine concentrations in brain. The rate of accumulation seems directly proportional to the rate of spermidine, and spermine degradation respectively, and since no compensatory changes of the polyamine synthetic enzymes, were induced by inhibition of polyamine oxidase, the rate of acetyl-polyamine accumulation is assumed to be a measure for polyamine turnover. The decrease of brain putrescine levels by 70 percent in the brains of MDL 72527-treated animals suggests the quantitative significance of putrescine reutilisation. Pretreatment of the animals with D, L--difluoromethylornithine, an irreversible inhibitor of ornithine decarboxylase reduced both, polyamine turnover rate and the extent of putrescine reutilization. Inhibition of GAPA-T produced a significant increase of polyamine turnover in brain, in agreement with the known induction of ornithine decarboxylase activity after treatment with inhibitors of GABA-T.     

Effects of MDL 72527, a Specific Inhibitor of Polyamine Oxidase, on Brain Edema, Ischemic Injury Volume, and Tissue Polyamine Levels in Rats After Temporary Middle Cerebral After Occlusion

Abstract The possible effects of the polyamine interconversion pathway on tissue polyamine levels, brain edema formation, and ischemic injury volume were studied by using a selective irreversible inhibitor, MDL 72527, of the interconversion pathway enzyme, polyamine oxidase. In an intraluminal suture occlusion model of middle coerebral artery in spontaneously hypertensive rats, 100 mg/kg MDL 72527 changed the brain edema formation from 85.7 ± 0.3 to 84.5 ± 0.9% in cortex ( P < 0.05) and from 79.9 ± 1.7 to 78.4 ± 2.0% in subcortex (difference not significant). Ischemic injury volume was reduced by 22% in the cortex ( P < 0.05) and 17% in the subcortex ( P < 0.05) after inhibition of polyamine oxidase by MDL 72527. There was an increase in tissue putrescine levels together with a decrease in spermine and spermidine levels at the ischemic site compared with the nonischemic site compared with the nonischemic site after ischemia-reperfusion injury. The increase in putrescine levels at the ischemic cortical and subcortical region was reduced by a mean of 45% with MDL 72527 treatment. These results suggest that the polyamine interconversion pathway has an important role in the postischemic increase ini putrescine levels and that blocking of this pathway can be neuroprotective against neuronal cell damage after temporary focal cerebral ischemia.     

Elevated N1-acetylspermidine levels in gerbil and rat brains after CNS injury

The polyamine system is very sensitive to different pathological states of the brain and is perturbed after CNS injury. The main modifications are significant increases in ornithine decarboxylase activity and an increase in tissue putrescine levels. Previously we have shown that the specific polyamine oxidase (PAO) inhibitor N1,N4-bis(2,3-butadienyl)-1,4-butanediamine (MDL 72527) reduced the tissue putrescine levels, edema, and infarct volume after transient focal cerebral ischemia in spontaneously hypertensive rats and traumatic brain injury of Sprague-Dawley rats. In the present study, N1-acetyl-spermidine accumulation was greater in injured brain regions compared with sham or contralateral regions following inhibition of PAO by MDL 72527. This indicates spermidine/spermine-N1-acetyltransferase (SSAT) activation after CNS injury. The observed increase in N1-acetylspermidine levels at 1 day after CNS trauma paralleled the decrease in putrescine levels after treatment with MDL 72527. This suggests that the increased putrescine formation at 1 day after CNS injury is mediated by the SSAT/PAO pathway, consistent with increased SSAT mRNA after transient ischemia.     

Inhibition of polyamine oxidase enhances the cytotoxicity of polyamine oxidase substrates. A model study with N1-(n-octanesulfonyl)spermine and human colon cancer cells

N(1)-(n-octanesulfonyl)spermine (N(1) OSSpm) is a substrate of polyamine oxidase. It shares several properties with spermine, such as antagonism of NMDA-type glutamate receptors, calmodulin antagonism, and cytotoxicity, but it is more potent by orders of magnitude in these regards than spermine. The human colon carcinoma-derived cell line CaCo-2 was used as a model to study the toxicity of N(1) OSSpm as a function of polyamine oxidase (PAO) activity and differentiation. If the formation of hydrogen peroxide and aminoaldehyde by the PAO-catalysed reactions was prevented by selective inactivation of the enzyme with MDL 72527, cytotoxicity of N(1)OSSpm was not diminished, but on the contrary, enhanced. Exponentially growing CaCo-2 cells were considerably more sensitive to N(1)OSSpm than differentiating cells. The results suggest that cytotoxic substrates of PAO exhibit enhanced cytotoxicity in cells, if PAO activity is inhibited. Since tumour cells are known to have lower polyamine oxidase activities than their normal counterparts, it will be interesting to explore whether cytotoxic substrates of polyamine oxidase, for which N(1)OSSpm is an example, are suited to preferentially kill tumour cells.     

Toxicity of enzymatic oxidation products of spermine to human melanoma cells (M14): sensitization by heat and MDL 72527

In situ formation of cytotoxic metabolites by an enzyme-catalyzed reaction is a recent approach in cancer chemotherapy. We demonstrate that multidrug resistant human melanoma cells (M14 ADR) are more sensitive than the corresponding wild type cells (M14 WT) to hydrogen peroxide and aldehydes, the products of bovine serum amine oxidase (BSAO)-catalyzed oxidation of spermine. Hydrogen peroxide was mainly responsible for the loss of cell viability. With about 20%, the aldehydes formed from spermine contribute also to cytotoxicity. Elevation of temperature from 37 degrees C to 42 degrees C decreased survival of both cell lines by about one log unit. Pre-treatment with N1,N4-bis(2,3-butadienyl)-1,4-butanediamine (MDL 72527), a lysosomotropic compound, sensitized cells to toxic spermine metabolites. MDL 72527 (at 300 microM) produced in M14 cells numerous cytoplasmic vacuoles which, however, disappeared by 24 h, even in the presence of the drug. Mitochondrial damage, as observed by transmission electron microscopy, correlated better with the cytotoxic effects of the treatment than vacuole formation. Since the release of lysosomal enzymes causes oxidative stress and apoptosis, we suggest that the lysosomotropic effect of MDL 72527 is the major reason for its sensitizing effect.     

A 30-angstrom-long U-shaped catalytic tunnel in the crystal structure of polyamine oxidase.

BACKGROUND: Polyamines are essential for cell growth and differentiation; compounds interfering with their metabolism are potential anticancer agents. Polyamine oxidase (PAO) plays a central role in polyamine homeostasis. The enzyme utilises an FAD cofactor to catalyse the oxidation of the secondary amino groups of spermine and spermidine. RESULTS: The first crystal structure of a polyamine oxidase has been determined to a resolution of 1.9 Angstroms. PAO from Zea mays contains two domains, which define a remarkable 30 Angstrom long U-shaped catalytic tunnel at their interface. The structure of PAO in complex with the inhibitor MDL72527 reveals the residues forming the catalytic machinery and unusual enzyme-inhibitor CH.O H bonds. A ring of glutamate and aspartate residues surrounding one of the two tunnel openings contributes to the steering of the substrate towards the inside of the tunnel. CONCLUSIONS: PAO specifically oxidizes substrates that have both primary and secondary amino groups. The complex with MDL72527 shows that the primary amino groups are essential for the proper alignment of the substrate with respect to the flavin. Conservation of an N-terminal sequence motif indicates that PAO is member of a novel family of flavoenzymes. Among these, monoamine oxidase displays significant sequence homology with PAO, suggesting a similar overall folding topology.     

Specific inhibition of polyamine oxidase in vivo is a method for the elucidation of its physiological role

N1-Methyl-N2-(2,3-butadienyl)-1,4-butanediamine (MDL 72521) and N1,N2-bis(2,3-butadienyl)-1,4-butanediamine (MDL 72527) are specific, potent, enzyme-activated, irreversible inhibitors of polyamine oxidase in vitro. These compounds are also capable of completely inhibiting polyamine oxidase in mouse tissues at intraperitoneal doses greater than 20 mg/kg. Enzyme activity reappears in the various organs within 2-3 days to 50% of the control values. Irreversible inhibition of polyamine oxidase in mice led to decreased putrescine (30-40%) and spermidine (10-20%) levels in liver and some other organs. At the same time N1-acetylspermidine and, to a lesser extent, N1-acetylspermine were accumulating at rates which are assumed to be related to the rates of polyamine degradation. Even after treatment with polyamine oxidase inhibitors over a period of 6 weeks at doses which produced complete inhibition of polyamine oxidase in all organs, including the brain, neither toxic effects nor changes in body weight or behaviour were observed.     

Inhibition of polyamine oxidase in rats improves the sensitivity of urinary polyamines as markers for cell death.

In this study we investigated polyamine metabolism during inhibition of two polyamine-catabolizing enzymes. This was performed by treating rats with aminoguanidine [an inhibitor of Cu-dependent amine oxidase (CuAO)], NN'-bis(buta-2,3-dienyl)butane-1,4-diamine [MDL 72527, an inhibitor of FAD-dependent polyamine oxidase (PAO)], tetrachloromethane (hepatotoxic agent) and combinations of these compounds. Emphasis was laid on the origin and possible clinical usefulness of two polyamine metabolites: acetylisoputreanine-gamma-lactam and N1N12-diacetylspermine. Acetylisoputreanine-gamma-lactam is a normal constituent of human and rat urine. Treatment of rats with aminoguanidine led to undetectable urinary levels of acetylisoputreanine-gamma-lactam, whereas MDL 72527 treatment resulted in a 12-fold increase. Under normal conditions this compound represents a minor CuAO catabolite of N1-acetylspermidine, but may become of more importance under CuAO-induced conditions. N1N12-diacetylspermine was undetectable in urine samples from non-pregnant adults and rats, but became detectable after treating rats with MDL 72527. Additional tetrachloromethane poisoning resulted in a 35-fold increase of N1N12-diacetylspermine in urine and its appearance in liver. Hence urinary excretion of N1N12-diacetylspermine during PAO inhibition may serve as a sensitive marker for cell death. This was confirmed by myeloid-leukaemia-bearing rats treated with MDL 72527, which also excreted N1N12-diacetylspermine in urine in relatively high amounts from at least day 14 until spontaneous death.     

Spermine cytotoxicity to human colon carcinoma-derived cells (CaCo-2)

Spermine is a constituent of all vertebrate cells. Nevertheless, it exerts toxic effects if it accumulates in cells. Spermine is a natural substrate of the FAD-dependent polyamine oxidase, a constitutive enzyme of many cell types. It has been reported that the toxicity of spermine was enhanced if polyamine oxidase was inhibited. We were interested to examine spermine toxicity to human colon carcinoma-derived CaCo-2 cells because, in contrast to most tumor cell lines, CaCo-2 cells undergo differentiation, which is paralleled by changes in polyamine metabolism. CaCo-2 cells were remarkably resistant to spermine accumulation, presumably because spermine is degraded by polyamine oxidase at a rate sufficient to provide spermidine for the maintenance of growth. Inactivation of polyamine oxidase increased the sensitivity to spermine. A major reason for the enhanced spermine cytotoxicity at low polyamine oxidase activity is presumably the profound depletion of spermidine, and the consequent occupation of spermidine binding sites by spermine. Hydrogen peroxide and the aldehydes 3-aminopropanal and 3-acetamidopropanal, the products of polyamine oxidase-catalyzed splitting of spermine and N ¹-acetylspermine, contribute little to spermine cytotoxicity. Activation of caspase by spermine was insignificant, and the formation of DNA ladders, another indicator of apoptotic cell death, could not be observed. Thus it appears that cell death due to excessive accumulation of spermine in CaCo-2 cells was mainly nonapoptotic. The content of brush border membranes did not change between days 6 and 8 after seeding, and it was not affected by exposure of the cells to spermine. However, the activities of alkaline phosphatase, sucrase, and aminopeptidase in nontreated cells were considerably enhanced during this period, but remained low if cells were exposed to spermine. These changes appear to indicate that differentiation is prevented by intoxication with spermine, although other explanations cannot be excluded. This revised version was published online in July 2006 with corrections to the Cover Date.     

Role of polyamine metabolism in kainic acid excitotoxicity in organotypic hippocampal slice cultures

Polyamines are ubiquitous cations that are essential for cell growth, regeneration and differentiation. Increases in polyamine metabolism have been implicated in several neuropathological conditions, including excitotoxicity. However, the precise role of polyamines in neuronal degeneration is still unclear. To investigate mechanisms by which polyamines could contribute to excitotoxic neuronal death, the present study examined the role of the polyamine interconversion pathway in kainic acid (KA) neurotoxicity using organotypic hippocampal slice cultures. Treatment of cultures with N1,N(2)-bis(2,3-butadienyl)-1,4-butanediamine (MDL 72527), an irreversible inhibitor of polyamine oxidase, resulted in a partial but significant neuronal protection, especially in CA1 region. In addition, this pre-treatment also attenuated KA-induced increase in levels of lipid peroxidation, cytosolic cytochrome C release and glial cell activation. Furthermore, pre-treatment with a combination of cyclosporin A (an inhibitor of the mitochondrial permeability transition pore) and MDL 72527 resulted in an additive and almost total neuronal protection against KA toxicity, while the combination of MDL 72527 and EUK-134 (a synthetic catalase/superoxide dismutase mimetic) did not provide additive protection. These data strongly suggest that the polyamine interconversion pathway partially contributes to KA-induced neurodegeneration via the production of reactive oxygen species.     

Catabolism of polyamines

Summary. Owing to the establishment of cells and transgenic animals which either lack or over-express acetylCoA:spermidine N¹-acetyltransferase a major progress was made in our understanding of the role of polyamine acetylation. Cloning of polyamine oxidases of mammalian cell origin revealed the existence of several enzymes with different substrate and molecular properties. One appears to be identical with the polyamine oxidase that was postulated to catalyse the conversion of spermidine to putrescine within the interconversion cycle. The other oxidases are presumably spermine oxidases, because they prefer free spermine to its acetyl derivatives as substrate. Transgenic mice and cells which lack spermine synthase revealed that spermine is not of vital importance for the mammalian organism, but its transformation into spermidine is a vitally important reaction, since in the absence of active polyamine oxidase, spermine accumulates in blood and causes lethal toxic effects.Numerous metabolites of putrescine, spermidine and spermine, which are presumably the result of diamine oxidase-catalysed oxidative deaminations, are known as normal constituents of organs of vertebrates and of urine. Reasons for the apparent contradiction that spermine is in vitro a poor substrate of diamine oxidase, but is readily transformed into N⁸-(2-carboxyethyl)spermidine in vivo, will need clarification.Several attempts were made to establish diamine oxidase as a regulatory enzyme of polyamine metabolism. However, diamine oxidase has a slow turnover. This, together with the efficacy of the homeostatic regulation of the polyamines via the interconversion reactions and by transport pathways renders a role of diamine oxidase in the regulation of polyamine concentrations unlikely. 4-Aminobutyric acid, the product of putrescine catabolism has been reported to have antiproliferative properties. Since ornithine decarboxylase and diamine oxidase activities are frequently elevated in tumours, it may be hypothesised that diamine oxidase converts excessive putrescine into 4-aminobutyric acid and thus restricts tumour growth and prevents malignant transformation. This function of diamine oxidase is to be considered as part of a general defence function, of which the prevention of histamine and cadaverine accumulation from the gastrointestinal tract is a well-known aspect.     

Purvalanol A is a strong apoptotic inducer via activating polyamine catabolic pathway in MCF-7 estrogen receptor positive breast cancer cells

Purvalanol A is a specific CDK inhibitor which triggers apoptosis by causing cell cycle arrest in cancer cells. Although it has strong apoptotic potential, the mechanistic action of Purvalanol A on significant cell signaling targets has not been clarified yet. Polyamines are crucial metabolic regulators affected by CDK inhibition because of their role in cell cycle progress as well. In addition, malignant cells possess impaired polyamine homeostasis with high level of intracellular polyamines. Especially induction of polyamine catabolic enzymes spermidine/spermine N1-acetyltransferase (SSAT), polyamine oxidase (PAO) and spermine oxidase (SMO) induced toxic by-products in correlation with the induction of apoptosis in cancer cells. In this study, we showed that Purvalanol A induced apoptosis in caspase- dependent manner in MCF-7 ER(+) cells, while MDA-MB-231 (ER?) cells were less sensitive against drug. In addition Bcl-2 is a critical target for Purvalanol A, since Bcl-2 overexpressed cells are more resistant to Purvalanol A-mediated apoptosis. Furthermore, exposure of MCF-7 cells to Purvalanol A triggered SSAT and PAO upregulation and the presence of PAO/SMO inhibitor, MDL 72,527 prevented Purvalanol A-induced apoptosis.     

Mechanism of the inhibition of cell growth by N1,N12-bis(ethyl)spermine.

The mechanism of the antiproliferation effect of N1,N12-bis(ethyl)spermine (BESPM) was studied in detail using mouse FM3A cells, since this polyamine analogue mimics the functions of spermine in several aspects [Igarashi, K., Kashiwagi, K., Fukuchi, J., Isobe, Y., Otomo, S. & Shirahata, A. (1990) Biochem. Biophys. Res. Commun. 172, 715-720]. Our results indicate that not only the decrease in sperimine and spermine caused by BESPM but also its accumulation play important roles on the inhibition of cell growth by BESPM, since BESPM accumulated in cells at a concentration fivefold that of spermidine in control cells. In comparison with the polaymine-deficient cells caused by alpha-difluoromethylornithine, an inhibitor of ornithine decarboxylase, and ethylglyoxal bis(guanylhydrazone), an inhibitor of S-adenosylmethionine decarboxylase, the behavior of polyamine-deficient cells caused by BESPM was different as follows: the inhibition of cell growth by BESPM was not abrogated by spermine or spermidine; polyamine uptake, which is stimulated during polyamine deficiency, was greatly inhibited, while spermidine/spermine N1-acetyltransferase activity, which is inhibited during polyamine deficiency, was enhanced in BESPM-treated cells; thymidine kinase activity did not decrease in BESPM-treated cells; inhibition of cell growth and macromolecule synthesis by BESPM correlated with the swelling of mitochondria and the decrease in ATP content; BESPM caused cell death when incubated together for several days. The role of BESPM accumulation on inhibition of cell growth is discussed.