Effects of alpha-difluoromethylornithine and methylglyoxal bis(guanylhydrazone) on the growth of experimental renal adenocarcinoma in mice

alpha-Difluoromethylornithine (DFMO) and methylglyoxal bis-(guanylhydrazone) (MGBG) were tested against a murine renal adenocarcinoma, because polyamines are necessary for neoplastic cell growth and because human renal adenocarcinomas contain higher levels of spermidine than do normal renal cells; MGBG inhibits spermidine synthesis and has some activity against human renal tumors; DFMO irreversibly inhibits ornithine decarboxylase, the first rate-limiting enzyme controlling polyamine biosynthesis; and DFMO promotes intracellular accumulation of MGBG in experimental tumor models and human leukemia. DFMO (2%) in drinking water, MGBG (15 mg/kg i.p.), or a combination of DFMO and MGBG was administered daily to BALB/c mice (n = 80) with intrarenal transplants of renal adenocarcinoma cells. At 28 days, renal carcinomas weighed 64 and 73% less, respectively, in DFMO- and DFMO-MGBG-treated mice than in control animals (p less than 0.01). MGBG alone had no antigrowth effect. DFMO-MGBG reduced the total metastatic index (total number of metastases/total number of animals) to 1.2 versus 3.6 in control animals (p less than 0.01) and increased survival by 12.3 +/- 1.5 (S.E.) days, from 30.8 to 42.5 days (p less than 0.05). Compared with control, DFMO-, or MGBG-treated animals, DFMO-MGBG exposure reduced tumor growth and the number of metastases, prevented metastases in some animals (47%), and increased survival of mice bearing renal adenocarcinomas. DFMO also appeared to selectively increase the uptake of [14C]MGBG by tumor tissue, which may help to explain the enhanced synergistic antigrowth effect of DFMO and MGBG against this murine renal adenocarcinoma.     

Modification of uptake and antiproliferative effect of methylglyoxal bis(guanylhydrazone) by treatment with alpha-difluoromethylornithine in rodent cell lines with different sensitivities to methylglyoxal bis(guanylhydrazone)

Uptake characteristics and growth-inhibitory effects of methylglyoxal bis(guanylhydrazone) (MGBG), a competitive inhibitor of S-adenosylmethionine decarboxylase, were investigated in 9L rat brain tumor cells and in V79 hamster lung cells. Proliferation of 9L cells was only slightly inhibited by treatment with 40 microM MGBG alone, but when used in combination with 0.5 mM alpha-difluoromethylornithine (DFMO), an irreversible inhibitor of ornithine decarboxylase, proliferation was much more effectively inhibited. The intracellular concentration of MGBG was approximately 2-fold higher 4 days after cells were treated with both DFMO and MGBG, either simultaneously or when MGBG was added after a 48-hr DFMO pretreatment, than that in cells treated with MGBG alone. Polyamine levels in DFMO- and MGBG-treated cells correlated with the antiproliferative effects of the drugs. Used either alone or in combination with 1 mM DFMO, 0.5 microM MGBG inhibited the growth of and eventually killed V79 cells. Simultaneous or sequential treatment with DFMO and MGBG increased intracellular concentrations of MGBG at 4 days by 2- and 3-fold, respectively, compared to treatment with MGBG alone. Intracellular polyamine levels did not correlate with the antiproliferative effect of the two drugs in V79 cells. In both cell lines, polyamines and MGBG share a common transport system. The net transport of polyamines and MGBG was more temperature dependent and up to 10-fold more active in V79 cells than in 9L cells. The Km and Vmax values for spermidine and MGBG measured 10 sec after addition (initial permeation) were not affected by DFMO pretreatment in either cell line. However, 1 hr after administration, the Vmax values for spermidine and MGBG uptake were doubled in V79 cells pretreated for 48 hr with DFMO; no significant change occurred in 9L cells. Mitochondrial function, assessed by pyruvate oxidation, was substantially impaired by MGBG in V79 cells but not in 9L cells when the intracellular concentrations of MGBG were equal in each cell line. Pretreatment with DFMO did not increase MGBG-induced inhibition of pyruvate oxidation in V79 cells. These results show that, compared with V79 cells, the decreased sensitivity of 9L cells to MGBG may be related to decreased intracellular MGBG accumulation but not to cellular permeation such as carrier transport. Results of measurements of both polyamine levels and mitochondrial function indicate that V79 cells may be more susceptible to nonpolyamine-dependent effects of MGBG than are 9L cells.     

Differential effect of alpha-difluoromethylornithine on the in vivo uptake of 14C-labeled polyamines and methylglyoxal bis(guanylhydrazone) by a rat prostate-derived tumor

The uptake of exogenously administered radiolabeled polyamines by a rat prostate-derived tumor line, the Dunning R3327 MAT-Lu, and various normal tissues was studied. Pretreatment of tumor cells in vitro with alpha-difluoromethylornithine (DFMO), a polyamine synthesis inhibitor, resulted in a markedly enhanced uptake of both [14C]putrescine and [14 C]spermidine. The in vitro uptake of [14C]putrescine by these cells was effectively inhibited by unlabeled spermine, spermidine, 1,8-diaminooctane, 1,7-diaminoheptane, 1,6-diaminohexane, 1,5-diaminopentane, 1,4-diaminopentane, and 1,4-diaminobutane, but less effectively by 1,4-diamino-2,3-butene and 1,4-diamino-2,3-butyne. The diamines, 1,3-diaminopropane and 1,2-diaminoethane, were ineffective in inhibiting [14C]putrescine uptake in vitro into the R3327 MAT-Lu cell line. When tumor-bearing animals were pretreated with DFMO or with DFMO and 5-alpha-dihydrotestosterone propionate, the tumor and prostate uptake of [14C]putrescine and [14C]-cadaverine was enhanced but not substantially increased in other tissues. In contrast to the in vitro results, spermidine and spermine were not enhanced substantially by DFMO pretreatment into any tissue, and their uptake into the tumor actually decreased. Ethylenediamine, which does not utilize the polyamine transport system, did not have its uptake increased into any tissue following DFMO pretreatment. The chemotherapeutic agent, methylglyoxal bis(guanylhydrazone), which utilizes the polyamine transport system for uptake into cells, exhibited uptake behavior different from that of the polyamines. Thus, methylglyoxal bis(guanylhydrazone) uptake into the tumor was not significantly increased or decreased by DFMO or by DFMO + 5-alpha-dihydrotestosterone propionate pretreatment, and only the ventral, but not the dorsal-lateral, lobe of the prostate showed increased uptake of methylglyoxal bis(guanylhydrazone) following DFMO + 5-alpha-dihydrotestosterone propionate pretreatment.     

Alterations in bone marrow and blood mononuclear cell polyamine and methylglyoxal bis(guanylhydrazone) levels: phase I evaluation of alpha-difluoromethylornithine and methylglyoxal bis(guanylhydrazone) treatment of human hematological malignancies

Nine patients with hematological malignancies were treated with difluoromethylornithine and methylglyoxal bis(guanylhydrazone). The number of circulating blast cells decreased in all of the patients treated with DFMO and MGBG for longer than 1 wk. Morphological evidence of myeloid maturation was evident in four patients with leukemia and the circulating M Protein decreased in one patient with multiple myeloma. The polyamine content of the mononuclear cells in both the peripheral blood and bone marrow was transiently increased after the initial MGBG dose. During administration of DFMO decreases were achieved in the peripheral blood mononuclear cell putrescine levels in 7 patients, spermidine levels in 5 patients, and spermine levels in 4 patients. Alterations in bone marrow mononuclear cell polyamine levels were similar to those which occurred in the peripheral cells. An average of 9 days of DFMO treatment was required to lower mononuclear cell polyamine levels. Three of the 4 evaluable patients receiving multiple MGBG doses had an increased mononuclear cell content of MGBG after DFMO pretreatment. Enhancement of cellular MGBG levels was not directly correlated to the degree of cellular polyamine depletion.     

Modulation of the tissue disposition of methylglyoxal bis(guanylhydrazone) in mice by polyamine depletion and by polyamine administration

Treatment of mice with DL-alpha-difluoromethylornithine (DFMO), an irreversible inhibitor of ornithine decarboxylase (EC 4.1.1.17), produced a significant spermidine depletion in liver, small intestine, and bone marrow among eight tissues studied. The accumulation of methylglyoxal bis(guanylhydrazone) (MGBG) was selectively enhanced in small intestine and in bone marrow cells in response to a prior DFMO treatment. In other tissues studied, i.e., brain, skeletal and cardiac muscle, liver, kidney, and spleen, a preceding treatment with DFMO had no effect on the accumulation of subsequently injected MGBG. When mice, primed with DFMO and then treated with a single injection of MGBG, were given nontoxic doses of spermidine or putrescine through a gastric tube, high concentrations of MGBG in the small intestine and in bone marrow cells were effectively reduced. In spite of the route of administration, bone marrow cells appeared to be more sensitive than intestinal tissue as regards the prevention of the tissue accumulation of MGBG by the polyamines. The different sensitivity of various tissues to the natural polyamines in this respect may offer a means to develop a tissue-specific "polyamine rescue concept" to be used in connection with MGBG treatment.     

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.     

Disposition of methylglyoxal bis(guanylhydrazone) (MGBG,NSC-32946) in man

Summary A high-pressure liquid chromatographic method was utilized to determine the concentration of the antileukemic agent methylgloxal bis(guanylhydrazone) (MGBG, NSC-32946) in tissue samples obtained at autopsy from patients who received MGBG. In a patient with cholangiocarcinoma who received one course of MGBG (500 mg/m²), the highest drug concentration was found in normal liver tissue. However, the drug concentration in intrahepatic tumor tissue was only 10% of that found in uninvolved liver. In a patient with acute myelogenous leukemia (AML) who received 12 courses of MGBG therapy, highly infiltrated lymph node tissue was found to contain the highest concentration of MGBG. High concentrations of the drug were also found in liver, spleen, kidney, adrenal, and thyroid. The drug penetrated well into normal brain tissue. After repeated administration, high drug concentrations were found in cerebral and cerebellar gray matter. These studies suggest that there is no selective uptake of MGBG into solid tumors early after drug administration and provide a pharmacologic rationale for testing this agent against endocrine and intracerebral tumors in man.The abbreviations used in this paper are MGBG methylglyoxal bis(guanylhydrazone) - TCA trichloroacetic acid - PCA perchloric acid - KOH potassium hydroxide - HPLC high-pressure liquid chromatography     

Isolation of variants resistant to methylglyoxal bis(guanylhydrazone) from adenovirus-transformed rat cells

It is presumed that proteins encoded by viral oncogenes interact with proteins encoded by cellular genes to bring about the transformed phenotype. To demonstrate the existence of such cellular genes we attempted to isolate mutants with a nontransformed phenotype from an adenovirus-transformed rat cell line (F4) which contains multiple copies of the transforming E1 region. F4 cells were mutagenized with ethyl methanesulfonate and variants resistant to the anticancer drug methylglyoxal bis(guanylhydrazone) were selected. The proportion of such variants was about one in 10(6) and increased 5-fold after mutagenesis. Two variant clones (G1 and G2) were isolated and characterized: they were 5-fold more resistant to methylglyoxal bis(guanylhydrazone); they had a stable phenotype; they showed decreased drug uptake; they had a reduced ability to grow in soft agar, low serum, and nude mice; there was no detectable change in the restriction pattern of integrated viral genes or in the expression of the E1a and E1b proteins. These properties suggest that selection for methylglyoxal bis(guanylhydrazone) resistance may result in the isolation of variants with phenotypic characteristics of nontransformed cells. It was likely that these variants were altered in a cellular function required for the maintenance of the transformed phenotype.     

Phase II trial of methylglyoxal bis (guanylhydrazone) (MGBG) in advanced head and neck cancer

Methylglyoxal bis (guanylhydrazone) (MGBG) is an inhibitor of polyamine synthesis. In vitro studies demonstrate the accumulation of some tumor cells in S and G2 phases of the cell cycle. Nineteen patients with advanced head and neck cancer were entered in a Phase II trial of MGBG. MGBG, 500 mg/M2, was administered as a brief intravenous infusion weekly for 4 weeks, then every 2 weeks. Dose modifications were based on cumulative toxicity after 2 weekly treatments. All but three patients had prior exposure to chemotherapy for disease recurrence. Of 17 patients evaluable for response and toxicity, one brief partial response was observed. The most common toxicities were mild to moderate nausea, vomiting, diarrhea, and stomatitis. Myelosuppression occurred in three patients. Dose modifications were required in four patients; a maximum dose of 700 mg/M2 was tolerated. The results of four other Phase II single and combination chemotherapy trials of MGBG in head and neck cancer are reviewed. The single agent response rate in 59 patients was 22% (range, 6%-41%). The poor response rate observed in this trial was similar to that in other trials in which a heavily pretreated group of patients was evaluated. It is concluded that single agent MGBG is not a useful drug in heavily pretreated recurrent disease patients. However, because of its biochemical effects, further testing in combination with cycle specific agents and in larger numbers of patients with minimal prior treatment may be warranted.     

Effect of antioxidants on the mitochondrial activity and toxicity of the cancer drug methylglyoxal bis (guanylhydrazone) in yeast and mammalian cells

Mitochondria of yeast cells were primary targets of methylglyoxal bis (guanylhydrazone) (MGBG) from the following criteria: (1) selective inhibition of growth of cells utilizing a non-fermentable energy source, (2) inhibition of mitochondrial protein synthesis compared with cytosolic protein synthesis and (3) selective mutagenesis of the mitochondrial genome compared with nuclear mutagenesis. Evidence of primary antimitochondrial activity of MGBG in mammalian cells was provided by greater potency of the drug in guinea pig keratinocyte cultures utilizing glutamine as carbon and energy source compared with fermentable glucose. Cell death was used as a measure of drug toxicity in both yeast and mammalian systems. The antioxidants glutathione, vitamin E and vitamin C reversed toxicity and antimitochondrial activity to a large extent implying that toxic free radical metabolites of the drug are of significance in cellular activity of MGBG.     

Quantitation of methylglyoxal bis(guanylhydrazone) in blood plasma and leukemia cells of patients receiving the drug

Methylglyoxal bis(guanylhydrazone), a cytostatic compound which apparently interferes with the metabolism and/or functions of the natural polyamines (spermidine and spermine), was effectively taken up by cultured human lymphocytic leukemia cells, rapidly resulting in the formation of a concentration gradient of up to 1,000-fold across the cell membrane in cells grown in the presence of micromolar concentrations of the drug. For an anti-proliferative effect on the leukemia cells, an intracellular concentration of more than 0.5 mm was required. The uptake of methylglyoxal bis-(guanylhydrazone) was critically dependent on the growth rate of the leukemia cells. Low intracellular concentrations of the drug were present in cells growing slowly, whereas in rapidly dividing cells the intracellular concentration of the drug approached 5 mm. When given as repeated intravenous infusions to two leukemic children, methylglyoxal bis(guanylhydrazone) exhibited sharp and transient peaks of plasma concentration, the drug having an apparent half-life in plasma of only 1-2 h. However, as in cultured cells, the drug was rapidly concentrated in the leukemia cells, reaching concentrations that were distinctly anti-proliferative. In contrast to the rapid disappearance of methylglyoxal bis(guanylhydrazone) from plasma, the circulation leukemia cells retained the drug for a period of several days with only minimal decrease in the initial concentrations. Methylglyoxal bis(guanylhydrazone) was given to the patients for 1 to 2 months as intravenous infusions, the timing of which was determined by regular assays of the drug concentrations in the leukemia cells. In agreement with the results obtained with the cultured cells, an intracellular concentration of about 0.5 to I mm was apparently required for growth-inhibitory action to occur. Regular determination of the cellular drug concentrations indicated that methylglyoxal bis(guanylhydrazone) could be given as weekly infusions. This treatment schedule represents much lower dosing of the drug than the earlier daily regimens which were commonly associated with unacceptable toxicity.     

Effect of ricin and abrin on survival of L1210 leukemic mice and on leukemic and normal bone-marrow cells

The effect of ricin and abrin on the survival of mice treated with L1210 leukemic cells intraperitoneally or intravenously was studied. In mice given 1 × 10⁵ L1210 leukemia cells intraperitoneally a single dose of ricin (2.1 μg/kg) intraperitoneally gave the best results, an increased life span (ILS) of 59%. Abrin also increased the life span of such animals although to a lesser extent. The effect of ricin was superior to that of 5-fluorouracil, but inferior to that of adriamycin, which gave a maximum ILS of 280%. In mice given L1210 cells intravenously no increase in life span was obtained with ricin, abrin or adriamycin, whereas 5-fluorouracil gave an ILS of 40–50%. In spleen colony assays the differential effect of ricin and abrin on the proliferative capacity of normal hematopoietic and leukemic colony-forming cells in bone marrow was studied. The differential effect of ricin was as good as that of adriamycin and considerably better than that of 5-fluorouracil. Abrin had a much smaller effect than ricin on both normal and leukemic cells. The effect of abrin on the leukemic cells was too small to be of therapeutic value. The results warrant exploration of the use of ricin in the treatment of human leukemia.