Inhibitory Effect Of DFMO Research Articles

Abstract 808: Chemoprevention of colon cancer by DFMO, sulindac and NO-sulindac administered individually or in combinations in male F344 rats

Abstract Different mode of action exerted by low-dose combination of agents represents a practical approach for improving the chemopreventive efficacy and minimizing unwanted toxicities. Recent clinical studies in high-risk sporadic colorectal cancer patients proven that DFMO combined with COX-1/COX-2 inhibitor, sulindac dramatically reduced polyps formation. Present study designed to address whether i) low-dose combinations DFMO and sulindac would provide the efficacy against rat colon adenocarcinomas in rat model representing human sporadic colon cancers; ii) test the advantages of nitric-oxide releasing (NO)-sulindac would provide colon cancer chemopreventive activity alone or combined with DFMO; and iii) understand the molecular changes associated in colon tumor inhibition by these combinations. Seven-week old, male F344 rats (36 rats/group) were fed the control AIN-76A diet and AOM (15 mg/kg b.w., once weekly for 2 weeks, at the age of 8 and 9 weeks) administered by s.c. Eight weeks after the last carcinogen treatment, rats were fed the diets containing 0 ppm, 500 ppm DFMO, 150 ppm Sulindac, 200 ppm NO-Sulindac individually or in combinations for 40 weeks. Rats were sacrificed and colon tumors were evaluated by histopathologically and colonic tissues were assayed for expression levels of COX-2, iNOS, p21WAF1/CIP1, apoptotic markers, and cell proliferation (BrdU) and ODC enzymatic activity. Bioassay results suggest that administration of 500 ppm DFMO and 150 ppm sulindac significantly suppressed total adenocarcinoma multiplicity by 50% (p<0.003) and 35.4% (p<0.03), respectively; whereas NO-sulindac had no significant effect on AOM-induced colon cancers. Importantly, combination of DFMO with sulindac or NO-Sulindac significantly suppressed colon adenocarcinoma incidence (42%p<0.0004; 32% p<0.001), and multiplicity (58%, p<0.0001; 52%, p<0.0018). We observed greater inhibitory effects of DFMO (63%), sulindac (52%) alone and more pronounced inhibitory DFMO combined with sulindac (78%) on adenocarcinomas with size of >0.5-mm. Colonic adenocarcinomas harvested from rats fed DFMO, sulindac or combinations showed significant inhibition of COX-2 and iNOS expression and activity compared to control diet fed rat colonic tumors. Also, DFMO and sulindac or combinations showed considerable enhancement of p21WAF1/CIP1, caspase-3 cleavage, down regulation of bcl-xL, bcl-2 and survivin in rat colonic tumors. Rats that were fed with the combination DFMO and sulindac showed reduced tumor cell proliferation (BrdU labeling index by 16-28%, p<0.05-0.01) in colonic tumors. These observations show that combination of low-dose DFMO and sulindac possesses significant colon tumor inhibitory properties; whereas 200 ppm NO-sulindac alone or combined with DFMO had modest colon tumor inhibitory effects. [Supported by NIH, NCI grants NO1-CN-35110 and R01 CA-102942)] Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 808. doi:10.1158/1538-7445.AM2011-808

Epidermal growth factor‐stimulated rabbit oesophageal mucosal growth: Role of polyamines

Factors operative in oesophageal mucosal growth are poorly characterized. Epidermal growth factor (EGF) has been shown to stimulate mucosal growth throughout the gastrointestinal tract. The present study was performed to test the hypothesis that polyamine biosynthesis is required for stimulation of epithelial growth by EGF in oesophageal mucosa. Using an in vitro explant model, oesophageal mucosal growth was quantified in the presence or absence of EGF and α‐difluoromethylornithine (DFMO), a specific inhibitor of polyamine synthesis. Administration of 50 nmol/L EGF significantly increased the rate of epithelial growth in oesophageal explants. Treatment with DFMO for 3 and 7 days not only depleted the tissue polyamines putrescine, spermidine and spermine, but also significantly impaired mucosal growth. The inhibitory effect of DFMO on mucosal growth was partially but significantly prevented when exogenous putrescine was given. Additionally, stimulation of epithelial growth by EGF was also blocked by depletion of cellular polyamines in DFMO‐treated oesophageal explants. These results indicate that EGF stimulates epithelial growth of oesophageal mucosa at least partly through a process involving polyamine biosynthesis.

Epidermal growth factor-stimulated rabbit oesophageal mucosal growth: Role of polyamines.

Factors operative in oesophageal mucosal growth are poorly characterized. Epidermal growth factor (EGF) has been shown to stimulate mucosal growth throughout the gastrointestinal tract. The present study was performed to test the hypothesis that polyamine biosynthesis is required for stimulation of epithelial growth by EGF in oesophageal mucosa. Using an in vitro explant model, oesophageal mucosal growth was quantified in the presence or absence of EGF and α-difluoromethylornithine (DFMO), a specific inhibitor of polyamine synthesis. Administration of 50 nmol/L EGF significantly increased the rate of epithelial growth in oesophageal explants. Treatment with DFMO for 3 and 7 days not only depleted the tissue polyamines putrescine, spermidine and spermine, but also significantly impaired mucosal growth. The inhibitory effect of DFMO on mucosal growth was partially but significantly prevented when exogenous putrescine was given. Additionally, stimulation of epithelial growth by EGF was also blocked by depletion of cellular polyamines in DFMO-treated oesophageal explants. These results indicate that EGF stimulates epithelial growth of oesophageal mucosa at least partly through a process involving polyamine biosynthesis.

α-Difluoromethylornithine (DFMO) as a Potent Arginase Activity Inhibitor in Human Colon Carcinoma Cells

α-Difluoromethylornithine (DFMO) is commonly used as a specific ornithine decarboxylase (ODC, EC4.1.1.17) irreversible inhibitor. ODC is the enzyme responsible for polyamine biosynthesis, which has been shown to be strictly necessary for cell proliferation. In HT-29 Glc −/+ cells, l-arginine is the major precursor of these molecules through the sequential actions of arginase, which leads to l-ornithine generation and ODC. l-ornithine, a substrate for ODC, retroinhibits arginase. Since DFMO is an ornithine analogue, we searched for a direct effect of this agent upon arginase. The flux of l-arginine through arginase in intact cells was inhibited by 51 ± 11% by 10 mM of DFMO whereas 10 mM of l-valine, a known potent arginase inhibitor, inhibited this flux by 73 ± 6%. DFMO equilibrated between extracellular and intercellular spaces and, when used at 10-mM concentration, was without effect on l-arginine net uptake. Measurement of arginase activity in HT-29 cell homogenates with increasing concentrations of DFMO and l-arginine led to an inhibition with a calculated K i (inhibitory constant) equal to 3.9 ± 1.0 mM. l-ornithine was less effective than DFMO in inhibiting arginase activity. Bovine liver arginase, used as another source of the enzyme, was also severely inhibited by DFMO. The inhibitory effect of DFMO upon arginase, one step upstream of the ODC reaction in the metabolic conversion of l-arginine to polyamines, is of potential physiological importance, since it could alter the production of ornithine and thus its metabolism in pathways other than the ODC pathway.

Role of nonmuscle myosin II in polyamine-dependent intestinal epithelial cell migration.

The current study determines whether nonmuscle myosin II is involved in the process requiring polyamines for the stimulation of cell migration in an in vitro model that mimics the early stages of epithelial restitution. Treatment with alpha-difluoromethylornithine (DFMO), a specific inhibitor of ornithine decarboxylase (ODC), for 4 days totally inhibited ODC activity and depleted intracellular polyamines in the IEC-6 cells. Nonmuscle myosin II concentrations in DFMO-treated cells were decreased by 75%, and stress fibers were sparse or absent. The most striking feature of DFMO-treated cells was the appearance of many small punctate foci of myosin II in the cell interior. Migration of DFMO-treated cells was reduced by 80%. In the presence of DFMO, exogenous putrescine not only returned nonmuscle myosin II levels and distribution toward normal but also restored cell migration to control levels. The administration of wortmannin, an inhibitor of myosin light chain kinase, significantly inhibited cell migration over the denuded area in control cells and in those treated with DFMO + polyamines. These results indicate that 1) polyamine depletion by DFMO is associated with decreased concentration and reorganization of nonmuscle myosin II in IEC-6 cells and 2) exogenous spermidine reverses the inhibitory effects of DFMO.

Selectivity of polyamine involvement in hormone action on normal and neoplastic target tissues of the rat

The present experiments were designed to evaluate the polyamine involvement in hormonal actions on proliferation and receptor content of neoplastic tissue (hormone-responsive breast cancer) as well as on growth of normal endocrine target tissue (uterus) in the same animals. Administration of estradiol and perphenazine (to stimulate endogenous prolactin release) stimulated N-nitrosomethyl-urea (NMU)-induced rat mammary tumor growth following ovariectomy-induced tumor regression. Such hormonal activation of breast cancer growth was completely abolished by treatment with alpha-difluoromethyl-ornithine (DFMO), a specific irreversible inhibitor of ornithine decarboxylase, which lowered tumor content of polyamines. The growth inhibitory effect of DFMO was partially reversible by exogenous putrescine administration. In contrast, the rise in cytosolic content of progesterone receptors induced by hormonal treatment was not affected by suppression of tumor polyamine levels by DFMO. Similarly, DFMO administration failed to influence the hormone-induced increase in uterine weight in the same animals. Thus, our data suggest selectivity of polyamine involvement in hormone actions, which, in our experimental system, seems to be restricted to the endocrine control of neoplastic cell proliferation.

Polyamines and autocrine control of tumor growth by prolactin in experimental breast cancer in culture.

We have previously observed that both polyamines and an autocrine mechanism are involved in the stimulation by ovine PRL (oPRL) of growth of the N-nitrosomethylurea-induced rat mammary tumor cultured in vitro in the soft agar clonogenic assay. These experiments were designed to test the interaction between these two pathways of oPRL action in this system. In agreement with previous findings in our laboratory, conditioned medium obtained from oPRL-treated (oPRL-CM), but not untreated, tumors consistently stimulated colony formation when added to N-nitrosomethylurea mammary tumors plated in soft agar under serum-free medium conditions. Administration of alpha-difluoromethyl-ornithine (DFMO), an irreversible inhibitor of polyamine biosynthesis, abolished the colony-stimulating effect of oPRL-CM. The inhibitory effect of DFMO was reversed in a dose-dependent fashion by the addition of spermidine, which entirely restored the growth-promoting action of oPRL-CM. In addition, the administration of increasing amounts of spermidine potentiated the colony-stimulating effect of suboptimal concentrations of oPRL-CM. In contrast, manipulation of the polyamine environment with DFMO and/or spermidine administration did not affect the number of colonies formed when conditioned medium from untreated tumors was added instead of oPRL-CM. We conclude that the polyamine pathways plays an essential role in the expression of autocrine control of tumor growth by oPRL.

Polyamines and estrogen control of growth of the NMU-induced rat mammary tumor.

Recent in vitro evidence suggests that polyamines play an important role in the growth of the N-nitrosomethyl-urea (NMU)-induced rat mammary tumor, and that they may be involved in mediating the effect of estrogens on tumor growth. In support of this hypothesis, we here show that inhibition of polyamine biosynthesis with alpha-difluoromethyl-ornithine (DFMO) blocks the mitogenic effect of estradiol-17 beta (E2) added to NMU-mammary tumors grown in soft agar in the presence of the antiestrogen tamoxifen (Tam). Exogenous polyamine administration reversed the inhibitory effect of DFMO and restored E2 action. Administration of polyamine inhibitors to NMU-tumor-bearing rats induced significant inhibition of tumor growth, although tumor ornithine decarboxylase (ODC) was not consistently suppressed. Under our experimental conditions, such treatment did not potentiate the antitumor effect of Tam. Tam alone was found to suppress tumor ODC, suggesting a possible involvement of the polyamine pathway in its antitumor action. These data suggest that the polyamines may play an important role in the hormonal control of the growth of this experimental breast cancer.

Polyamines as Mediators of the Effect of Prolactin and Growth Hormone on the Growth of <italic>N</italic>-Nitroso-<italic>N</italic>-methylurea-Induced Rat Mammary Tumor Cultured in Vitro in Soft Agar<xref ref-type="fn" rid="FN1">2</xref>

This study evaluated the role of polyamines in mediating the effect of ovine prolactin (oPRL) and ovine growth hormone (oGH) on the growth of the N-nitroso-N-methylurea [(NMU) CAS:684-93-5]-induced rat (Sprague-Dawley) mammary cancer cultured in soft agar. oPRL and oGH had a dose-related growth-promoting effect when added to NMU-induced mammary tumors cultured in soft agar in the absence of serum. The effect of oPRL and oGH was blocked by alpha-difluoromethylornithine (DFMO), a suicide inhibitor of ornithine decarboxylase, the rate-limiting enzyme of polyamine biosynthesis. Exogenous administration of polyamines reversed the inhibitory effect of DFMO and completely restored the action of oPRL and oGH. Our results indicated that polyamines are essential in mediating the growth of the NMU-induced mammary tumor under these experimental conditions.

The role of polyamines in somatomedin-stimulated differentiation of L6 myoblasts.

The somatomedins are potent stimulators of proliferation and differentiation of cultured myoblasts. In studies on the mechanism(s) of these actions, we have measured the activities of ornithine decarboxylase (ODC), an enzyme associated with rapid cell proliferation, and creatine kinase (CK), a biochemical marker for muscle differentiation, after treatment of L6 myoblast cultures with Multiplication Stimulating Activity (MSA), a member of the somatomedin family of insulinlike growth factors. ODC levels reached a peak 24 hours after MSA addition (before any detectable differentiation of the myoblasts) and then decreased as differentiation commenced and CK activity increased. Addition of alpha-difluoromethylornithine (DFMO), an irreversible inhibitor of ODC, caused a dramatic decrease in differentiation. Measurement of 3H-thymidine incorporation, DNA content, and cell number established that the effect of DFMO on differentiation was not a simple consequence of its antiproliferative actions. Cellular levels of putrescine and spermidine (but not spermine) decreased substantially following addition of DFMO to the cultures. The inhibitory effects of DFMO were abolished upon addition of exogenous polyamines to the medium. However, addition of polyamines in the absence of MSA or DFMO did not mimic the stimulation of differentiation by MSA. We conclude that polyamines play an essential role in the stimulation of L6 myoblast differentiation by somatomedins, but they are not sufficient to effect this stimulation.

Polyamines as mediators of estrogen action on the growth of experimental breast cancer in rats.

The role of polyamines as mediators of the mitogenic effect of 17 beta-estradiol (E2) was investigated in the N-nitrosomethylurea [(NMU) CAS: 684-93-5; N-methyl-N-nitrosourea]-induced Sprague-Dawley rat mammary tumor grown in the soft agar clonogenic assay under serum-free medium conditions. Inhibition of polyamine biosynthesis with alpha-difluoromethylornithine (DFMO) (1 mM), an irreversible inhibitor of ornithine decarboxylase, completely blocked the growth-promoting effect of E2 (10(-8) M) in this system. Exogenous administration of putrescine (2.5 mM), spermidine (0.1 mM), and spermine (0.1 mM) reversed the inhibitory effect of DFMO and completely restored the action of E2. These polyamines, however, had no effect on tumor growth when they were added alone in identical concentrations. The results indicate that polyamines are essential but probably not sufficient in mediating the E2 effect on the growth of the NMU-induced mammary tumor under these experimental conditions.