Differential sensitivity of various human tumors to inhibition of polyamine biosynthesis in vivo

Polyamines are essential for normal and neoplastic growth. Ornithine decarboxylase (ODC) is the first and rate-limiting enzyme in the polyamine biosynthetic pathway. alpha-Difluoromethylornithine (DFMO) is an enzyme-activated irreversible inhibitor of ODC, and a known anti-neoplastic agent. The purpose of this study was to examine the susceptibility of various human cancers to inhibition by DFMO in vivo. We have studied three human pancreatic adenocarcinomas, designated CAV, SKI, and PGER, two human colon adenocarcinomas (LS-180 and WIDR), and three metastatic cell lines of a human gastric adenocarcinoma (BHM, BMM, BLM) that were growing in congenitally athymic (nude) Balb/c mice. Mice bearing each tumor were divided into two groups; one group served as controls and the other group received DFMO 3% in drinking water. Tumor growth and weight, and content of DNA, RNA, protein and polyamines were determined and correlated. DFMO significantly inhibited the growth of three of the three gastric tumors, two of the three pancreatic tumors and neither of the two colon tumors. The tumor content of DNA, RNA and protein exhibited a pattern that was parallel to tumor growth. The tumor polyamine concentration did not correlate with sensitivity to DFMO. These findings provide clear evidence for important differences in the sensitivity of various human cancers to growth inhibition by DFMO and indicate that endogenous polyamine levels alone do not predict the sensitivity of the tumors to DFMO.     

Therapy with inhibitors of polyamine biosynthesis in refractory prostatic carcinoma. An experimental and clinical study

Transplantable prostate adenocarcinoma were treated with polyamine biosynthetic inhibitors. alpha-difluoromethylornithine (alpha-DFMO), an inhibitor of ornithine decarboxylase and by s-methylglyoxal-bisguanylhydrazone (MGBG), an inhibitor of s-adenosylmethionine decarboxylase. The therapeutic regimen of 0.8-1.11 g/kg DFMO reduced the tumor growth by 40% whilst the combination with 10.5 mg/kg MGBG completely destroyed the prostate adenocarcinomas in the tumor-bearing animals. The polyamine content of spermidine and spermine in the cancerous tissues is significantly lower whereas the putrescine levels remain unchanged. The MGBG therapy distinctly stimulates the activity of ornithine decarboxylase and increases the putrescine concentration up to toxic levels. The application of alpha-DFMO prevented the toxic accumulation of putrescine and allowed higher doses of MGBG. Clinical trials with polyamine antimetabolites appeared useful due to pathological polyamine excretion of patients with metastatic prostate cancer. The therapy with 0.2-0.3 g/kg DFMO in patients with hormone-resistent prostate cancer and metastasis displayed a moderate anti-tumor activity following 2 months additional treatment. High levels of side effects, however, were registered and were similar to those of other cytotoxic compounds. A combined therapy with DFMO/MGBG in a patient with metastatic anaplastic prostate cancer did not improve the survival rate but showed regressive effects of the histological pattern.     

Alpha-difluoromethylornithine-induced inhibition of growth of autochthonous experimental colonic tumors produced by azoxymethane in male F344 rats

Ornithine decarboxylase, the first regulatory enzyme in polyamine biosynthesis, is inhibited by alpha-difluoromethylornithine (DFMO), a specific enzyme-activated irreversible inhibitor. DFMO has been shown previously to inhibit experimental colonic tumorigenesis in rodents given the large bowel carcinogen azoxymethane or dimethylhydrazine. Therefore, we assessed the effects of DFMO on growth of established autochthonous experimental colonic tumors. Ten-wk-old male F344 rats were given 10 weekly s.c. injections of azoxymethane, 10 mg/kg. Starting 5 wk after the last dose, colonoscopy to the splenic flexure was performed weekly with a pediatric fiberoptic bronchoscope. When a tumor was visualized, its growth was assessed by computer image analysis of weekly colonoscopic photographs which included a scale. After two measurements for baseline tumor growth, the tumor-bearing rats were assigned in predetermined alternating sequence to the DFMO group (n = 26) or control group (n = 28). DFMO, 30 mg/ml (3%), was given in drinking water for 4 wk, resulting in mean weekly intake of 16 +/- 1 (SE) to 18 +/- 1 mg of DFMO/g of body weight. Control rats were pair-fed, resulting in reduced body weights comparable to DFMO rats. DFMO dramatically inhibited tumor growth, beginning in the first week of administration: mean tumor volume of DFMO rats reached only 7.0 +/- 2.0 mm3 compared with 17.4 +/- 3.2 mm3 in controls (P less than 0.02); and tumors in three DFMO rats disappeared. Mean change in tumor volume in DFMO rats was less than controls during all 4 wk of administration, although there was a suggestion of escape from DFMO suppression of tumor growth in the last 2 wk. At necropsy, tumor ornithine decarboxylase activity was 115 +/- 22 pmol/h/mg of protein in DFMO rats as compared with 842 +/- 576 in controls. There was a suggestion of greater tumor desmoplasia in DFMO rats, but tumor differentiation, depth of invasion, inflammation, and labeling index with tritiated thymidine showed no statistically significant differences between the DFMO and control groups. Our findings suggest that (a) ornithine decarboxylase plays a key role in growth of autochthonous experimental colonic tumors, and (b) DFMO may have potential for chemotherapy and chemoprophylaxis of colorectal neoplasms in human beings.     

Combined therapy of polyamine antimetabolite and nitrosourea in human gastric cancer

Antitumor therapy using the polyamine antimetabolites, alpha-difluoromethylornithine (DFMO) and methylglyoxal-bis-guanylhydrazone (MGBG), combined with ACNU was studied in human gastric cancer xenotransplanted into nude mice. DFMO 1,000 mg/kg (in two divided doses) and MGBG 50 mg/kg were given i.p. for 6 successive days from the time when the xenotransplanted tumor weighed about 100 mg, and ACNU 20 mg/kg was given i.p. every other day from the same time. Antitumor efficacy was assessed by the time course of tumor weight as well as of DNA biosynthesis and polyamine levels in tumor tissue. Tumor weight was estimated using Battelle's Columbus Institute protocol and DNA biosynthesis was assayed biochemically by 3H-TdR injection at a prescribed interval after termination of therapy. Furthermore, tumoral polyamine levels were assayed by HPLC. This three-drug regimen showed a favorable antitumor effect, compared to those of the other two therapies with DFMO plus MGBG as well as ACNU only. These data suggest that this combined regimen may have a synergistic efficacy judging from the action mechanisms of these three drugs.     

Chemoprevention of prostate carcinogenesis by alpha-difluoromethylornithine in TRAMP mice

Development of effective chemopreventive agents for human consumption requires conclusive evidence of their efficacy in animal models that have relevance to human diseases. Transgenic adenocarcinoma mouse prostate (TRAMP) is an excellent model of prostate cancer that mimics progressive forms of human disease inasmuch as 100% of males develop histological PIN by 8-12 weeks of age that progress to adenocarcinoma with distant site metastases by 24-28 weeks of age. In these animals, ornithine decarboxylase (ODC) activity (>3-fold) as well as protein expression (>4-fold) was found to be markedly higher in the dorsolateral prostate as compared with the nontransgenic littermates, suggesting their suitability to determine the chemopreventive effect of alpha-difluoromethylornithine (DFMO), an enzyme-activated irreversible inhibitor of ODC, against prostate cancer. Using male TRAMP mice, we studied the effect of oral consumption of DFMO on development of prostate carcinogenesis and surrogate end point biomarkers related to prostate cancer progression. In two independent experiments, each consisting of 8 animals on test, the cumulative incidence of prostatic cancer development at 28 weeks of age in 16 untreated TRAMP mice was 100% (16 of 16), whereas 94% (15 of 16) and 69% (11 of 16) of the animals exhibited distant site metastases to lymph nodes and lungs, respectively. Oral consumption of 1% DFMO (w/v) in the drinking water to TRAMP mice from 8 to 28 weeks of age resulted in a significant decrease in (a) weight (59%) and volume (66%) of prostate, (b) genitourinary weight (63%), and (c) ODC enzyme activity (52%) in the dorsolateral prostate. Importantly, in none of the DFMO-fed TRAMP mice were any distant metastases to lymph node and lungs observed. Furthermore, DFMO treatment resulted in the marked reduction in the protein expression of proliferation cell nuclear antigen, ODC, and probasin in the dorsolateral prostate. The protein expression of antimetastases markers, i.e., E-cadherin and alpha- and beta-catenin, was found to be restored in DFMO-fed animals as compared with the non-DFMO-fed mice. These chemopreventive effects of DFMO were further confirmed by immunohistochemical analysis of the dorsolateral prostate. Histological analysis of the dorsolateral prostate of DFMO-fed animals displayed marginal epithelial stratification, a small number of cribriform structures, elongated hyperchromatic epithelial nuclei, and a significant increase in apoptotic index. Non-DFMO-fed animals, on the other hand, displayed extensive epithelial stratification with profound cribriform structures accompanied with marked thickening, remodeling, and hypercellularity of the fibromuscular stroma. In nontransgenic littermates fed with DFMO, no significant alterations in the above parameters were evident. These data demonstrate that ODC represents a promising and rational target for chemoprevention of human prostate cancer and that TRAMP mice are excellent models for screening of novel drugs and chemopreventive regimens for potential human use.     

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.     

Effects of alpha-difluoromethylornithine on the growth of experimental Wilms' tumor and renal adenocarcinoma

Because polyamines are essential for cellular growth and differentiation, and because human renal carcinomas have spermidine levels that are higher than those in normal renal tissue, effects of 2-difluoromethylornithine (DFMO) on the growth of experimental renal tumors were investigated. DFMO is a specific enzyme-activated irreversible inhibitor of ornithine decarboxylase, the rate-limiting enzyme controlling polyamine biosynthesis. DFMO (2%) in drinking water was administered to BALB/c mice with intrarenal transplants of a renal adenocarcinoma cell suspension and to Wistar/Furth rats with s.c. transplants of a Wilms' tumor. At 28 days, renal carcinomas in DFMO-fed mice weighed 72% less than those in control animals (p less than 0.001). Wilms' tumor weight was not affected by DFMO feeding. DFMO caused 72 to 75% inactivation of ornithine decarboxylase activity and reduced putrescine levels in renal carcinoma and Wilms' tumor, reduced spermidine levels in Wilms' tumor, and apparently raised spermine levels in the latter as a consequence. DNA content was not affected by DFMO feeding. The mean number of lung metastases in DFMO-fed, renal carcinoma-bearing mice was 0.1 and in controls was 1.4 (p less than 0.001). DFMO feeding increased survival of mice bearing renal carcinomas by 3.0 +/- 0.8 (S.E.) days (p less than 0.05), i.e., from 30.5 +/- 0.8 days to 33.5 +/- 1.2 days. DFMO did not affect the growth of Wilms' tumor; however, in renal adenocarcinoma, it reduced growth, prevented lung metastases, and increased survival.     

Interspecies analysis of the chemopreventive efficacy of dietary alpha-difluoromethylornithine

The anticarcinogenic efficacy of the polyamine biosynthesis inhibitor, alpha-difluoromethylornithine (DFMO), was assessed in three rodent models of human epithelial cancer. In DMBA-induced female, Sprague-Dawley rats, DMFO treatment (3.2 or 6.4 g/kg diet) for 180 days significantly inhibited mammary carcinogenesis and reduced tumor-related intercurrent mortality compared to untreated controls. In male, C57BL/6x DBA/2F1 mice induced with N-butyl-N(4-hydroxybutyl)nitrosamine (OH-BBN), DFMO treatment (2 or 4 g/kg diet) concurrent with the period of carcinogen administration significantly reduced the incidence and severity of urinary bladder carcinomas. In methylnitrosourea (MNU)-induced male Syrian golden hamsters, DFMO (3.2 g/kg diet) numerically reduced the incidence and size of tracheal carcinoma relative to untreated controls. DFMO-mediated toxicity was not evident in any of the animals on study, although a slight reduction in mean body weight gain was evident in rats and mice.     

Inhibitory action of alpha-difluoromethylornithine on N-butyl-N-(4-hydroxybutyl)nitrosamine-induced rat urinary bladder carcinogenesis

We previously have shown that urine components capable of stimulating ornithine decarboxylase activity of urothelium can enhance rat urinary bladder carcinogenesis, and that alpha-difluoromethylornithine (DFMO), an irreversible inhibitor of ornithine decarboxylase, suppresses carcinogen-initiated rat urinary bladder carcinogenesis. The present investigation was conducted to determine whether DFMO's suppressive effect is stage specific during carcinogenesis and whether the suppressive effect lasts with its continued use. Following initiation with 0.05% N-butyl-N-(4-hydroxybutyl)-nitrosamine in drinking water for 6 wk, male Fischer 344 rats initially weighing 125 to 150 g were randomly divided into two groups, the first receiving 0.2% DFMO in drinking water ad libitum and the second receiving tap water only. Groups of animals were killed at regular intervals until the completion of the experiment at 75 wk. The effect of DFMO was evaluated by monitoring the incidence of tumors, the mean number of tumors per rat, the mean volume of individual tumors, and the mean total tumor volume per rat. The results showed that continuous treatment with DFMO significantly reduced tumor formation until 60 wk (P less than 0.017). The effect was only of borderline significance (0.017 less than P less than 0.035) at 75 wk. Discontinuation of DFMO treatment at 40 wk resulted in the loss of protective effect in all comparisons except for the borderline effect on the tumor number and total tumor volume per rat. DFMO had no significant effect on the incidence or development of preneoplastic early lesions. Mucosal polyamine (spermidine and spermine) levels were reduced and correlated well with the reduction in tumor growth, suggesting that the reduction in tumor growth rate by DFMO may be due to its ability to reduce polyamine levels in urothelium. There were no side effects attributable to DFMO treatment. DFMO may be a useful chemopreventive agent to retard the recurrence of human superficial bladder cancer.     

Role of polyamines in the growth of hormone-responsive experimental breast cancerin vivo

Summary We have provided evidence for a critical role of polyamines in the growth of the hormone-responsive N-nitrosomethyl-urea (NMU)-induced rat mammary tumorin vitro. The present experiments were designed to test whether polyamines are involved in the growth of this experimental tumorin vivo. To test this hypothesis, groups of rats bearing NMU-induced mammary cancers were randomly allocated to receive no treatment or escalating doses of the polyamine biosynthesis inhibitor -difluoromethyl-ornithine (DFMO) (0.5%, 1%, 2%, 3% in drinking water). DFMO inhibited tumor growth in a dose-dependent fashion and consistently reduced tumor putrescine level. To evaluate the time dependency of this effect, additional groups of rats received either no treatment or 2% DFMO for 3, 7, 14, and 21 days. At all times DFMO suppressed tumor putrescine level as well as spermidine to spermine ratio. Finally, exogenous administration of putrescine (200 mg/kg/i.p./day × 21 days) given concomitantly with DFMO restored tumor growth, partially repleted tumor putrescine level, and raised the spermidine to spermine ratio to control levels. Putrescine, given alone, had no significant effect on either tumor polyamine levels or tumor growth. Except for modest weight loss, no major toxicity was encountered. These results indicate that polyamines play an important role in the growth of the NMU rat mammary tumorin vivo. The interaction between polyamines and hormones in supporting NMU mammary tumor growthin vivo remains to be elucidated.     

Alpha-difluoromethylornithine induction of apoptosis: a mechanism which reverses pre-established cell proliferation and cancer initiation in esophageal carcinogenesis in zinc-deficient rats.

Alpha-difluoromethylornithine (DFMO) is an irreversible inhibitor of ornithine decarboxylase, the first enzyme in polyamine synthesis. Previous work showed simultaneous administration of DFMO and a zinc-deficient (ZD) diet to weanling rats from the beginning inhibited the onset of zinc-deficiency-induced esophageal cell proliferation by activating apoptosis and reduced the incidence of N-nitrosomethylbenzylamine (NMBA)-induced esophageal cancer. Because esophageal cancer initiation by NMBA is very rapid in ZD rats, this study determined whether DFMO is effective in preventing esophageal carcinogenesis when administered after the establishment of a carcinogenic environment. Weanling rats were given a ZD diet for 5 weeks to establish sustained increased esophageal cell proliferation and then an intragastric dose of NMBA. Thereafter, 20 rats were switched to DFMO-containing water while nine control ZD animals remained on deionized water; all of the animals continued on the ZD diet. Esophagi were collected 15 weeks later. The upper portion was processed for immunohistochemical analysis of cell proliferation, apoptosis, and expression of related genes, and the lower was processed for polyamine content. DFMO substantially reduces the levels of esophageal putrescine and spermidine and esophageal tumor incidence from 89 to 10% in ZD rats. Importantly, DFMO-treated ZD esophagi display increased rate of apoptosis accompanied by intense bax expression and greatly reduced cell proliferation by proliferating cell nuclear antigen expression. In addition, the p16(ink4a)/retinoblastoma control at G1 to S, deregulated in ZD esophagi, is restored after DFMO treatment. These results demonstrate that DFMO, a highly effective chemopreventive agent in esophageal carcinogenesis, reverses and counteracts esophageal cell proliferation/cancer initiation in ZD animals by way of stimulating apoptosis.     

Combination therapy with 2-difluoromethylornithine and a polyamine transport inhibitor against murine squamous cell carcinoma

Using a recently developed autochthonous mouse model of squamous cell carcinoma (SCC), a combination therapy targeting polyamine metabolism was evaluated. The therapy combined 2-difluoromethylornithine (DFMO), an inhibitor of ornithine decarboxylase (ODC), and MQT 1426, a polyamine transport inhibitor. In 1 trial lasting 4 weeks, combination therapy with 0.5% DFMO (orally, in the drinking water) and MQT 1426 (50 mg/kg i.p., bid) was significantly more effective than with either single agent alone when complete tumor response was the endpoint. In the combination group, 72% of SCCs responded completely vs. 21 and 0% for DFMO and MQT 1426, respectively. A second trial involved a 4-week treatment period followed by 6 weeks off-treatment. With apparent cures as an endpoint, combination therapy was again more effective than either agent alone: a 50% apparent cure rate was observed in the combination group vs. 7.7% in the DFMO group. MQT 1426 had no inhibitory effect on SCC ODC activity nor did it enhance the inhibition by DFMO, but SCC polyamine levels declined more rapidly when treated with combination therapy vs. DFMO alone. The apoptotic index in SCCs was transiently increased by combination therapy but not by DFMO alone. Thus, targeting both polyamine biosynthesis and polyamine transport from the tumor microenvironment enhances the efficacy of polyamine-based therapy in this mouse model of SCC.     

Follow-up of tumor development in the colons of living rats and implications for chemoprevention trials: assessment of aspirin-difluoromethylornithine combination

We aimed to establish a reliable procedure allowing the follow-up of tumor development by computed tomographic (CT) colonography in an animal model of colon carcinogenesis in order to assess the chemopreventive efficacy of aspirin and difluoromethylornithine (DFMO) given in combination. Fischer rats received an intraperitoneal injection (25 mg/kg) of dimethylhydrazine (DMH) once a week for two weeks in order to initiate colon carcinogenesis. Five months after the last injection of DMH, a first CT colonography was performed and rats were then randomly separated into two groups (control and experimental). The experimental group received a 0.1% mixture of aspirin and DFMO in drinking water. CT colonography was performed at 6, 7 and 8 months. Data showed a precise correlation between location and size of tumors found at autopsy and those detected by CT colonography at 8 months. All tumors were also detected on the CT views obtained previously. Animals of the aspirin/DFMO group exhibited an inactivation of ornithine decarboxylase, a key enzyme in polyamine biosynthesis, and a two-fold reduction in the prostaglandin E2 content of the colonic mucosa (p<0.01). In rats with tumors at the start of the aspirin/DFMO treatment, a significant slow-down of tumor development was observed. In contrast, in rats where no tumors were detected at the start of the treatment, tumor formation was inhibited. Our data show that CT colonography represents a reliable method to assess in a living animal the efficacy of chemopreventive agents.     

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.     

Effect of tamoxifen and D,L-2-difluoromethylornithine on the growth, ornithine decarboxylase activity and polyamine content of mammary carcinomas induced by 1-methyl-1-nitrosourea

The effect of combined treatment with D,L-2-difluoromethylornithine(DFMO) and tamoxifen on the growth status, ornithine decarboxylase(ODC) activity and polyamine content of established 1-methyl-l-nitrosourea(MNU)-induced mammary tumors was investigated. DFMO treatment,a 0.125% solution provided as drinking water, inhibited therate of tumor occurrence and reduced the number of mammary tumorsinduced by a high dose of MNU (50 mg/kg body weight) duringthe first 120 days post-carcinogen treatment. Tamoxifen wasadministered daily via s.c. injection (25 µ/100 g bodyweight) to tumor-bearing rats in both treatment groups, i.e.control and DFMO-treated, for a 30-day period beginning 120days after carcinogen. Tamoxifen treatment induced tumor regressionbut the percentage of regressing, static or growing tumors wasno different in the presence or absence of DFMO. Whereas themammary tumors of DFMO-treated rats had reduced ODC activityand lower polyamine concentrations in comparison to the tumorsof untreated animals, tamoxifen had no effect on these parametersindependent of its effect on tumor growth status. DFMO did notincrease the efficacy of tamoxifen in inducing tumor regression.     

Combined therapy with polyamine biosynthesis inhibitors and mitomycin C

An attempt was made to analyse tumor growth after cessation of combined therapy with the polyamine biosynthesis inhibitors, alpha-difluoromethylornithine (DFMO) and methylglyoxal-bis-guanylhydrazone (MGBG), as well as mitomycin C (MMC). DFMO 1000 mg/kg, MGBG 50 mg/kg and/or MMC 2 mg/kg were given intraperitoneally to BALB/c nu/nu mice xenotransplanted human gastric cancer, and its growth as well as DNA biosynthesis were measured daily, after cessation of these combined treatments. Histological observation of the tumor was also performed by hematoxylin-eosin staining. The combination with DFMO and MGBG stunted tumor growth during the treatment, but 3 days later its growth and DNA biosynthesis were accelerated distinctly. MMC injection halted tumor growth, and 5 days after termination of MMC injection its growth rate and DNA biosynthesis almost completely recovered. The microscopic findings on the 4th day after termination of MMC injection were similar to those of DFMO + MGBG treatment. The combination DFMO, MGBG and MMC suppressed not only tumor growth during the treatment, but also tumor growth and DNA biosynthesis over 7 days. The histologic observation 4 days later revealed extensive damage.     

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.     

New approaches to cancer chemoprevention with difluoromethylornithine and selenite

New approaches to enhancing D,L-alpha-difluoromethylornithine (DFMO) inhibition of DMBA-induced mammary tumorigenesis were investigated. It is reasoned that perturbation of a second regulatory element in polyamine biosynthesis, i.e., the generation of propylamine groups from S-adenosylmethionine (AdoMet), would potentiate the effectiveness of DFMO. Precursor availability for AdoMet was limited when rats were fed a low methionine diet. The diet of control rats was supplemented with 0.3% methionine. DFMO, when added to the diet at 0.1%, suppressed tumorigenesis, regardless of methionine levels, although its efficacy was significantly enhanced by the low methionine diet. Selenite, another effective chemopreventive agent in this tumor model, also requires AdoMet for its metabolism. However, the anticarcinogenic action of selenite is not compromised by low dietary methionine. The differential sensitivity of DFMO and selenium chemoprevention to low dietary methionine, therefore, provides an opportunity to test for an additive effect. Results of the combination regimen study showed that the incidence and yield of DMBA-induced mammary tumors were significantly lower in the two-agent protocol compared to either DFMO or selenite alone. The mechanism(s) that accounts for the heightened efficacy of combined DFMO and selenite chemoprevention will be discussed.     

Antitumor efficacy of polyamine antimetabolites and mitomycin C under polyamine-free diet

Treatment of nude mice xenografted with human gastric cancer was carried out by polyamine antimetabolites combined with mitomycin C (MMC) and polyamine-free diet. Polyamine antimetabolites, alpha-difluoromethylornithine (DFMO) and ethylglyoxal-bis-guanylhydrazone (EGBG), were given ip in a daily dose of 1,000 mg/kg and 20 mg/kg, respectively, for 6 consecutive days. MMC 2.0 mg/kg was administered every other day. The polyamine-free diet was given from 4 days before start of the treatment through the end of the study. Although the tumor growth rate of the control group given polyamine-free diet was similar to that given normal diet, in the mice treated with EGBG, DFMO plus MMC, the antitumor effect in the polyamine-free diet group was superior to the normal diet group. In comparison with tumor growth suppression due to EGBG plus DFMO or MMC only, the polyamine-free diet group showed better result than the normal diet group to some extent. In mice treated with EGBG, DFMO plus MMC, tumor tissue spermine levels in the polyamine-free diet group were significantly depressed, compared to the normal diet group. Furthermore, marked suppression of DNA biosynthesis was observed in mice given EGBG, DFMO plus MMC together with the polyamine-free diet. These results suggest that combined treatments of polyamine antimetabolites and MMC revealed a marked enhancement of antitumor effects, under conditions of polyamine depletion, which may be responsible for the alteration in DNA structure.     

Phospho-sulindac (OXT-328) combined with difluoromethylornithine prevents colon cancer in mice

The nonsteroidal anti-inflammatory drug (NSAID) sulindac and the ornithine decarboxylase (ODC) antagonist difluoromethylornithine (DFMO), individually and together, are effective inhibitors of colon carcinogenesis. However, chronic use of sulindac is associated with significant side effects. We evaluated the chemopreventive efficacy of phospho-sulindac (P-S, OXT-328), an apparently safe derivative of sulindac, together with DFMO, in HT-29 human colon cancer xenografts. Nude mice were divided into four groups as follows: group 1 received vehicle (corn oil); group 2 received P-S (100 mg/kg/d) by oral gavage; group 3 received DFMO (2% in drinking water); and group 4 received P-S (100 mg/kg/d) by gavage plus DFMO (2% in drinking water; P-S/DFMO). Eighteen days after implantation, compared with controls, tumor volume was inhibited 65.9% by P-S, 52.9% by DFMO, and 70.9% by P-S/DFMO (P < 0.01 for all). P-S/DFMO reduced cell proliferation 27.1% and increased apoptosis 38.9% compared with controls (P < 0.05 for both). Compared with controls, P-S reduced the levels of thioredoxin-1 (Trx-1) and thioredoxin reductase (TrxR), whereas DFMO reduced polyamine content (putrescine and spermidine) and TrxR levels. Importantly, P-S/DFMO decreased putrescine and spermidine levels and the expression of Trx-1, TrxR, and cyclooxygenase (COX) 2. Of these molecular targets, TrxR most consistently correlated with tumor growth. Study results show that P-S/DFMO is an efficacious drug combination for colon cancer prevention and also show the safety of P-S, which may overcome the limiting side effects of conventional sulindac. P-S/DFMO has an intricate mechanism of action extending beyond polyamines and including the thioredoxin system, an emerging regulator of chemoprevention. P-S/DFMO merits further evaluation.