Spermidine regulates insulin synthesis and cytoplasmic Ca(2+) in mouse beta-TC6 insulinoma cells.

In order to assess the functional role of the polyamines spermidine and spermine in pancreatic beta-cells, we examined the effect of spermidine and spermine synthase inhibitors, trans-4-methylcyclohexylamine (MCHA) and N-(3-aminopropyl)cyclohexylamine (APCHA), on cellular polyamine and insulin contents, insulin secretion, and cytoplasmic Ca(2+) concentration ([Ca(2+)](i)) in mouse insulin-secreting Beta-TC6 cells. The cellular spermidine and spermine contents were reduced 90% and 64% by cultivation of cells in the presence of MCHA and APCHA for 3 days, respectively. Addition of spermidine or spermine reversed the polyamine level reduced by MCHA or APCHA, respectively. Insulin secretion was decreased 40~60% in the cells treated with MCHA or APCHA. The reduction by MCHA was reversed to the untreated level by adding spermidine exogenously, while the effect of APCHA was not reversed by treatment with spermine. The cellular insulin content was also reduced by treatment with MCHA but not the expression of insulin 1 and 2 genes, suggesting that spermidine was involved in the translation of insulin mRNAs. The elevation of [Ca(2+)](i), a key event triggering insulin secretion induced by glucose, was reduced in Beta-TC6 cells by MCHA treatment. The spermidine synthase inhibitor also augmented the sustained [Ca(2+)](i) rise induced by carbamylcholine but not by a high concentration of KCl or nicotine. These results suggested that spermidine rather than spermine plays an important role in the regulation of insulin synthesis and the glucose-induced [Ca(2+)](i) rise in Beta-TC6 cells.

Antitumor effect of a new multienzyme inhibitor of polyamine synthetic pathway, methylglyoxal-bis(cyclopentylamidinohydrazone), against human and mouse leukemia cells.

Methylglyoxal-bis(cyclopentylamidinohydrazone) (MGBCP) has been synthesized as a multienzyme inhibitor for the polyamine-synthesizing pathway. This drug inhibited S-adenosylmethionine decarboxylase (EC 4.1.1.50), spermine synthase and spermidine synthase activities, competitively with S-adenosylmethionine, spermidine, and putrescine, respectively. MGBCP inhibited the growth of human leukemia Molt 4B and K 562 cells at 10 to 100 microM concentrations. Spermidine and spermine levels were markedly depressed in these MGBCP-treated leukemic cells, and the synthesis of protein, but not of DNA or RNA, was significantly diminished. In in vivo experiments, MGBCP depleted spermidine and spermine in the P388 leukemic ascites cells, and prolonged the survival time of mice bearing P388 leukemia. The S-adenosylmethionine decarboxylase-stabilizing effect of MGBCP in mouse liver, Molt 4B and K 562 cells was much less than that of the parent inhibitor methylglyoxal-bis(guanylhydrazone). Induction of ornithine decarboxylase activity by MGBCP in the cultured leukemic cells was also much less than that by methylglyoxal-bis(guanylhydrazone).

Antiproliferative effect of spermine depletion by N-cyclohexyl-1,3-diaminopropane in human breast cancer cells.

Spermine is often the most abundant polyamine in human tumors such as breast carcinomas. However, its specific role in tumor biology is still uncertain, since inhibitors of ornithine decarboxylase such as alpha-difluoromethylornithine depress cell growth while leaving spermine content mostly unaffected. We have assessed the specific role of spermine in breast cancer cell growth using N-cyclohexyl-1,3-diaminopropane (C-DAP), a potent spermine synthase inhibitor. In ZR-75-1 cells, C-DAP decreased net cell growth after 14 days by 65% at 50 microm, with an IC50 of about 5 microM, and was about 10 times more potent than N-(n-butyl)-1,3-diaminopropane, another spermine synthase inhibitor. C-DAP acted as a specific inhibitor of spermine biosynthesis, since (a) it depleted spermine content while causing an equal or greater accumulation of spermidine on a molar basis, (b) it rapidly induced S-adenosylmethionine decarboxylase activity and the accumulation of its products due to relief of spermine-dependent inhibition of enzyme expression, and (c) exogenous spermine (1 microM) completely reversed C-DAP-induced growth inhibition. C-DAP and related compounds were accumulated, at least in part, through a mechanism distinct from the polyamine transport system, while also blocking putrescine and spermidine uptake with various potencies. Reversibility of C-DAP-induced growth inhibition by exogenous spermine was progressively lost on prolonged treatment, in association with marked morphological changes. In 4 different human breast cancer cell lines (ZR-75-1, T47-D, MCF-7, and MDA-MB-231), relative growth sensitivity to C-DAP was inversely related to the extent of spermidine accumulation caused by spermine synthase inhibition, suggesting that spermidine overaccumulation can functionally replace spermine. Interestingly, C-DAP strongly potentiated growth inhibition caused by alpha-difluoromethylornithine in all cell lines tested by preventing conversion of residual spermidine to spermine, indicating that spermine synthesis limits alpha-difluoromethylornithine action and that under some critical threshold, spermidine cannot fulfill cellular needs for spermine. Thus, spermine plays specific and important functions in breast tumor growth, and spermine synthase inhibitors could markedly improve the therapeutic effectiveness of existing polyamine depletion strategies, especially in spermine-rich tumors.

Studies on the metabolic effects of methylthioformycin.

5'-Methylthioformycin, a structural analog of 5'-methylthioadenosine in which the N-C glycosidic bond is substituted by a C-C bond, has been synthesized by a newly developed procedure. Membrane permeability of the molecule has been compared to that of methylthioadenosine in intact human erythrocytes and Friend erythroleukemia cells. The formycinyl compound is taken up with a rate significantly lower than that of 5'-methylthioadenosine and is not metabolized by the cells. 5'-Methylthioformycin inhibits Friend erythroleukemia cells' growth: the effect is dose-dependent, fully reversible and not caused by cytotoxicity. Several enzymes related to methylthioadenosine metabolism are inhibited by methylthioformycin. Rat liver methylthioadenosine phosphorylase is competitively inhibited with a Ki value of 2 microM. Among the propylamine transferases tested only rat brain spermine synthase is significantly inhibited, while rat brain spermidine synthase is less sensitive. Rat liver S-adenosylhomocysteine hydrolase is irreversibly inactivated with 50% inhibition at 400 microM methylthioformycin. 5'-Methylthioformycin does not exert any significant effect on protein carboxyl-O-methyltransferase. Inferences about the mechanism of the antiproliferative effect of the drug have been drawn from the above results.

Thirty years of polyamine-related approaches to cancer therapy. Retrospect and prospect. Part 1. Selective enzyme inhibitors.

As soon as the natural polyamines (PAs), putrescine (Put), spermidine (Spd) and spermine (Spm), were recognized as ubiquitous constituents of eukaryotic cells, their involvement in growth-related processes attracted particular interest. The high activities of ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (AdoMetDC) in rapidly growing tissues and cells, particularly in tumour cells, suggested PA biosynthesis as a target for antineoplastic therapy. In the course of the years selective inhibitors have been developed for literally all enzymes of PA metabolism. Some became important as tools in the elucidation of the PA metabolic system, but only few of them were efficient as inhibitors of tumour growth. A major reason for the inefficacy of selective enzyme inhibitors as anticancer drugs is the sophistication of the system, which regulates intracellular PA pools. Selective blockade of a single enzyme induces changes of metabolism and transport, which compensate for the deficit. The selective impairment of tumour growth is in addition hampered by the ubiquitous occurrence of the PAs, their importance in normal functions of nearly all mammalian cells, and the ability or the mammalian organism to utilize exogenous (gastrointestinal) PAs. Among the inhibitors of PA-related enzymes, the ODC inactivator (R, S)-2-(difluoromethyl)ornithine (DFMO) became most famous. Although it was disappointing in most therapeutic attempts to use it as single drug, it has--based on its low toxicity--considerable potential in cancer chemoprevention, and it turned out to be a highly efficient anti-trypanosome agent. Very likely DFMO is suitable to improve the efficacy of some of the current cytotoxic drugs, and it may allow one to create new therapies in combination with other PA-directed drugs. Some of the less selective enzyme inhibitors, particularly those, which inhibit two or more enzymes of PA metabolism, appear to have had a chance to become practically useful, but they have not been developed energetically. Disregarding DFMO, the AdoMetDC inhibitor SAM486A is the only compound for which clinical trials were published. The future of this drug is unclear at present; presumably phase III clinical trials have been discontinued. One of the lessons that had to be learned from the work on selective enzyme inhibitors was that PA metabolism is a much more difficult target, than has been expected on the basis of the simplicity of the PA structures, and the simple reactions involved in their biosynthesis. In order to inhibit tumour growth several reactions or regulatory functions of PA metabolism have to be impaired at the same time. Recent efforts devoted to the development new types of anticancer drugs, which are based on the perturbation of PA metabolism by structural analogues of the natural PAs, take this message into account. These approaches are the topic of the 2nd part of this overview.

Synthesis and evaluation of some stable multisubstrate adducts as specific inhibitors of spermidine synthase.

A new series of aminopropyltransferase inhibitors has been designed in which the nuclephilic aminopropyl acceptor is attached to the aminopropyl donor, S-adenosyl-1-(methylthio)-3-propylamine (decarboxylated S-adenosylmethionine), to form a "multisubstrate adduct". In the present case, S-adenosyl-1,8-diamino-3-thiooctane (2b) and the corresponding methysulfonium salt (3b) have been synthesized. Several compounds of this type were assayed as inhibitors of spermidine synthase, and both 2b and 3b were found to be potent inhibitors of the enzyme. The thioether 2b is the most potent inhibitor of spermidine synthase described to date and is almost totally devoid of inhibitory activity against the closely related aminopropyltransferase, spermine synthase. This type of compound should have use as a specific inhibitor of spermidine biosynthesis in vivo.

Inhibitors of polyamine biosynthesis. 9. Effects of S-adenosyl-L-methionine analogues on mammalian aminopropyltransferases in vitro and polyamine biosynthesis in transformed lymphocytes.

Seven analogues of S-adenosyl-L-methionine were studied as inhibitors or substrates for mammalian spermidine and spermine synthases. One of these, S-(5'-deoxy-5'-adenosyl)-(+/-)-1-methyl-3-(methylthio)propylamine (5), showed a unique spectrum of activities on the polyamine biosynthesis enzymes. It was an inhibitor of S-adenosyl-L-methionine decarboxylase from rat liver and spermine synthase from bovine brain and rat ventral prostate. This compound was a substrate for the spermidine synthases from bovine brain and rat ventral prostate but not a substrate for the spermine synthases from these same sources. At concentrations of 0.2 mM and higher, compound 5 blocked the increases in polyamine levels and in [3H]thymidine incorporation induced by concanavalin A in cultured mouse lymphocytes. At approximately a 0.5 mM concentration of 5, the cellular polyamine levels and the rate of thymidine incorporation were similar to those of the unstimulated lymphocytes. Lower concentrations of 5 (0.02-0.1 mM) produced a dose-dependent increase in thymidine incorporation. A dose-dependent decrease in the cellular polyamine levels was observed in the range of 0.05-0.5 mM of the inhibitor. These results suggest that the effects of 5 on transformed lymphocytes are complex and may not be solely due to the inhibition of polyamine biosynthesis by this compound.

Synthesis and biological evaluation of S-adenosyl-1,12-diamino-3-thio-9-azadodecane, a multisubstrate adduct inhibitor of spermine synthase.

As part of a continuing search for specific inhibitors of the enzymes involved in polyamine biosynthesis, we have designed and synthesized a multisubstrate adduct inhibitor, S-adenosyl-1,12-diamino-3-thio-9-azadodecane (AdoDATAD), in which critical portions of the nucleophilic aminopropyl acceptor are covalently linked to critical portions of the electrophilic aminopropyl donor to form a potent and specific inhibitor of spermine synthase. In addition, the corresponding desamino analogue which was designed to lack activity against spermine synthase on the basis of substrate structure-activity data has been synthesized as a control. Preliminary biological results demonstrate that AdoDATAD is a potent and specific inhibitor of mammalian spermine synthase in vitro, while being almost completely devoid of inhibitory activity toward the closely related aminopropyltransferase spermidine synthase. The desamino analogue, as predicted, showed no inhibitory activity against either enzyme. AdoDATAD represents an important addition to the arsenal of specific enzyme inhibitors available for blockade of the polyamine biosynthetic pathway at specific sites.

Effects of inhibitors of spermidine synthase and spermine synthase on polyamine synthesis in rat tissues.

Several inhibitors of aminopropyltransferases, developed recently in this laboratory, were tested for their specificity by measuring their effects on six enzyme activities related to polyamine biosynthesis and interconversion. Two of them, trans-4-methylcyclohexylamine (4MCHA) and N-(3-aminopropyl)cyclohexylamine (APCHA), selectively and potently inhibited the activities of spermidine synthase and spermine synthase, respectively. They were subjected to in vivo studies using rats. Oral administration of 4MCHA or APCHA dissolved in drinking water (0.02 and 0.1%) available ad lib. for a period of 10 days or 4 months caused a specific and marked decrease in spermidine or spermine in tissues (such as a 95% decrease) with a compensatory increase of spermine or spermidine, respectively, but without any observable change in the growth of the treated rats. Also, with extreme reduction of spermidine or spermine, when their sum was approximately constant, the activity of S-adenosyl-methionine decarboxylase in these tissues was enhanced significantly with no change in the activity of ornithine decarboxylase. These results suggested a separate role for spermidine or spermine in the in vivo enhancement of S-adenosylmethionine decarboxylase activity.

Putrescine or spermidine binding site of aminopropyltransferases and competitive inhibitors.

A model of the active site of aminopropyltransferases was proposed based on the study of a number of monoamino and diamino compounds as potential inhibitors and substrates, respectively, of spermidine synthase purified from pig liver. The active site seems to have a relatively large hydrophobic cavity adjacent to a negatively charged site, to which a protonated amino group of putrescine binds, with another amino group of putrescine being situated in the hydrophobic cavity as a free form to be aminopropylated by decarboxylated S-adenosylmethionine. On the basis of the above-mentioned model, another modified one was proposed for spermine synthase, and several compounds mentioned model, another modified one was proposed for spermine synthase, and several compounds designed according to the modified model were found to potently inhibit spermine synthase, purified from rat brain, in competition with spermidine. The newly developed inhibitors were about two orders of magnitude more potent in vitro than a known inhibitor of spermine synthase, dimethyl(5'-adenosyl)sulfonium perchlorate.

Effect of N-(n-butyl)-1,3-diaminopropane on polyamine metabolism, cell growth and sensitivity to chloroethylating agents.

The effects of N-(n-butyl)-1,3-diaminopropane (BDAP) on cell growth and polyamine content were examined in L1210, SV-3T3 and HT-29 cells. In all cases, BDAP was a specific and highly effective inhibitor of spermine synthesis, and spermine levels were greatly suppressed in the presence of 50 microM BDAP. At the same time, there was a parallel increase in spermidine, which equalled or exceeded the fall in spermine so that total polyamine levels were not reduced. Cell growth was not affected in short-term experiments but culture of L1210 cells for 72-144 hr in the presence of BDAP did lead to an effect on growth that was reversed by the addition of spermine. These results suggest that, in the short term, a normal growth rate is maintained by spermidine but that a function or cellular component critically dependent on spermine becomes depleted at longer times. BDAP was a weak inducer of spermidine/spermine-N1-acetyltransferase and this enzyme may be responsible for excretion or degradation of the inhibitor. The reduction of spermine produced by BDAP led to a substantial increase in the activity of S-adenosylmethionine decarboxylase (AdoMetDC) showing that the repression of this enzyme by spermine is greater than the repression by spermidine. Although higher concentrations were required, BDAP was as effective an inhibitor of spermine synthesis as the mechanism-based inhibitor, S-adenosyl-1,12-diamino-3-thio-9-azadodecane (AdoDATAD), and produced similar decreases in spermine and increases in AdoMetDC. Prior treatment of HT-29 human colon carcinoma cells with BDAP increased the killing by chloroethylating agents but to a much smaller extent than the increase brought about by the DNA repair inhibitor, O6-benzylguanine. The effect of BDAP is likely to be due to an increased interaction of chloroethylating drugs with nuclear DNA in the absence of spermine since BDAP treatment sensitized cells even in the presence of O6-benzylguanine, which prevents repair of these lesions.

Specific depletion of spermidine and spermine in HTC cells treated with inhibitors of aminopropyltransferases.

The effects of a potent spermidine synthase inhibitor, trans-4-methylcyclohexylamine (4MCHA), and a spermine synthase inhibitor, N-(3-aminopropyl)cyclohexylamine (APCHA), on polyamine biosynthesis and cell growth have been studied in rat hepatoma cells (HTC cells) in culture. Treatment of HTC cells with 4MCHA or APCHA caused a marked decrease of spermidine or spermine with a compensatory increase of putrescine and spermine or spermidine, respectively, in a dose-dependent manner, suggesting specific and potent inhibition of each target enzyme. When 250 microM 4MCHA or APCHA was administered to the cells for 8 days, spermidine was decreased to 2% of control culture or spermine below 1%, respectively, while total polyamine (sum of putrescine, spermidine, and spermine) remained almost unchanged during the culture. There were no significant changes in the growth rate during treatment with the inhibitors at 250 microM concentration. The results suggest that in the growth of HTC cells, putrescine and spermine can be substituted for most of the fraction of cellular spermidine, and spermidine for most of the fraction of cellular spermine. Of five enzymatic activities involved in polyamine biosynthesis and interconversion, S-adenosylmethionine decarboxylase activity increased 8-fold with 250 microM 4MCHA, and 3-fold with 250 microM APCHA during the treatment. This increase was partially due to the increase of half-life of the enzyme. Separate roles for spermidine and spermine in the biosynthesis of the enzyme protein were also suggested.

Aminooxy analogues of spermidine as inhibitors of spermine synthase and substrates of hepatic polyamine acetylating activity.

Aminooxy analogues of spermidine, 1-aminooxy-3-N-[3-aminopropyl]- aminopropane (AP-APA) and N-[2-aminooxyethyl]-1,4-diaminobutane (AOE-PU), were tested as substrates or inhibitors of the enzymes involved in methionine and polyamine metabolism. Both compounds were good competitive inhibitors and poor substrates of spermine synthase, good substrates of cytosolic polyamine acetyltransferase, inactivators of S-adenosylmethionine decarboxylase and inhibitors of ornithine decarboxylase. AP-APA and AOE-PU showed K1-values of 1.5 and 186 microM as inhibitors of purified spermine synthase, and Km-values of 1.4 and 2.1 mM as substrates of the crude hepatic polyamine acetyltransferase activity. AP-APA was more potent than AOE-PU in crude enzyme preparations. Neither drug had any significant effect at 1 mM concentration on the activities of spermidine synthase, methionine adenosyltransferase, S-adenosylhomocysteine hydrolase, and methylthioadenosine phosphorylase. The results suggest that compounds of this type are valuable tools in unraveling the physiology of polyamines.

Inhibition of cell growth by combination of alpha-difluoromethylornithine and an inhibitor of spermine synthase.

The inhibitory effect on cell growth of a combination of alpha-difluoromethylornithine (DFMO) and an inhibitor of aminopropyl transferase was examined. N-(3-aminopropyl)cyclohexylamine (APCHA) and trans-4-methylcyclohexylamine (4MCHA) were used as inhibitors of spermine and spermidine synthases, respectively. Combination of DFMO and APCHA showed strong inhibitory effects on the growth of FM3A cultured cells and P388 leukemia cells in mice, compared with DFMO alone. The prolongation of survival time of P388 leukemia-bearing mice by DFMO (1,500 mg/kg) was 1.12-fold, while that by DFMO (1,500 mg/kg) plus APCHA (25 mg/kg) was 1.30-fold. The prolongation of survival time nearly paralleled the decrease of P388 leukemia cells in mice. However, the antiproliferative effect of DFMO was not strengthened by 4MCHA in the above two experimental systems. In the FM3A cell culture system, both putrescine and spermidine contents were decreased by DFMO, but spermine content did not decrease significantly. When APCHA was added to the medium with DFMO, spermine content was decreased greatly but a compensatory increase in spermidine was observed. Spermidine content in P388 leukemia cells was also decreased by DFMO, the increase in spermine was suppressed but a compensatory increase in spermidine was observed. Nevertheless, the spermidine content remained significantly low compared with the value in non-treated P388 leukemia cells. Thus, the results indicate that the antiproliferative effect of DFMO was strengthened by APCHA due to the decrease in spermine content, and that the decrease in total amount of spermidine and spermine, especially the decrease in spermine, is necessary for inhibition of cell growth.

Specific multisubstrate adduct inhibitors of aminopropyltransferases and their effect on polyamine biosynthesis in cultured cells.

We have designed and synthesized multisubstrate adduct inhibitors for each of the three enzymes in the polyamine biosynthetic pathway (equation 1). The specific aminopropyltransferase inhibitors AdoDATO (2b) and AdoDATAD (2d) have been used to study the effects of specific polyamine depletion on cell growth. As shown in Table 2, these compounds effectively modulate the biosynthesis of Spd and Spm in vitro. However, tight regulation of the biosynthetic and degradative pathways results in little or no change in total polyamine levels in the presence of a single inhibitor (Table 2). Further studies with these aminopropyltransferase inhibitors in combination with other specific inhibitors of polyamine biosynthesis or degradation (e.g. DFMO) should shed light on the mechanism(s) underlying this tight biological regulation.

15-Deoxyspergualin, an antiproliferative agent for human and mouse leukemia cells shows inhibitory effects on the synthetic pathway of polyamines.

The mechanism of the antitumor action of 15-deoxyspergualin (DSG) was investigated. DSG inhibited spermidine synthase noncompetitively with putrescine, spermine synthase competitively with spermidine and polyamine oxidase in vitro. Induction of ornithine decarboxylase (ODC) activity observed after subculture of human leukemia cells was blocked by the addition of DSG to the culture medium. In DSG-treated leukemia cells, putrescine, spermidine and spermine levels were markedly depressed. The synthesis of protein was also greatly diminished in these polyamine-depleted leukemic cells, whereas the depressions of DNA and RNA syntheses were minimum. In in vivo experiments, DSG depressed polyamine levels in P388 leukemic ascites cells, and prolonged the survival times of mice bearing the leukemia cells. These results suggest that inhibition of polyamine and protein biosyntheses by DSG is substantially responsible for its antitumor action on the tumor cells.

Kinetic studies on inhibition of aminopropyltransferases by aurintricarboxylic acid in vitro.

Activities of aminopropyltransferases (spermidine synthase and spermine synthase) were inhibited by aurintricarboxylic acid (ATA). Spermidine synthase was slightly more sensitive to the inhibitor than spermine synthase. These inhibitions were not prevented by 0.15 M NaCl. Inhibition by ATA of spermidine synthase was 'uncompetitive' with respect to putrescine and that of spermine synthase was 'non-competitive' with respect to spermidine. When the amount of spermidine synthase or spermine synthase was varied, inhibition ratio hardly changed on either case implying no appreciable interaction between ATA and these enzymes.

An intercellular polyamine transfer via gap junctions regulates proliferation and response to stress in epithelial cells.

In the organism, quiescent epithelial cells have the potential to resume cycling as a result of various stimuli, including wound healing or oxidative stress. Because quiescent cells have a low polyamine level, resuming their growth requires an increase of their intracellular polyamine levels via de novo polyamine synthesis or their uptake from plasma. Another alternative, explored here, is an intercellular exchange with polyamine-rich cycling cells via gap junctions. We show that polyamines promote gap junction communication between proliferating cells by promoting dynamical microtubule plus ends at the cell periphery and thus allow polyamine exchange between cells. In this way, cycling cells favor regrowth in adjacent cells deprived of polyamines. In addition, intercellular interactions mediated by polyamines can coordinate the translational response to oxidative stress through the formation of stress granules. Some putative in vivo consequences of polyamine-mediated intercellular interactions are also discussed regarding cancer invasiveness and tissue regeneration.