Pauli Seppänen

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.

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.

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.

Methylglyoxal bis(guanylhydrazone) stimulates the cellular transport system of the polyamines

1. INTRODUCTION Methylglyoxal bis(guanylhydrazone) (MGBG), an inhibitor of polyamine biosynthesis [l], is ap- parently transported into the interior of the cell (by virtue of its structural resemblance to spermidine) via an inducible transport system used by the natu- ral polyamines spermidine and spermine [2-51. Cellular uptake of polyamines, and hence also of MGBG, is strikingly enhanced under conditions of intracellular putrescine and spermidine depletion, as a result of the use of inhibitors of ornithine de- carboxylase (EC 4.1.1.17) [5]. Even though the lat- ter phenomenon is obviously aimed at normalizing the reduced intracellular polyamine pools, it also offers a means of enhancing the cellular accumula- tion of the antiproliferatively acting MGBG when profound growth inhibition is desired [6]. Although intracellular polyamine depletion trig- gers a variety of compensatory mechanisms, such as induction and stabilization of ornithine and ad- enosylmethionine (EC 4.1.150) decarboxylases (for ref. see [7]), the greatly enhanced transport of extracellular polyamines can apparently alone abolish the antiproliferative effects of polyamine antimetabolites (inhibitors of polyamine bio- synthesis) in the presence of only trace amounts of exogenous polyamines. This fact may be prac- tically important since polyamine antimetabolites [MGBG and 2-difluoromethylornithine (DFMO)] have already been used alone (for ref. see [6]) or in combination [8] in the treatment of human malig- nancies. We will show here that when cultured Ehrlich ascites carcinoma cells were exposed to MGBG for not longer than a few hours, the uptake of poly- amines, especially that of diamines, was greatly en- hanced. Exposure to MGBG likewise stimulated the uptake of polyamines by tumor cells that have been brought to the state of severe polyamine de- privation with DFMO, a condition characterized by strikingly increased uptake of polyamines (51. The enhanced uptake of polyamines induced by the antimetabolites was apparently responsible for the appearance of substantial amounts of cadaver- ine (and putrescine) in L1210 leukemia cells har- vested from mice treated with MGBG alone or in combination with DFMO. 2. MATERIALS AND METHODS 2.1.

Inhibition of long-chain fatty acid oxidation by methylglyoxal bis(guanylhydrazone)

Methylglyoxal bis(guanylhydrazone) (MGBG), an inhibitor of spermidine and spermine biosynthesis and clinically used anti-cancer drug, powerfully inhibited carnitine-dependent fatty acid oxidation in heart muscle homogenates. Equipotent inhibition was also produced by spermine whereas spermidine and putrescine were less effective. MGBG appeared to act as a competitive inhibitor in respect to carnitine. Even though MGBG and spermine equally effectively depressed palmitate oxidation in muscle homogenates in vitro, a striking difference existed between the compounds as regards their effects on fatty acid oxidation in cultured tumor cells. Micromolar concentrations of MGBG distinctly impaired palmitate utilization also in cultured L 1210 leukemia cells, whereas similar concentrations of spermine markedly enhanced the oxidation of the fatty acid. The inhibitory effect of MGBG in cultured tumor cells was, at least partly, reversed upon addition of exogenous carnitine. The finding indicating that MGBG impairs fatty acid utilization may be an explanation for the known hypoglycemic effect produced by the drug in most animal species as well as for some of the side-effects associated with its clinical use, most notably severe myalgia.

A rapid chemiluminescence-based method for the determination of total polyamines in biological samples

The metabolism of the natural polyamines putrescine, spermidine and spermine is closely associated with cell proliferation [l]. Although in human cancer, the concentrations of polyamines in serum and urine are mostly higher than in healthy persons, the determination of extracellular polyamines obviously does not represent a reliable method for early detection of cancer (see [2]). However, an acute rise in serum polyamine levels or in their urinary excretion in response to successful anti-cancer therapy [3], as the result of the release from damaged cells of these strictly intracellular compounds, may be helpful for the evaluation of the efficacy of a given anti-cancer regimen. In addition, serial determinations of extracellular polyamines have a distinct value in the surveillance of tumor regression or relapse in certain tumor types, especially in medulloblastomas, where an increase in putrescine in cerebrospinal fluid predicts the regrowth of the tumor well before it is detected by any other diagnostic techniques [4]. A number of sensitive methods are available for determination of polyamines in biological samples including extracellular fluids. Dansylation of polyamines followed by chromatography on thin layer plates [S] has the drawback of being time-consuming. High performance liquid chromatography [6] and liquid ion exchange chromatography [7] are sensitive methods but somewhat cumbersome for efficient handling of large numbers of samples. Radioimmunological methods utilizing antibodies to the polyamines [8] are useful for rapid analyses, but require a good quality antibody. We have now developed a rapid (up to 20 samples/h) and sensitive (in the pmol range) method for the determination of total polyamines (putrescine + spermidine +