Leena Alhonen-Hongisto

Sensitive Enzymic determination of methylglyoxal bis(guanylhydrazone) in cultured cells and in animal tissues

Methylglyoxal bis(guanylhydrazone) (MGBG) is an antiproliferative compound used clinically in cancer chemotherapy [l-3]. Earlier the mechanism of action of MGBG was often related to the metabolism of polyamines, especially because its antiproliferative effects were counteracted by spermidine 141: it was only in [5] that MGBG was shown to be a remarkably potent and specific inhibitor of eukaryotic S-adenosylL-methionine decarboxylase (EC 4.1 .1.50), an enzyme directly involved in the synthesis of spermidine and spermine. Since then the compound has been widely used to produce intracellular polyamine deprivation in a number of experimental systems including cultured cells and animal tissues (for references see [6]). In all systems employed, the growth-inhibitory effect of MGBG can be prevented by PM levels of spermidine and spermine [6]. This finding has been taken as evidence indicating that the antiproliferative action exerted by MGBG is mediated through an inhibition of intracellular accumulation of spermidine and spermine. However, a straightforward interpretation of these studies is complicated by the fact that MGBG, spermidine and spermine appear to compete for a common cellular uptake system [7,8]. It is thus possible that the prevention of MGBG-induced toxicity by polyamines could partly result from a reduction of the intracellular concentration of the drug, and is not necessarily due to a normalization of cellular polyamine pattern. With the possible exception of a few studies [9 ,101, the effect of polyamines on the intracellular concentrations of MGBG has not been checked properly. This apparently is related to the lack of sensitive methods for determination of

Polyamine deprivation-induced enhanced uptake of methylglyoxal bis(guanylhydrazone) by tumor cells

1. Putrescine and spermidine depletion produced by alpha-difluoromethylornithine, an irreversible inhibitor or ornithine decarboxylase (EC 4.1.1.17), resulted in a strikingly enhanced cellular uptake of methylglyoxal bis(guanylhydrazone) in cultured Ehrlich ascites carcinoma cells and human lymphocytic leukemia cells. 2. A prior priming of the cells with difluoromethylornithine followed by a short exposure of the cells to methylglyoxal bis(guanylhydrazone) rapidly established intracellular concentrations of the latter drug approaching 10 mM. 3. The enhanced transport of methylglyoxal bis(guanylhydrazone) into the tumor cells apparently required metabolic energy as the uptake of extracellular drug rapidly ceased and intracellular methylglyoxal bis(guanylhydrazone) was excreted into the medium when the glycolysis of the tumor cells was inhibited by iodoacetate. 4. A sequential treatment of cultured tumor cells with difluoromethylornithine until established polyamine depletion followed by an addition of low concentrations of methylglyoxal bis(guanylhydrazone) produced an antiproliferative action not achieved with either of the drugs alone. 5. A similar treatment schedule, i.e a priming of mice inoculated with Ehrlich ascites cells with difluoromethylornithine for a few days, likewise enhanced the uptake of methylglyoxal bis(guanylhydrazone) by the carcinoma cells, but only marginally increased the drug concentration in the liver and small intestine of the animals.

S-adenosylmethionine decarboxylase as target of chemotherapy

Although ornithine decarboxylase under most conditions is the rate-controlling enzyme of polyamine biosynthesis and thus the most logical target for chemical intervention, the inhibition of the enzyme triggers a series of compensatory reactions all aimed to circumvent the inhibition. These include secondary induction of adenosylmethionine decarboxylase, enhanced accumulation of extracellular polyamines and an overproduction of ornithine decarboxylase resulting from enhanced expression and gene amplification. Thus chemotherapy based on an intervention of polyamine formation has also to be directed to reactions other than the decarboxylation of ornithine. Adenosylmethionine decarboxylase is the second natural target for chemotherapy. Virtually all effective inhibitors of this enzyme are members of the family of bis(guanylhydrazones). Small modifications, such as increased hydrophobicity at the glyoxal portion of the parent compound glyoxal bis(guanylhydrazone), greatly enhance the inhibition of adenosylmethionine decarboxylase and diminish the undesirable inhibition of diamine oxidase. However, although ethylglyoxal and propylglyoxal bis(guanylhydrazone) appear to utilize the putative polyamine carrier for their cellular entry, their cellular accumulation, in contrast to that of glyoxal and methylglyoxal bis(guanylhydrazone), is not stimulated by putrescine and spermidine deprivation produced by inhibitors of ornithine decarboxylase. It is obvious that the cellular accumulation of each of the bis(guanylhydrazones) is determined by their different efflux rates: GBG and MGBG are effectively retained whereas EGBG is rapidly excreted by the tumor cells. GBG and MGBG, but possibly not EGBG, behave as mitochondrial poisons and rapidly produce extensive morphological damage of the mitochondria. The bis(guanylhydrazones) likewise inhibit carnitine-dependent mitochondrial oxidation of long-chain fatty acids, competitively in respect to carnitine. It is possible that this inhibition has something to do with the mitochondrial damage, as carnitine protects tumor cells from the early mitochondrial damage produced by MGBG. Carnitine also protects experimental animals from MGBG-induced acute toxicity and death.

Difluoromethylornithine-induced amplification of ornithine decarboxylase genes in Ehrlich ascites carcinoma cells.

Stepwise increments of the concentration of 2-difluoromethylornithine, a mechanism-based irreversible inhibitor of mammalian ornithine decarboxylase (EC 4.1.1.17), resulted in a selection of cultured Ehrlich ascites carcinoma cells capable of growing in the presence of up to 50 mM difluoromethylornithine. Dialyzed extracts of drug-resistant tumor cells exhibited a very high ornithine decarboxylase activity and contained large excess of immunoreactive ornithine decarboxylase protein. Hybridization analyses with cloned complementary DNA revealed that the difluoromethylornithine-resistant tumor cells also expressed mRNA of the enzyme at greatly enhanced rate. The overproduction of ornithine decarboxylase by the tumor cells grown under the pressure of difluoromethylornithine was at least partly attributable to a 10 to 20-fold increase in the total gene dosage of ornithine decarboxylase involving an amplification of several genes of the gene family. The gene amplification developed appeared to be stable, as the gene dosage only slowly (during a period of several months) returned towards the normal level upon the removal of difluoromethylornithine. The overproduction of ornithine decarboxylase was accompanied by an enhanced resistance of the enzyme towards difluoromethylornithine in vitro.

Replacement of natural polyamines by cadaverine and its aminopropyl derivatives in Ehrlich ascites carcinoma cells

Abstract Ehrlich ascites carcinoma cells were cultured in the presence of difluoromethyl ornithine (DFMO) and micromolar concentrations of cadaverine for several months. This treatment resulted in a complete disappearance of putrescine and spermidine and reduced spermine content to traces of its normal content. The natural polyamines were replaced by cadaverine (about 40% of total polyamines), N-(3-aminopropyl)cadaverine (about 50%) and N,N′-bis(3-aminopropyl)cadaverine (about 5%). In comparison with untreated cells or cells grown in the presence of DFMO and putrescine, the “cadaverine cells” grew definitely slower, their protein synthesis was depressed while DNA and RNA syntheses proceeded at near normal rate. In spite of the high intracellular concentrations of cadaverine and its aminopropyl derivatives, the tumor cells grown in the presence of DFMO and cadaverine, behaved exactly like cells severly depleted of putrescine and spermidine. Though exposed to DFMO, ornithine decarboxylase activity was almost 10 times higher than that in untreated cells. S-Adenosyl-L-methionine decarboxylase activity was likewise strikingly elevated, and these cells transported methylglyoxal strikingly elevated, and these cells transported methylglyoxal bis(guanylhydrazone) (MGBG) at a rate that was more than 5 times faster than that in untreated cells. Furthermore, these cells exhibited arginase activity, which was less than one fifth of that found in untreated cells.

Polyamine depletion induces enhanced synthesis and accumulation of cadaverine in cultured Ehrlich ascites carcinoma cells

Abstract An exposure of cultured Ehrlich ascites carcinoma cells to DL-α-difluoromethyl ornithine, an irreversible inhibitor of ornithine decarboxylase (EC 4.1.1.17), rapidly depleted the tumor cells of putrescine and spermidine. The decrease in the cellular concentrations of these two natural polyamines, however, was accompanied by a striking appearance of two new major amines: cadaverine and a compound tentatively identified as N -3-aminopropyl-1,5-diaminopentane (aminopropylcadaverine). When the cultures were grown in the presence of uniformly labeled [ 14 C]lysine, tumor cells exposed to difluoromethyl ornithine converted lysine to cadaverine and aminopropyl cadaverine at strikingly enhanced rate. The difluoromethyl ornithine-induced accumulation and synthesis of cadaverine and aminopropylcadaverine were totally prevented by the presence of micromolar concentrations of spermidine (or spermine) in the culture media.

Effect of dexamethasone on the activity and expression of ornithine decarboxylase in rat liver and thymus.

A single intraperitoneal injection of the synthetic glucocorticoid dexamethasone into rats resulted in a marked stimulation (more than 60-fold) of hepatic ornithine decarboxylase (ODC) at 4 h after the injection, whereas the enzyme activity in thymus was almost totally (about 95%) depressed at the same time. The stimulation of ODC activity in liver was in all likelihood attributable to a greatly enhanced accumulation of mRNA species for the enzyme as revealed by Northern blot and dot-blot hybridization analyses. ODC activity in thymus, in response to dexamethasone, was only 5% of that found in control animals, but this decrease was apparently not accompanied by similar reductions of the levels of ODC message, which was in fact decreased only by 50% at the maximum. In addition to two mRNA species (2.1 and 2.6 kilobases; kb), typical to mouse cells, rat tissues seemed to contain a third hybridizable message for ODC, smaller (1.6 kb) than the above-mentioned species and not seen in samples obtained from mouse or human cells. Interestingly, these smaller poly(A)+ RNA sequences, hybridizable with cDNA complementary to mouse ODC mRNA, were apparently constitutively expressed, as the treatment with glucocorticoid altered the amount of these sequences only slightly.

Combined use of 2-difluoromethylornithine and methylglyoxal bis(guanylhydrazone) in normal and leukemia-bearing mice

Mice were treated with daily injections of methylglyoxal bis(guanyl-hydrazone) (MGBG) without or with concurrent administration of 2-difluoromethylornithine (DFMO) in drinking water for 15 days. Analysis of 10 different tissues for their MGBG content during the treatment revealed little evidence for a tissue specific cumulative accumulation of the drug given either alone or in combination with DFMO. On the contrary, tissue MGBG levels tended to increase until the 4th to 7th day of the treatment, whereafter a gradual decline or a plateau was obvious in most tissues. The concomitant DFMO treatment produced a consistent elevation of tissue MGBG concentrations in bone marrow cells and possibly also in intestinal tissue. In L1210 leukemia-bearing DBA mice, MGBG was most actively taken up by the ascitic leukemia cells. A priming of the tumor-bearing mice with DFMO for a few days before the start of MGBG injections resulted in a strikingly enhanced accumulation of the latter drug in the leukemia cells and also in the spleen, which was apparently heavily infiltrated by tumor cells. In liver, small intestine and in bone marrow cells of tumor-bearing animals the concentration of MGBG was not influenced by the DFMO treatment. In DBA mice without the L1210 tumor, DFMO only insignificantly increased the level of MGBG in bone marrow cells whereas no increase was seen in the spleen, in contrast to the same organ obtained from tumor-bearing mice. This combined treatment, in comparison with DFMO or MGBG alone, also produced the best therapeutic response as revealed by marked reduction of the tumor mass.

Diethylglyoxal bis(guanylhydrazone): a novel highly potent inhibitor of S-adenosylmethionine decarboxylase with promising properties for potential chemotherapeutic use.

Diethylglyoxal bis(guanylhydrazone) (DEGBG), a novel analog of the antileukemic agent methylglyoxal bis(guanylhydrazone) (MGBG) was synthesized. It was found to be the most powerful inhibitor of yeast S-adenosylmethionine decarboxylase (AdoMetDC) so far studied (Ki approx. 9 nM). This property, together with the finding that the compound is a weaker inhibitor of intestinal diamine oxidase than are MGBG and its glyoxal, ethylglyoxal and ethylmethylglyoxal analogs, makes the compound a promising candidate as a polyamine antimetabolite for chemotherapy studies. DEGBG was also found to potentiate the antiproliferative effect of the ornithine decarboxylase inhibitor alpha-difluoromethyl ornithine against mouse L1210 leukemia cells in vitro. DEGBG increased several-fold the intracellular putrescine concentration of cultured L1210 cells, just as MGBG and its ethylglyoxal analog are known to do. The results strongly suggest that DEGBG is worth further studies. Combined with previous studies, they also made possible the construction of some empirical rules concerning the structure-activity relationships of bis(guanylhydrazone) type inhibitors of AdoMetDC. The identity of DEGBG was confirmed by a single-crystal X-ray analysis and by 1H- and 13C-NMR spectroscopy. It consisted of the same isomer as MGBG and several of its analogs are known to consist of.

Biochemical Properties and Crystal Structure of Ethylmethylglyoxal Bis(guanylhydrazone) Sulfate — an Extremely Powerful Novel Inhibitor of Adenosylmethionine Decarboxylase

Ethylmethylglyoxal bis(guanylhydrazone) (EMGBG) sulfate, an analog of the well-known antileukemic drug methylglyoxal bis(guanylhydrazone), was synthesized. It was shown to be an extremely powerful competitive inhibitor of eukaryotic S-adenosyimethionine decarboxylase, with an apparent Ki value 12 nᴍ. Thus, it appears to be the most powerful known inhibitor of the enzyme, being almost an order of magnitude more powerful than the corresponding ethylglyoxal derivative. It neither inhibited the proliferation of mouse L1210 leukemia cells in vitro, nor did it potentiate the growth inhibition produced by α-difluoromethyl ornithine. In this respect, its properties are closely related to those of dimethylglyoxal, ethylglyoxal and propylglyoxal bis(guanylhydrazones), while in striking contrast to those of the antiproliferative glyoxal and methylglyoxal analogs. EMGBG also inhibited intestinal diamine oxidase activity (Ki 0.7 μᴍ). EMGBG sulfate was crystallized from water, giving orthorhombic crystals (space group Pbcn). Their crystal and molecular structure was determined by X-ray diffraction methods. The carbon-nitrogen double bonds between the ethylmethylglyoxal part and the aminoguanidine moieties were found to have the same configuration as they are known to have in the salts of glyoxal. methylglyoxal and propylglyoxal bis(guanylhydrazones). The glyoxal bis(guanylhydrazone) chain of the EMGBG cation deviated strongly from planarity, thus differing dramatically from the corresponding chains of the glyoxal, methylglyoxal and propylglyoxal analogs.