mitoguazone and Carcinoma--Ehrlich-Tumor

This investigation was designed to determine whether cell death plays a role in the antiproliferative action exerted by polyamine synthesis inhibitors. To estimate the rate of tumor cell death, we measured the loss of 125I from mice harboring Ehrlich ascites tumor cells in which DNA was labeled with 5-125I-iodo-2'-deoxyuridine. DL-alpha-difluoromethylornithine (0.85 mumoles/g body weight/6 h), and enzyme-activated irreversible inhibitor of ornithine decarboxylase, and methylglyoxal-bis(guanylhydrazone) (45 nmoles/g body weight/6 h), an inhibitor of S-adenosylmethionine decarboxylase, were both found to increase the rate of 125I excretion. Our data suggest that these polyamine synthesis inhibitors provoke an increase in the rate of tumor cell death beyond that normally occurring during growth, methylglyoxal-bis(guanylhydrazone) being considerably more potent than DL-alpha-difluoromethylornithine. These in vivo data were corroborated by a study where the host-mediated responses did not have to be considered. Thus, Ehrlich ascites tumor cells were adapted for suspension growth in culture and treated with methylglyoxal-bis(guanylhydrazone) or DL-alpha-difluoromethylornithine. The growth kinetics and the colony forming efficiency of the drug-treated cells clearly show that polyamine synthesis inhibitors not only slow the growth rate but also cause an increase in tumor cell death.

Topics: Adenosylmethionine Decarboxylase; Animals; Antineoplastic Agents; Carcinoma, Ehrlich Tumor; Cell Division; Cell Survival; Eflornithine; Guanidines; Male; Mice; Mice, Inbred Strains; Mitoguazone; Ornithine; Ornithine Decarboxylase Inhibitors; Polyamines

Treatment of Ehrlich-ascites-carcinoma-bearing mice with methylglyoxal bis(guanylhydrazone) alone or in combination with 2-difluoromethylornithine greatly enhanced the transfer of intragastrically administered radioactive putrescine and cadaverine into the carcinoma cells. Difluoromethylornithine alone did not have any effect on the accumulation of intestine-derived diamines in the tumour cells. The frequently reported restoration of difluoromethylornithine-induced polyamine depletion on administration of methylglyoxal bis(guanylhydrazone) is in all likelihood attributable to a profound inhibition of intestinal diamine oxidase (EC 1.4.3.6), resulting in an enhanced entry of intestinal (bacterial) diamines into general circulation and finally into tumour cells.

Topics: Animals; Cadaverine; Carcinoma, Ehrlich Tumor; Diamines; Eflornithine; Guanidines; Mice; Mitoguazone; Ornithine; Putrescine

Glyoxal bis(guanylhydrazone), the parent compound of methylglyoxal bis(guanylhydrazone), was synthesized and tested for its ability to inhibit the biosynthesis of polyamines. It was found to be a powerful competitive inhibitor of adenosylmethionine decarboxylase (EC 4.1.1.50), yet the lack of the methyl group at the glyoxal portion increased the apparent Ki value for the enzyme by about 30-fold in comparison with methylglyoxal bis(guanylhydrazone). Glyoxal bis(guanylhydrazone) inhibited diamine oxidase (EC 1.4.3.6) activity as effectively as did methylglyoxal bis(guanylhydrazone). The cellular accumulation curves of glyoxal bis(guanylhydrazone) in L1210 cells were practically superimposable with those of methylglyoxal bis(guanylhydrazone), and the uptake of both compounds was distinctly stimulated by a prior treatment with 2-difluoromethylornithine. The drug decreased the concentration of spermidine in a dose-dependent manner and, in contrast with methylglyoxal bis(guanylhydrazone), without a concomitant accumulation of putrescine. The fact that putrescine concentrations were decreased in cells exposed to glyoxal bis(guanylhydrazone) was, at least in part, attributable to an inhibition of ornithine decarboxylase (EC 4.1.1.17) activity in cells treated with the compound. Under these experimental conditions equivalent concentrations of methylglyoxal bis(guanylhydrazone) [1,1'-[(methylethanediylidine)dinitrilo]diguanidine] elicited large increases in the enzyme activity. When combined with difluoromethylornithine, glyoxal bis(guanylhydrazone) potentiated the growth-inhibitory effect of that drug. Taking into consideration the proven anti-leukaemic activity of glyoxal bis(guanylhydrazone), its effectiveness to inhibit spermidine biosynthesis (without raising the concentration of putrescine) as well as its suitability for combined use with inhibitors of ornithine decarboxylase, this drug is apparently worthy of further testing in tumour-bearing animals, especially in combination with difluoromethylornithine or related inhibitors of ornithine decarboxylase.

Topics: Adenosylmethionine Decarboxylase; Amine Oxidase (Copper-Containing); Animals; Carcinoma, Ehrlich Tumor; Cells, Cultured; DNA, Neoplasm; Eflornithine; Female; Guanidines; Leukemia L1210; Mice; Mitoguazone; Neoplasm Proteins; Ornithine; Polyamines

Topics: Animals; Biological Transport, Active; Carcinoma, Ehrlich Tumor; Cells, Cultured; Female; Guanidines; Leukemia L1210; Mice; Mice, Inbred DBA; Mitoguazone; Polyamines

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.

Topics: Animals; Antineoplastic Agents; Carcinoma, Ehrlich Tumor; Cell Line; Eflornithine; Female; Guanidines; Humans; Iodoacetates; Iodoacetic Acid; Leukemia, Lymphoid; Mice; Mitoguazone; Ornithine; Putrescine; Spermine

Topics: Animals; Biological Transport; Carcinoma, Ehrlich Tumor; Cell Survival; Cells, Cultured; Eflornithine; Guanidines; Mitoguazone; Ornithine; Polyamines

Intracellular polyamine deprivation, produced by DL-alpha-difluoromethylornithine (DFMO), resulted in a striking enhancement of cellular transport of the natural polyamines and methylglyoxal bis(guanylhydrazone) (MGBG). In addition to the natural polyamines and MGBG, the uptake of other diamines and triamines was likewise enhanced in response to DFMO, although longer than three-carbon backbone was required for about 10-fold stimulation to occur. Intracellular deprivation of polyamines did not increase the affinity of the transport system for MGBG but greatly enhanced the maximum velocity of the drug transport. The uptake process of MGBG was temperature dependent and the activation energy (Ea = 67.5 kJ) for the uptake system was the same for both the polyamine-depleted tumour cells and for the untreated cells. The uptake of the drug appeared to be more dependent on the Na+-linked uptake, as indicated by the inhibitory effect of ouabain, than on energy production. Deprivation of putrescine and spermidine changed the intracellular distribution of MGBG since a major portion of the drug was concentrated in the microsomal fraction in polyamine-depleted cells.

Topics: Animals; Carcinoma, Ehrlich Tumor; Cells, Cultured; Eflornithine; Guanidines; Iodoacetates; Iodoacetic Acid; Mice; Mitoguazone; Neoplasms, Experimental; Ornithine; Ouabain; Polyamines; Putrescine; Spermidine

Topics: Adenosylmethionine Decarboxylase; Amine Oxidase (Copper-Containing); Animals; Biological Transport; Carcinoma, Ehrlich Tumor; Guanidines; Humans; Kidney; Kinetics; Leukemia, Lymphoid; Liver; Methods; Mice; Mitoguazone; Putrescine; Rats; Spermidine; Spermine; Spleen

Inhibition of polyamine synthesis by alpha-difluoromethylornithine in cultured Ehrlich ascites-carcinoma cells rapidly enhanced the uptake of exogenous putrescine, spermidine and spermine from the culture medium. In tumour cells exposed to the drug for 2 days, the intracellular concentration of spermidine was decreased to less than 10% of that found in untreated cells. However, the strikingly stimulated transport system brought the concentration of spermidine to the control values in less than 2h after supplementation of the cells with micromolar concentrations of the polyamine. In the absence of polyamine deprivation, tumour cells did not accumulate extracellular polyamines to any appreciable extent. Ascites-tumour cells deprived of putrescine and spermidine likewise concentrated methylglyoxal bis(guanylhydrazone) [1,1'-[methylethanedylidine)dinitrilo]diguanidine] at a greatly enhanced rate. A previous "priming of tumour cells with difluoromethylornithine followed by an exposure of the cells to methylglyoxal bis(guanylhydrazone) resulted in a marked and rapid anti-proliferative effect.

Topics: Adenosylmethionine Decarboxylase; Animals; Carcinoma, Ehrlich Tumor; Cells, Cultured; Eflornithine; Guanidines; Mitoguazone; Ornithine; Ornithine Decarboxylase; Putrescine; Spermidine

The anti-proliferative effects of 1,1'-[(methylethanediylidene)dinitrilo]diguanidine [methylglyoxal bis(guanylhydrazone)] and 1,1'-[(metHYLETHANEDIYLIDENE)dinitrilo]bis-(3-aminoguaNIDINE) HAVE BEEN STUDIED IN Ehrlich ascites carcinoma cells grown in suspension cultures. Both compounds are potent inhibitors of S-adenosyl-L-methionine decarboxylase from the tumour cells. In the presence of putrescine (but not in its absence), the inhibition produced by 1,1'-[methylethanediylidene)dinitrilo]bis-(3-aminoguanadine) was apparently irreversible, as judged by persistent depression of the enzyme activity even after extensive dialysis. The two compounds produced similar increases in adenosylmethionine decarboxylase activity, which resulted from a striking stabilization of the enzyme in cells grown in the presence of the drugs. The inhibitory effect of the two diguanidine derivatives on the synthesis of DNA and protein became evident after an exposure of 4--8 h. At that time, the only change seen in tumour polyamines in cells grown in the presence of the inhibitors was an increase in cellular putrescine. To find out whether the compounds initially interfered with the energy production of the tumour cells, the cultures were grown in the presence of uniformly labelled glucose, and the formation of lactate, as well as the oxidation of the sugar into CO2, were measured. The activation of glycolysis upon dilution of the tumour cells with fresh medium and the subsequent formation of labelled CO2 were siliar in control cells and in cells exposed to methylglyoxal bis(buanylhydrazone), 1,1'-[(methylethanediylidene)dinitrilo]bis-(3-aminoguanidine) or diaminopropanol. Only a marginal decrease in the cellular content of ATP was found in cells exposed to the inhibitors for 24 h. The diguanidine-induced growth inhibition was fully reversed by low concentrations of exogenous polyamines. However, the possibility remained that the reversal by polyamines was due to a decrease of intracellular diguanidine concentration. Our results indicate that the mode of action of 1,1'-[(methylethanediylidene)dinitrilo]bis-(3-aminoguanidine) is fully comparable to that of methylglyoxal bis(guanylhydrazone), as regards stabilization of adenosylmethionine decarboxylase and the appearance of growth inhibition in Ehrlich ascites cells. The data tend to support the view that both compounds apparently have an early anti-proliferative effect unrelated to polyamine metabolism.

Topics: Adenosylmethionine Decarboxylase; Animals; Carcinoma, Ehrlich Tumor; Cell Division; Cells, Cultured; DNA; Female; Glucose; Guanidines; Mice; Mitoguazone; Polyamines; Propanolamines

Enzymic determinations of intracellular concentrations of methylglyoxal-bis(guanylhydrazone), an anticancer drug which inhibits the synthesis of the polyamines spermidine and spermine, in cultured tumor cells revealed that the drug was remarkably effectively concentrated inside the cell. A concentration gradient across the cell membrane as great as 500--1000-fold was formed in cells exposed to the drug for 1-2 days. An exposure of cultured Ehrlich ascites carcinoma cells to increasing concentrations of the drug indicated that the cells could tolerate intracellular concentrations up to 1 mM with only slight changes in their proliferation rate. Micromolar concentrations of spermidine or spermine, but not putrescine, effectively blocked the uptake of methylglyoxal-bis(guanylhydrazone) and reduced the intracellular concentration of the drug below the levels required for growth inhibition. Analysis of cellular polyamine contents in Ehrlich ascites cells exposed to rising concentrations of methylglyoxal-bis(guanylhydrazone) gave little support to the view that the drug-induced growth inhibition was solely produced by an intracellular polyamine deprivation. Not only was the uptake of the drug inhibited in the presence of spermidine and spermine, but it was likewise washed out by polyamines from the cells that had been previously exposed to the drug and then transferred into drug-free medium in the presence of polyamines. For the inhibition of methylglyoxal-bis(guanylhydrazone) uptake by amines, three or more amino/imino groups were apparently required, since low concentrations of aliphatic diamines were either without any effect (short-chain diamines) or only marginally prevented (long-chain diamines) the uptake of the drug. High concentrations of Mg2+ ions, however, markedly inhibited the transport of the drug into Ehrlich ascites tumor cells.

Topics: Animals; Carcinoma, Ehrlich Tumor; Cell Division; Cells, Cultured; Dose-Response Relationship, Drug; Female; Guanidines; Humans; Magnesium; Mice; Mitoguazone; Spermidine; Spermine

Topics: Adenosylmethionine Decarboxylase; Animals; Carboxy-Lyases; Carcinoma, Ehrlich Tumor; Diamines; DNA, Neoplasm; Female; Guanidines; Mice; Mitoguazone; Ornithine; Ornithine Decarboxylase; Ornithine Decarboxylase Inhibitors; Putrescine; Spermidine; Spermine; Time Factors

Population kinetics of Ehrlich ascites tumor cells grown in vivo were studied following treatment with methylglyoxal-bis(guanylhydrazone) (MeGAG; NSC-32946), a potent chemotherapeutic agent and a specific polyamine synthesis inhibitor. MeGAG-treatment resulted in a continuous accumulation of cells in the S and G2 phases of the cell cycle, and, as a consequence, the rate of cell proliferation decreased. This finding emphasizes the importance of the polyamines for progression through the cell cycle.

Topics: Adenosylmethionine Decarboxylase; Animals; Carcinoma, Ehrlich Tumor; Cell Division; DNA, Neoplasm; Guanidines; Kinetics; Mitoguazone