Antigrowth effect of some inhibitors of polyamine synthesis on transplantable prostate cancer

Inhibitors of polyamine synthesis were tested for therapeutic effectiveness on transplantable prostate cancer. Inhibition of either ornithine decarboxylase or S-adenosyl-L-methionine decarboxylase (AMDC) by alpha-difluormethylornithine (DFMO) or methylglyoxal-bis[guanylhydrazone] (MGBG), respectively, was associated with significant antitumor effect. The combination of DFMO with MGBG was not only more effective but no more toxic than MGBG alone. Combination of MGBG with 9-B-D-arabinofuranosyladenine, an indirect effector of SAMDC, failed to increase therapeutic effectiveness of MGBG.     

Effects of inhibitors of polyamine biosynthesis on the growth and melanogenesis of murine melanoma cells

Both 2-difluoromethylornithine (DFMO), an irreversible inhibitor of ornithine decarboxylase (EC 4.1.1.17), and methylglyoxal bis(guanylhydrazone) (MGBG), a competitive inhibitor of S-adenosylmethionine decarboxylase (EC 4.1.1.50), strikingly stimulated melanotic expression of murine Cloudman S91 melanoma cells. The stimulation of tyrosinase (EC 1.10.3.1) activity and melanin formation by DFMO was closely associated with intracellular depletion of putrescine and spermidine developed in response to the drug. However, little or no evidence was obtained indicating that enhanced melanogenesis in response to MGBG was mediated through an inhibition of polyamine biosynthesis. Indirect inhibitors of ornithine decarboxylase, such as 1,3-diaminopropane and 1,3-diaminopropan-2-ol, but not putrescine, likewise inhibited the growth of the melanoma cells and stimulated their melanin production. The stimulation of melanogenesis by polyamine antimetabolites was not mediated by cyclic adenosine 3':5'-monophosphate, in contrast to the effect elicited by alpha-melanotropin. It is also unlikely that MGBG or the diamines acted as lysosomotropic agents capable of stimulating tyrosinase activity in situ, since the enzyme activity was stimulated by the drugs irrespective of whether assayed in cultured cells or using cell-free homogenates. None of the agents stimulated tyrosinase activity in vitro. The effect of DFMO and MGBG on melanoma cell proliferation was reversible, but the restoration of normal growth and melanin formation, especially in cells exposed to DFMO, was remarkably slow. The present results represent a further experimental model, in which the inhibition of polyamine accumulation is accompanied by signs of terminal differentiation.     

Combined use of alpha-difluoromethylornithine and an inhibitor of S-adenosylmethionine decarboxylase in mice bearing P388 leukemia or Lewis lung carcinoma

The antitumor and antimetastatic effects of alpha-difluoromethylornithine (DFMO), an inhibitor of ornithine decarboxylase, combined with an inhibitor of S-adenosylmethionine decarboxylase, either methylglyoxal bis(guanylhydrazone) (MGBG) or ethylglyoxal bis(guanylhydrazone) (EGBG), were studied in mice bearing P388 leukemia or Lewis lung carcinoma. Although EGBG is a more specific inhibitor of polyamine biosynthesis than the widely used MGBG, the antitumor effect of the DFMO-EGBG combination on P388 leukemia-bearing mice was less than that of the DFMO-MGBG combination. The prolongation of survival time by the DFMO(1000 mg/kg)-MGBG(25 mg/kg) combination was 2.65-fold, while that of the DFMO(1000 mg/kg)-EGBG(50 mg/kg) combination was 1.34-fold. When mice were fed a polyamine-deficient diet, stronger antitumor effects were exerted; the prolongation of survival time by the DFMO-MGBG and the DFMO-EGBG combinations was 2.89-fold and 2.03-fold, respectively. The antitumor effect of combined use of the two polyamine antimetabolites with mice on normal and polyamine-deficient diets correlated with a decrease of polyamine charge contents in the tumor cells. The above in vivo results were confirmed clearly in the KB cell culture system. The antimetastatic activity of DFMO on Lewis lung carcinoma-bearing mice was strengthened by the addition of MGBG or EGBG. The antimetastatic activity of the DFMO-MGBG or DFMO-EGBG combination did not parallel the polyamine charge contents in the primary tumor and blood.     

Effects of cyclosporin A and alpha-difluoromethylornithine on the growth of hamster pancreatic cancer in vitro

The growth and survival of hamster H2T pancreatic ductal adenocarcinoma in vitro are known to be significantly reduced by inhibitors of polyamine biosynthesis. alpha-Difluoromethylornithine (alpha-DFMO) is a specific and irreversible inhibitor of ornithine decarboxylase, the rate-limiting enzyme in polyamine biosynthesis. alpha-DFMO treatment inhibits the growth of H2T pancreatic cancer cells and decreases H2T cell survival in vitro and in vivo. In the present study, the effects of cyclosporin A (CsA) were examined on growth, survival, and polyamine levels in H2T pancreatic ductal adenocarcinoma in vitro. CsA had inhibitory effects on H2T pancreatic cancer growth similar to those of alpha-DFMO; these effects were blocked by the addition of the polyamine putrescine. Polyamine levels were found to be significantly altered in cells treated with CsA and/or alpha-DFMO. The combination of CsA (8.3 X 10(-4) mM) and alpha-DFMO (0.5 mM or 1.0 mM) inhibited H2T cell survival to a greater extent than either agent alone. These results suggest that CsA in combination with other agents that inhibit polyamine synthesis may be useful for the treatment of pancreatic cancer.     

The effect of difluoromethylornithine on decreasing prostate size and polyamines in men: results of a year-long phase IIb randomized placebo-controlled chemoprevention trial.

BACKGROUND: Prostate cancer is a major health issue, and prevention of prostate cancer and/or its progression will yield benefits for men. Difluoromethylornithine (DFMO) is an antiproliferative agent, inhibiting ornithine decarboxylase, the first enzyme in the polyamine pathway, and has been studied as a therapeutic and chemopreventive agent. The prostate has high levels of tissue polyamines and has shown sensitivity to DFMO both in vitro and in vivo. METHODS: Eighty-one men participated in a 1-year randomized trial of placebo or DFMO. Prostate volume determination and biopsy of the prostate for histology and polyamine content were done at baseline and after 12 months. Other biomarker variables were assessed, including total and free prostate-specific antigen and prostate-specific antigen doubling time. RESULTS: Compared with baseline, men receiving DFMO had a smaller increase in prostate volume (0.14 cm(3)) than those on placebo (2.95 cm(3); P = 0.0301) at 1 year. In addition, DFMO caused a 60.8% reduction of prostate putrescine levels compared with a 139.5% increase in the placebo arm (P = 0.0014). Stratification by ornithine decarboxylase genotype showed that DFMO reduced prostate volume (P = 0.029) and putrescine levels (P = 0.0053) in the AA + GA group but not in the GG group. There were no grade 3 or 4 toxicities. There was no clinical ototoxicity, with one subclinical grade 2 hearing decline on audiogram. CONCLUSION: In this randomized placebo-controlled trial, DFMO induced a decrease of prostate putrescine levels and rate of prostate growth. The potential of this compound for prostate cancer or hyperplasia should be further studied.     

Potentiation of methylglyoxal-bis-guanylhydrazone by alpha-difluoromethylornithine in rat prostate cancer

The polyamines, putrescine, spermidine, and spermine, are fundamentally related to both normal and neoplastic cell proliferation. The prostate gland and prostatic tumors in man and rodents contain large amounts of polyamines. This suggests that inhibition of polyamine biosynthetic enzymes, ornithine decarboxylase (ODC) and S-adenosyl-methionine decarboxylase (SAMDC) may retard the growth of prostatic cancer. Since alpha-difluoromethylornithine (DFMO) and methylglyoxal-bis-guanylhydrazone (MGBG) are irreversible and competitive inhibitors of ODC and SAMDC, respectively, they were tested as single agents and in combination on a transplantable rapidly growing and hormone-resistant G subline of the Dunning R-3327 rat prostatic adenocarcinoma. Groups of rats bearing tumors were treated with various regimens of DFMO, MGBG, and DFMO plus MGBG, daily for 21 days. Analysis of differences in tumor growth between treatment groups and controls showed DFMO had no antitumor effect but was well tolerated, MGBG retarded growth rate significantly but resulted in drug deaths in over 50% of the animals, and the combination of DFMO and MGBG resulted in rapid decline in tumor growth rates after 5 to 9 days of treatment with reduced toxicity. At 21 days, or death, 38 of 60 (63%) rats had no viable tumor on histologic study, whereas tumor was present in each of the animals in the other groups. Alpha-difluoromethylornithine increased the intracellular uptake of MGBG and potentiated the antigrowth activity of MGBG on a hormone refractory rat prostatic tumor with less toxicity than MGBG alone.     

Combined Use of α-Difluoromethylornithine and an Inhibitor of S-Adenosylmethionine Decarboxylase in Mice Bearing P388 Leukemia or Lewis Lung Carcinoma

The antitumor and antimetastatic effects of α-difluoromethylornithine (DFMO), an inhibitor of ornithine decarboxylase, combined with an inhibitor of S-adenosylmethionine decarboxylase, either methylglyoxal bis(guanylhydrazone) (MGBG) or ethylglyoxal bis(guanylhydrazone) (EGBG), were studied in mice bearing P388 leukemia or Lewis lung carcinoma. Although EGBG is a more specific inhibitor of polyamine biosynthesis than the widely used MGBG, the antitumor effect of the DFMO-EGBG combination on P388 leukemia-bearing mice was less than that of the DFMO-MGBG combination. The prolongation of survival time by the DFMOC1000 mg/kg)-MGBG(25 mg/kg) combination was 2.65-fold, while that of the DFMO(1000 mg/kg)-EGBG(50 mg/kg) combination was 1.34-fold. When mice were fed a polyamine-deficient diet, stronger antitumor effects were exerted; the prolongation of survival time by the DFMO-MGBG and the DFMO-EGBG combinations was 2.89-fold and 2.03-fold, respectively. The antitumor effect of combined use of the two polyamine antimetabolites with mice on normal and polyamine-deficient diets correlated with a decrease of polyamine charge contents in the tumor cells. The above in vivo results were confirmed clearly in the KB cell culture system. The antimetastatic activity of DFMO on Lewis lung carcinoma-bearing mice was strengthened by the addition of MGBG or EGBG. The antimetastatic activity of the DFMO-MGBG or DFMO-EGBG combination did not parallel the polyamine charge contents in the primary tumor and blood.     

Urinary polyamine levels in cancer patients treated with D,L-alpha-difluoromethylornithine, an inhibitor of polyamine biosynthesis

The polyamine biosynthesis inhibitor D,L-alpha-Difluoromethylornithine hydrochloride monohydrate (DFMO) has cytostatic and cytotoxic effects against various human tumor cell lines in vitro. We measured levels of the polyamines putrescine and spermidine in the urine of cancer patients undergoing "conventional" chemotherapy in a two-arm randomized phase I-II study with and without additional DFMO administered orally at a dose of 1.7 g/sq.m. t.i.d. The study group included 38 patients with carcinoma of the breast, stomach, prostate, or female genital organs or metastatic carcinoma of unknown origin. A control group of 32 patients with similar malignancies received "conventional" chemotherapy without DFMO. Polyamine levels were determined periodically in the urine of all patients. In DFMO-treated patients, a significant decrease in putrescine and spermidine levels was observed after 3 weeks of DFMO therapy (the first time point evaluated) that usually persisted throughout the course of treatment. Significant differences in polyamine levels between DFMO-treated and control patients were observed for patients in remission. Less significant differences were noted, however, for patients with static or progressive disease between DFMO-treated and control groups. DFMO activity appears to be reflected by a long-term decrease in urinary polyamine levels.     

Synergistic antileukemic effect of two polyamine synthesis inhibitors. Host survival and cell-cycle kinetic analysis

alpha-Difluoromethylornithine (DFMO), an enzyme-activated irreversible inhibitor of ornithine decarboxylase, was used alone and in combination with multiple doses of methylglyoxal-bis(guanylhydrazone) (MGBG) to treat mice with systemic L1210 leukemia. Used as a single agent (administered p.o. as a 3% solution in tap water), DFMO exerted a weak therapeutic effect against this tumor. The therapeutic effect of MGBG (administered i.p. at 50 mg/kg/day) was only slightly better. However, 1-3 days of pretreatment with DFMO strongly potentiated the effect of MGBG treatment. Thus, mice treated with the combination exhibited an increase in life span of up to 138%. The prolonged survival of leukemic mice treated with a combination of DFMO and MGBG was associated with inhibition of polyamine synthesis and a marked decrease in the spermidine and spermine content of the tumor cells as compared to untreated controls. As a consequence, there was a continuous decrease in the S- and G2-phase fractions with a concomitant increase in G1. Used singly, DFMO and MGBG had no significant effect on the cell-cycle distribution. The effects of the combination of DFMO and MGBG on the cell-cycle distribution are consistent with the contention that polyamine deficiency primarily interferes with initiation of DNA synthesis. However, the possibility that selective S-phase kill partly contributes to this change in cell-cycle distribution cannot be excluded.     

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.     

Phase I–II clinical trial with alpha-difluoromethylornithine—An inhibitor of polyamine biosynthesis

Alpha-difluoromethylornithine (DFMO) is an enzyme-activated, irreversible inhibitor of ornithine decarboxylase, the first enzyme in the synthesis of the polyamines putrescine, spermidine and spermine. DFMO has been shown to have a cytostatic and cytotoxic effect against various human tumor cell lines. The present study was designed to evaluate the toxicity and efficacy of this compound when administered orally at a dose of 1.7 g/m sq. t.i.d. added to conventional chemotherapy to 38 patients with carcinoma of the breast, stomach, prostate, female genital organs or metastatic carcinoma of unknown origin. A control group of 32 patients with similar malignancies received conventional chemotherapy only.Gastrointestinal, hematologic and biochemical abnormalities caused by DFMO were negligible. Reasonable ototoxicity was the major toxic effect caused by DFMO and resulted in discontinuation of therapy in 6 of 38 patients (15.8%). No differences in disease progression were seen between those patients receiving DFMO plus conventional chemotherapy and those receiving only conventional chemotherapy.     

Inhibition of the development of metastatic squamous cell carcinoma in protein kinase C epsilon transgenic mice by alpha-difluoromethylornithine accompanied by marked hair follicle degeneration and hair loss

The role of 12-O-tetradecanoylphorbol-13-acetate (TPA)-stimulated polyamine biosynthesis in the development of metastatic squamous cell carcinoma (mSCC) in protein kinase C epsilon (PKC epsilon) transgenic mice was determined. TPA treatment induced epidermal ornithine decarboxylase (ODC) activity and putrescine levels approximately 3-4-fold more in PKC epsilon transgenic mice than their wild-type littermates. Development of mSCC by the 7,12-dimethylbenz(a)anthracene (100 nmol)-TPA (5 nmol) protocol in PKC epsilon transgenic mice was completely prevented by administration of the suicide inhibitor of ODC alpha-difluoromethylornithine (DFMO, 0.5% w/v) in the drinking water during TPA promotion. However, DFMO treatment led to marked hair loss in PKC epsilon transgenic mice. DFMO treatment-associated hair loss in PKC epsilon transgenic mice was accompanied by a decrease in the number of intact hair follicles. These results indicate that TPA-induced ODC activity and the resultant accumulation of putrescine in PKC epsilon transgenic mice are linked to growth and maintenance of hair follicles, and the development of mSCC. Severe hair loss observed in PKC epsilon transgenic mice on DFMO during skin tumor promotion has not been reported before in the prevention of cancer in other animal models or in human cancer prevention trials.     

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.     

Putrescine-dependent invasive capacity of rat ascites hepatoma cells.

The effects of inhibitors of polyamine synthesis on the invasive capacity of rat ascites hepatoma (LC-AH) cells were examined by in vitro assay of penetration of the LC-AH cells through a monolayer of calf pulmonary arterial endothelial (CPAE) cells. Pretreatment of LC-AH cells with alpha-difluoromethylornithine (DFMO), an inhibitor of ornithine decarboxylase, before seeding them onto a CPAE cell monolayer and culturing them for 24 h in the absence of DFMO decreased the number of penetrating tumor cells time and dose dependently (about 35% of the maximal inhibition) without affecting their viability or proliferative activity. DFMO treatment caused a marked decrease in the intracellular level of putrescine but not of spermidine or spermine. The DFMO-induced decreases in invasive capacity and putrescine level were almost completely reversed by the addition of putrescine to the medium during pretreatment with DFMO or invasion assay but were not affected by exogenous spermidine or spermine. No change in the invasive capacity was observed when the CPAE cells were treated with DFMO and the LC-AH cells with methylglyoxal-bis(guanylhydrazone), an inhibitor of S-adenosylmethionine decarboxylase, which depressed the spermidine and spermine levels but increased the putrescine level in the LC-AH cells. These results suggest that intracellular putrescine modulates the in vitro invasive capacity of LC-AH cells.     

Antitumor effects of two polyamine antimetabolites combined with mitomycin C on human stomach cancer cells xenotransplanted into nude mice

The antitumor effects of alpha-difluoromethylornithine (DFMO), methylglyoxal-bis-guanylhydrazone (MGBG) and mitomycin C (MMC), administered separately or in various combinations, on human stomach cancer cells xenotransplanted into BALB/c nude mice were studied using the protocol of Battelle's Columbus Laboratories (Ovejera et al., 1978). DFMO (1,000 mg/kg in 2 divided doses) and MGBG (50 mg/kg) were given intraperitoneally (i.p.) for 7 consecutive days from the time when the tumor weighed about 100 mg. MMC (2 mg/kg) was given i.p. every other day from the same time. Animals treated with either DFMO or MGBG alone displayed tumor growth comparable to that seen in untreated controls. In mice treated with DFMO plus MGBG with or without MMC, or in mice treated only with MMC, tumor growth was significantly lower than in untreated mice. In the group which received only combined DFMO/MGBG there was a rapid regrowth of the tumor after termination of therapy. Tumor putrescine levels decreased within 4 days following the administration of DFMO; however, spermidine levels did not decline with either DFMO or MGBG treatment even after 7 days. When combined DFMO/MGBG was given, there was a significant decline in spermidine levels 7 days after the initiation of treatment. In contrast, when MMC alone was administered, putrescine and spermidine levels in the tumor did not differ from those in control mice. Spermine decreased markedly in tumor with the combined administration of DFMO/MGBG as well as with combined DFMO/MGBG/MMC, but decreased only slightly when MMC alone or MMC plus either DFMO or MGBG was administered. By the 7th treatment day, DNA biosynthesis in the tumor had dropped markedly in all groups except those receiving DFMO or MGBG alone.     

Individual and combined effects of alpha-difluoromethylornithine and ovariectomy on the growth and polyamine milieu of experimental breast cancer in rats

Despite considerable evidence suggesting a critical role of polyamines in the hormonal control of breast cancer growth in vitro, their role in in vivo tumor growth is not established. In these experiments, we evaluated the individual and combined effects of the polyamine biosynthesis inhibitor alpha-difluoromethylornithine (DFMO) and ovariectomy on the growth and cellular levels of ornithine decarboxylase (ODC) and polyamines of N-nitrosomethylurea-induced rat mammary tumors. Despite a similar suppressive effect on ODC activity, the two treatments had a different effect on polyamine levels. As expected, DFMO selectively suppressed putrescine, whereas spermidine and spermine levels were minimally or not affected at all. Since quantitatively putrescine contributes the least to overall polyamine pools, the DFMO effect on this latter parameter was modest. In contrast, ovariectomy, by suppressing the more abundant spermidine and spermine, produced a more profound suppression of total polyamine pools. This finding is in agreement with the notion that hormones not only control ODC activity, but also other enzymes involved in the synthesis of the distal polyamines. Ovariectomy was also more potent than DFMO administration in inhibiting N-nitrosomethylurea-induced mammary tumor growth. No major additive/synergistic effects were observed between DFMO and ovariectomy on tumor growth and cellular levels of ODC activity and polyamines. DFMO administration lowered the tumor level of progesterone receptors and appeared to potentiate the suppressive effect of ovariectomy. In contrast, neither treatment, alone or in combination, altered tumor levels of estrogen receptors. DFMO administration did not affect circulating levels of estradiol and prolactin or uterine and ovarian weights, thus suggesting that its effects were not indirectly mediated through alterations of the endocrine milieu of the host.     

Inhibition of ornithine decarboxylase with 2-difluoromethylornithine: reduced incidence of dimethylhydrazine-induced colon tumors in mice

2-Difluoromethylornithine (DFMO) was administered to 1,2-dimethylhydrazine (DMH)-treated mice to reduce colonic polyamine levels and mucosal hyperplasia. Mice received 1% DFMO in drinking water throughout the experiment and were given injections of DMH (20 mg/kg) weekly for 28 weeks. DFMO inactivated 93% of colonic ornithine decarboxylase activity. Although DMH treatment did not induce colonic ornithine decarboxylase activity by Week 28, the putrescine content was increased 31% in DMH-treated mice (p less than 0.01). Concurrent treatment with DFMO depressed putrescine content (42 to 63%) and spermidine content (27 to 38%), but it increased spermine content (18 to 22%). At Week 28 of treatment with DMH alone, RNA content was increased 8.6% (p less than 0.01), DNA content 10% (p less than 0.01), DNA specific activity 24% (p less than 0.01), and crypt depth 20% (p less than 0.01), but not in mice receiving DMH and DFMO. At 28 weeks, 13 of 17 mice (76%) treated with DMH alone had histologically confirmed colon cancers; of mice treated with DMH and DFMO, two of 18 (11%) had colonic tumors. Throughout the experiment, 50 colon cancers developed in 16 DMH-treated mice (mean, 3.12 tumors/mouse); three mice treated with DMH and DFMO developed three colon cancers total (p less than 0.001). Reduction of colonic polyamine levels after DFMO treatment prevents proliferative changes induced by DMH and reduces the incidence of tumors.     

Alpha-difluoromethylornithine as treatment for metastatic breast cancer patients.

DFMO (alpha-difluoromethylornithine) is an oral irreversible inhibitor of ornithine decarboxylase, the first rate-limiting enzyme in polyamine synthesis. DFMO has been shown to have antiproliferative effects against several human cancers, and some studies have suggested that DFMO may have pro-apoptotic and anti-invasive properties as well. DFMO is well tolerated with minimal toxicity but has been associated with ototoxicity with prolonged daily administration. We conducted a Phase I/II tolerability, pharmacokinetic, and efficacy study of high-dose DFMO in metastatic breast cancer patients. Twenty-one patients were treated with 4800 mg of DFMO p.o. three times a day for 14 days, followed by a 2-week drug holiday on a 28-day cycle. Urinary polyamine and blood DFMO levels were measured at multiple time points during therapy. High-dose DFMO was well tolerated, and no clinically significant ototoxicity was noted. No patient achieved an objective antitumor response; however, one patient with heavily pretreated liver metastases has achieved stable disease for 18 months to date on DFMO. Putrescine, spermine, and spermidine urinary levels were suppressed with DFMO treatment and remained low during the 2-week drug holiday. High-dose DFMO on a schedule of 2 weeks on treatment followed by 2 weeks off is well tolerated, is not associated with ototoxicity, and leads to sustained suppression of urinary polyamine levels. Although not an active cytotoxic agent for metastatic breast cancer, the intriguing prolonged growth arrest of liver metastases in one patient highlights the potential clinical growth inhibitory properties of DFMO. We believe that DFMO is worthy of study as adjuvant therapy in primary breast cancer patients and as a chemopreventive agent.     

Combined efficacy of polyamine antimetabolites and cis-diamminedichloroplatinum

The combined antitumor effects of the polyamine antimetabolites, alpha-difluoro methylornithine (DFMO) and methylglyoxal-bis-guanylhydrazone (MGBG), with CDDP were studied using human gastric cancer cells xenotransplanted into nude mice. DFMO (1000 mg/kg in two divided doses) and MGBG (50 mg/kg) were given IP for six consecutive days from the time when the xenotransplanted tumor weighted about 100 mg, and CDDP (3.0 mg/kg) was given IP every other day from the same time. Animals treated with DFMO plus MGBG with or without CDDP as well as with CDDP only displayed suppressed tumor growth, compared to untreated mice. In mice treated with these three drugs, however, tumor growth was rather rapid compared to those treated with CDDP only, although tumoral CDDP levels in animals given DFMO, MGBG and CDDP were higher than those given CDDP only. When DFMO, MGBG and CDDP or DFMO and MGBG were administered, tumoral spermidine and spermine levels decreased markedly. On the other hand, tumor DNA biosynthesis in the CDDP only group dropped markedly 24 hours after the termination of therapy. These results suggest that an alteration in the DNA structure caused by polyamine deficiency may prevent cross-link formation in DNA by CDDP.     

Glucocorticoids and polyamine inhibitors synergize to kill human leukemic CEM cells

Glucocorticoids are well-known apoptotic agents in certain classes of lymphoid cell malignancies. Reduction of intracellular polyamine levels by use of inhibitors that block polyamine synthesis slows or inhibits growth of many cells in vitro. Several such inhibitors have shown efficacy in clinical trials, though the toxicity of some compounds has limited their usefulness. We have tested the effects of combinations of the glucocorticoid dexamethasone (Dex) and two polyamine inhibitors, difluoromethylornithine (DFMO) and methyl glyoxal bis guanylhydrazone (MGBG), on the clonal line of human acute lymphoblastic leukemia cells, CEM-C7-14. Dex alone kills these cells, though only after a delay of at least 24 hours. We also evaluated a partially glucocorticoid-resistant c-Myc-expressing CEM-C7-14 clone. We show that Dex downregulates ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine synthesis. Pretreatment with the ODC inhibitor DFMO, followed by addition of Dex, enhances steroid-evoked kill slightly. The combination of pretreatment with sublethal concentrations of both DFMO and the inhibitor of S-adenosylmethionine decarboxylase, MGBG, followed by addition of Dex, results in strong synergistic cell kill. Both the rapidity and extent of cell kill are enhanced compared to the effects of Dex alone. These results suggest that use of such combinations in vivo may result in apoptosis of malignant cells with lower overall toxicity.