Role of dietary polyamines in a phase III clinical trial of difluoromethylornithine (DFMO) and sulindac for prevention of sporadic colorectal adenomas.

BACKGROUND: The polyamine-inhibitory regimen difluoromethylornithine (DFMO)+sulindac has marked efficacy in preventing metachronous colorectal adenomas. Polyamines are synthesised endogenously and obtained from dietary sources. Here we investigate dietary polyamine intake and outcomes in the DFMO+sulindac colorectal adenoma prevention trial. METHODS: Dietary polyamine data were available for 188 of 267 patients completing the study. Total dietary polyamine content was derived by the sum of dietary putrescine, spermine and spermidine values and categorised into two groups: highest (>75-100%) vs the lower three quartiles (0-25, 25-50 and 50-75%). Baseline tissue polyamine concentration and ODC1 genotype were determined. Logistic regression models were used for risk estimation. RESULTS: A significant interaction was detected between dietary polyamine group and treatment with regard to adenoma recurrence (P=0.012). Significant metachronous adenoma risk reduction was observed after DFMO+sulindac treatment in dietary polyamine quartiles 1-3 (risk ratio (RR) 0.19; 95% confidence interval (CI) 0.08-0.42; P<0.0001) but not in quartile 4 (RR 1.51; 95% CI 0.53-4.29; P=0.44). However, a lower number of events in the placebo group within dietary quartile 4 confound the aforementioned risk estimates. CONCLUSION: These preliminary findings reveal complex relationships between diet and therapeutic prevention, and they support further clinical trial-based investigations where the dietary intervention itself is controlled.

Rationale for, and design of, a clinical trial targeting polyamine metabolism for colon cancer chemoprevention.

Polyamine metabolic genes are downstream targets of several genes commonly mutated in colon adenomas and cancers. Inhibitors of ornithine decarboxylase, such as difluoromethylornithine (DFMO), and agents that stimulate polyamine acetylation and export, such as non-steroidal anti-inflammatory drugs (NSAIDS), act at least additively to arrest growth in human cell models and suppress intestinal carcinogenesis in mice. These preclinical studies provided the rationale for colon cancer prevention trials in humans. A Phase IIb clinical study comparing the combination of DFMO and the NSAID sulindac versus placebo was conducted. Endpoints were colorectal tissue polyamine and prostaglandin E2 contents and overall toxicity to participants. Participants in the Phase IIb study served as a vanguard for a randomized, placebo-controlled prospective Phase III trial of the combination of DFMO and sulindac with the primary study endpoint the prevention of colon polyps. Seventy percent of participants will have completed the three years of treatment in December 2006.

Impact of dietary amino acids and polyamines on intestinal carcinogenesis and chemoprevention in mouse models.

Colon cancer in humans is influenced by both genetic and dietary risk factors. The majority of colon cancers have somatic mutations in the APC (adenomatous polyposis coli) tumour-suppressor gene. Dietary arginine enhances the risk of APC-dependent colon carcinogenesis in mouse models by a mechanism involving NOS2 (nitric oxide synthase 2), as elimination of NOS2 alleles suppresses this phenotype. DFMO (difluoromethylornithine), a specific inhibitor of polyamine synthesis, also inhibits dietary arginine-induced colon carcinogenesis in C57BL/6J-Apc(Min)/J mice. The primary consequence of dietary arginine is to increase the adenoma grade in these mice. Either loss of NOS2 alleles or inhibition of polyamine synthesis suppresses the arginine-induced increase in adenoma grade. In addition to promoting intestinal carcinogenesis, polyamines can also reduce the efficacy of certain intestinal cancer chemopreventive agents. The NSAID (non-steroidal anti-inflammatory drug) sulindac is a potent inhibitor of intestinal carcinogenesis in the C57BL/6J-Apc(Min)/J mouse model and is used to treat humans with FAP (familial adenomatous polyposis). Dietary putrescine reduces the ability of sulindac to suppress intestinal tumorigenesis in the mouse model. These data suggest that reducing polyamine metabolism and dietary polyamine levels may enhance strategies for colon cancer chemoprevention.

Polyamine-dependent gene expression.

The polyamines spermidine and spermine along with the diamine putrescine are involved in many cellular processes, including chromatin condensation, maintenance of DNA structure, RNA processing, translation and protein activation. The polyamines influence the formation of compacted chromatin and have a well-established role in DNA aggregation. Polyamines are used in the posttranslational modification of eukaryotic initiation factor 5A, which regulates the transport and processing of specific RNA. The polyamines also participate in a novel RNA-decoding mechanism, a translational frame-shift, of at least two known genes, the TY1 transposon and mammalian antizyme. Polyamines are crucial for their own regulation and are involved in feedback mechanisms affecting both polyamine synthesis and catabolism. Recently, it has become apparent that the polyamines are able to influence the action of the protein kinase casein kinase 2. Here we address several roles of polyamines in gene expression.

Increased thioredoxin-1 inhibits SSAT expression in MCF-7 human breast cancer cells.

Spermidine/spermine N(1)-acetyltransferase (SSAT) regulates polyamine catabolism. Thioredoxin-1 (Trx-1) is a redox protein that is overexpressed in human cancer leading to increased cell proliferation, decreased apoptosis, and decreased patient survival. We report that SSAT mRNA expression is decreased in Trx-1 transfected MCF-7 human breast cancer cells. There is also a decrease in SSAT enzyme activity and lower putrescine levels but no change in spermine or spermidine levels. The expression of SSAT is regulated by the NF-E2-related factor 2 (Nrf-2) and polyamine modulated factor-1 (PMF-1) transcription factor complex. Trx-1 transfected MCF-7 cells showed decreased Nrf-2/PMF-1 DNA binding without a change in Nrf-2 or PMF-1 protein expression. The results suggest that Trx-1 may play a role in the redox regulation of SSAT expression and polyamine homeostasis that could contribute to the biological effects of Trx-1.

Polyamines as modifiers of genetic risk factors in human intestinal cancers.

Polyamines are downstream mediators of genetic risk factors in human intestinal cancers. The adenomatous polyposis coli (APC) tumour-suppressor gene, which is mutated in essentially all human colon cancers, regulates the expression of several e-box transcription factors. These factors, in turn, regulate the transcription of ornithine decarboxylase (ODC), the first enzyme in polyamine synthesis. The Kirsten ras ( K-ras ) oncogene regulates the expression of several genes, including suppressing the expression of peroxisomal proliferator-activated receptor gamma (PPARgamma). This PPAR, in turn, activates the expression of the spermidine/spermine-N(1)-acetyltransferase (SSAT), the first enzyme in polyamine catabolism. The non-steroidal anti-inflammatory drug (NSAID) sulindac induces the transcription of SSAT via activation of PPARgamma. Inactivation of the APC tumour-suppressor gene, and the activation of K-ras, have a combined effect on increasing tissue polyamine contents due to increased synthesis and decreased catabolism of the polyamines. Pharmacological strategies for suppressing ODC (e.g. the enzyme-activated inhibitor alpha-difluoromethylornithine) and activating SSAT (e.g. NSAIDs) are potent inhibitors of intestinal carcinogenesis in rodent models. Clinical trials combining these classes of agent in humans with risk factors for colon cancer are in progress.

Chemoprevention by difluoromethylornithine: correlation of an in vitro human cell assay with human clinical data for biomarker modulation.

The efficacy of difluoromethylornithine (DFMO) as a chemopreventive agent has been tested in vitro using a human epidermal cell (HEC) assay with growth inhibition and involucrin induction as endpoints. Suppression of polyamine content is currently being utilized as a biomarker in clinical trials for the chemopreventive efficacy of DFMO against colon cancer formation. We have now examined the effects of DFMO on suppression of polyamine content in the HEC assay. The findings indicate 1) the % change in spermidine to spermine ratio and the depletion of putrescine show excellent correlation with chemopreventive efficacy in vitro; 2) the effective concentrations in vitro overlap the plasma concentrations in the clinical trial. These observations serve as further validation of the usefulness of the HEC assay as a screen for chemopreventive efficacy.

Sulindac sulfone inhibits K-ras-dependent cyclooxygenase-2 expression in human colon cancer cells.

Both the sulfide and sulfone metabolites of sulindac, a nonsteroidal anti-inflammatory drug, display anticarcinogenic effects in experimental models. Sulindac sulfide inhibits cyclooxygenase (COX) enzyme activities and has been reported to suppress ras-dependent signaling. However, the mechanisms by which sulindac sulfone suppresses cancer growth are not as defined. We studied the effects of these sulindac metabolites in human colon cancer-derived Caco-2 cells that have been transfected with an activated K-ras oncogene. Stable transfected clones expressed high levels of COX-2 mRNA and protein, compared with parental cells. K-ras-transfected cells formed tumors more quickly when injected into severe combined immunodeficiency disease mice than parental cells, and this tumorigenesis was suppressed by treatment with sulindac. Sulindac sulfone inhibited COX-2 protein expression, which resulted in a decrease in prostaglandin synthase E2 production. Sulindac sulfide had little effect on COX-2 in this model, but did suppress prostaglandin synthase E2 production, presumably by inhibiting COX enzyme activity. These data indicate that the sulfide and sulfone derivatives of sulindac exert COX-dependent effects by distinct mechanisms.

Influence of K-ras activation on the survival responses of Caco-2 cells to the chemopreventive agents sulindac and difluoromethylornithine.

The nonsteroidal anti-inflammatory drug sulindac and the ornithine decarboxylase inhibitor difluoromethylornithine (DFMO) are both potent inhibitors of colon carcinogenesis in experimental models of this disease. The combination of these two agents is undergoing evaluation as a strategy for colon cancer chemoprevention in humans with resected colon polyps. We evaluated the effects of the major sulfide and sulfone metabolites of sulindac and DFMO alone, or in combinations, on the growth and survival of Caco-2 colon cancer-derived cells and in clones of these cells transfected with an activated K-ras oncogene. Both the sulfide and sulfone metabolites of sulindac reduced cell viability, measured by colony-forming assays, primarily by inducing apoptosis. Expression of an activated K-ras oncogene caused cells treated with either sulindac sulfide or sulfone to undergo apoptosis earlier than nontransfected controls. However, clonogenic survival, measured 2 weeks after drug treatment, was the same in both Caco-2 and ras-transfected Caco-2 cells treated with sulindac metabolites. A 24-h treatment with DFMO caused a dose-dependent decrease in the colony-forming ability of cells expressing an activated K-ras but had no effect on the viability of the parental Caco-2 cells. The DFMO-dependent decrease in colony formation in K-ras-activated cells occurred in the absence of apoptosis. Assessment of cell survival by colony-forming assays indicated that these two agents acted in an additive manner when combined. These data indicate that K-ras can influence the kinetics of apoptosis induction by sulindac metabolites and cell survival in response to DFMO. However, cytotoxicity induced by these agents occurs via unique mechanisms. These studies suggest that the combination of DFMO and sulindac may be useful in human cancer prevention strategies.

Increased susceptibility of cells to inducible apoptosis during growth from early to late log phase: an important caveat for in vitro apoptosis research.

The physiologic mode of cell death known as apoptosis has become a major focus of scientific research due to its biologic importance. Much of this research involves cells grown in culture, where induction of apoptosis is achieved through a variety of agents. We report that cell cultures in late log growth phase exhibit an increased susceptibility to apoptosis compared with cultures in early log growth phase when apoptosis is induced by sodium deoxycholate (NaDOC), anti-Fas antibody and cytosine-b-D-arabino-furanoside (Ara-C), three agents which induce apoptosis through different upstream mechanisms. We show that this phenomenon occurs in Jurkat lymphocytes, HT-29 and HCT-116 colon epithelial cells. We also present evidence that cell density alone does not affect NaDOC-induced apoptosis, but rather that the growth media plays a key role in increased susceptibility of cells in late log growth phase to NaDOC-induced apoptosis. These results indicate that growth phase is a variable that must be controlled in order to obtain reliable apoptosis data.

Heat-inducible vectors for use in gene therapy.

The objectives of this study were to quantity and compare the activities of a minimal heat shock (HS) promoter and other promoters used in gene therapy applications, and to identify strategies to amplify the heat inducibility of therapeutic genes. Human tumour cells were transiently or stably transfected with the HS promoter driving expression of reporter genes. HS promoter activity was induced transiently, with maximum activity 16-24 h after HS, and was dependent on temperature. The activity of the minimal HS promoter was similar, after 42 degrees C HS for 1 h, to that of the cytomegalovirus (CMV) promoter. To determine if the HS promoter could be used to activate a second conditional promoter, cells were transiently transfected with vectors containing both the HS and human immunodeficiency virus type 1 (HIV1) promoters. When the IL-2 gene was placed downstream of the HIV1 promoter. IL-2 production was temperature-independent. The addition of the HIV tat gene downstream of the HS promoter caused IL-2 to be induced more than 3 fold after a single 42 degrees C HS. These data indicate that the minimal HS promoter, following activation by clinically attainable temperatures (< or = 42 degrees C), can drive expression of therapeutic genes at levels comparable to the CMV promoter and be used in conjunction with a second conditional promoter to drive temperature-dependent, gene expression.

APC-dependent changes in expression of genes influencing polyamine metabolism, and consequences for gastrointestinal carcinogenesis, in the Min mouse.

The colorectal mucosa of pre-symptomatic individuals with familial adenomatous polyposis (FAP) contains elevated levels of the proliferation-associated polyamines. The Min mouse, like humans with FAP, expresses an abnormal genotype for the APC tumor suppressor gene. In order to determine how APC mutation influences intestinal tissue polyamine content, we measured steady-state RNA levels of ornithine decarboxylase (ODC), the first enzyme in polyamine synthesis, antizyme (AZ), a protein which negatively regulates ODC, and the spermidine/spermine N(1)-acetyltransferase (SSAT), the first enzyme in polyamine catabolism. RNA content was increased 6- to 8-fold in both the small intestine and colon for ODC, decreased significantly in the small intestine but not the colon for AZ and was not statistically different in either intestinal tissue for SSAT in Min mice compared with normal littermates. Consistent with the changes in ODC and AZ gene expression, small intestinal, but not colonic, polyamine content was elevated in Min mice compared with normal littermates. Treatment of Min mice with the specific ODC inhibitor difluoromethylornithine (DFMO) suppressed small intestinal, but not colonic, polyamine content and tumor number. These data indicate that small intestinal tissue polyamine content is elevated in Min mice by a mechanism involving APC-dependent changes in ODC and AZ RNA. Further, ODC enzyme activity, which is influenced by both ODC and AZ RNA levels and inhibited by DFMO, is consequential for small intestinal tumorigenesis in this model. In the FAP population, DFMO may be of value in the chemoprevention of small intestinal adenocarcinoma that remains a risk following colectomy.

Development of difluoromethylornithine (DFMO) as a chemoprevention agent.

D,L-alpha-difluoromethylornithine (DFMO) was synthesized over 20 years ago. It was hoped that this enzyme-activated, irreversible inhibitor of ornithine decarboxylase, the first enzyme in polyamine synthesis, would be effective as a chemotherapy for hyperproliferative diseases, including cancer and/or infectious processes. DFMO was generally found to exert cytostatic effects on mammalian cells and tissues, and its effectiveness as a therapeutic agent has been modest. DFMO was also found to cause treatment-limiting (but reversible) ototoxicity at high doses. This side effect, along with its minimal therapeutic activity, contributed to the loss of interest by many clinicians in further developing DFMO as a cancer therapeutic agent. However, DFMO was subsequently shown to inhibit carcinogen-induced cancer development in a number of rodent models, and interest in developing this compound as a preventive agent has increased. The rationale for the inhibition of ornithine decarboxylase as a cancer chemopreventive agent has been strengthened in recent years because this enzyme has been shown to be transactivated by the c-myc oncogene in certain cell/tissue types and to cooperate with the ras oncogene in malignant transformation of epithelial tissues. Recent clinical cancer chemoprevention trials, using dose de-escalation designs, indicate that DFMO can be given over long periods of time at low doses that suppress polyamine contents in gastrointestinal and other epithelial tissues but cause no detectable hearing loss or other side effects. Current clinical chemoprevention trials are investigating the efficacy of DFMO to suppress surrogate end point biomarkers (e.g., colon polyp recurrence) of carcinogenesis in patient populations at elevated risk for the development of specific epithelial cancers, including colon, esophageal, breast, cutaneous, and prostate malignancies.

Characterization of a novel spermidine/spermine acetyltransferase, BltD, from Bacillus subtilis.

Overexpression of the BltD gene in Bacillus subtilis causes acetylation of the polyamines spermidine and spermine. BltD is co-regulated with another gene, Blt, which encodes a multidrug export protein whose overexpression facilitates spermidine export [Woolridge, Vazquez-Laslop, Markham, Chevalier, Gerner and Neyfakh (1997) J. Biol. Chem. 272, 8864-8866]. Here we show that BltD acetylates both spermidine and spermine at primary propyl amine moieties, with spermine being the preferred substrate. In the presence of saturating concentrations of acetyl CoA, BltD rapidly acetylates spermine at both the N1 and N12 positions. The Km (app) values for spermine, spermidine and N1-acetylspermine are

Reactive nitrogen species in colon carcinogenesis.

The role of reactive nitrogen species (RNS) in colon carcinogenesis is multifactorial and affects diverse processes, such as proliferation, apoptosis, differentiation, tumorigenesis, and metastases. This review describes the stages in colon carcinogenesis where nitric oxide (NO) and inducible NO synthase (NOS2) may influence the progression of a normal mucosa to overt metastatic cancer. Overexpression of NOS2 and an increase in the generation of NO and other RNS may lead to apoptosis resistance, DNA damage, mutation, up-regulation of COX-2, increased proliferation, an increase in oxidative stress and an increase in tumor vascularity and metastatic potential. Therefore, future goals are to establish mechanistically based biomarkers to assess individuals at risk for colon cancer and to implement chemopreventive and dietary strategies that reduce colon cancer risk. An understanding of NO signaling pathways in colon epithelial cells should provide the basis for novel biomarker development. Colon cancer prevention may be achieved effectively by chemically interfering with key components of the NO signaling pathways, changing dietary habits to reduce fat and increase antioxidant-containing vegetables, and dietary supplementation to increase DNA repair.

Different bile acids exhibit distinct biological effects: the tumor promoter deoxycholic acid induces apoptosis and the chemopreventive agent ursodeoxycholic acid inhibits cell proliferation.

Epidemiological studies have suggested that the concentration and composition of fecal bile acids are important determining factors in the etiology of colon cancer. However, the mechanism by which these compounds influence tumor development is not understood. To begin to elucidate their mechanism of action, four bile acids, cholic acid, chenodeoxycholic acid, deoxycholic acid (DCA), and ursodeoxycholic acid, were examined for their effects on the growth of several different tumor cell lines. We found that incubating cells with chenodeoxycholic acid or DCA caused morphological changes, seen by electron and light microscopy, that were characteristic of apoptosis, whereas incubating cells with ursodeoxycholic acid inhibited cell proliferation but did not induce apoptosis. Cholic acid had no discernible effect on cells. Notably, the apoptosis induced by DCA could be suppressed by inhibiting protein kinase C activity with calphostin C. These results indicate that different bile acids exhibit distinct biological activities and suggest that the cytotoxicity reported for DCA may be due to its capacity to induce apoptosis via a protein kinase C-dependent signaling pathway.

Effect of alpha-difluoromethylornithine on rectal mucosal levels of polyamines in a randomized, double-blinded trial for colon cancer prevention.

BACKGROUND: Polyamines (e.g., putrescine, spermidine, and spermine) are required for optimal cell growth. Inhibition of polyamine synthesis suppresses carcinogen-induced epithelial cancers, including colon cancer, in animal models. In a short-term phase IIa trial, we determined that low doses of alpha-difluoromethylornithine (DFMO), an inhibitor of ornithine decarboxylase (an enzyme involved in polyamine synthesis), reduced the polyamine content of normal-appearing rectal mucosa of subjects with a prior history of resected colon polyps. In a follow-up study, we have attempted to determine the lowest dose of DFMO that can suppress the polyamine content of rectal mucosa over a course of 1 year with no or minimal side effects. METHODS: Participants were randomly assigned to daily oral treatment with a placebo or one of three doses (0.075, 0.20, or 0.40 g/m2) of DFMO. Baseline and serial determinations of polyamine levels in rectal mucosa and extensive symptom monitoring (including audiometric measurements, since DFMO causes some reversible hearing loss at higher doses) were performed over a 15-month period. RESULTS: DFMO treatment reduced putrescine levels in a dose-dependent manner. Following 6 months of treatment, doses of 0.20 and 0.40 g/m2 per day reduced putrescine levels to approximately 34% and 10%, respectively, of those observed in the placebo group. Smaller decreases were seen in spermidine levels and spermidine:spermine ratios. Polyamine levels increased toward baseline values after discontinuation of DFMO. Although there were no statistically significant differences among the dose groups with respect to clinically important shifts in audiometric thresholds and nonaudiologic side effects, statistically significant higher dropout and discontinuation rates were observed in the highest dose group. CONCLUSIONS: Polyamine levels in rectal mucosa can be continuously suppressed by daily oral doses of DFMO that produce few or no side effects. A dose of 0.20 g/m2 can be used safely in combination phase IIb or single-agent phase III chemoprevention trials.

Characterization of a diamine exporter in Chinese hamster ovary cells and identification of specific polyamine substrates.

Export of the diamine putrescine was studied using inside-out plasma membrane vesicles prepared from Chinese hamster cells. Putrescine uptake into vesicles was a saturable and an ATP- and antizyme-independent process. Excess amounts of a series of diamines or monoacetyl spermidine, but not monoacetyl putrescine, spermidine, or spermine, inhibited putrescine transport. Putrescine uptake into vesicles prepared at pH 7.4 was suppressed at pH 5, compared with pH 7.4; was stimulated approximately 2.5-fold at pH 7.4 in vesicles prepared at pH 6.25, compared with vesicles prepared at pH 7.4; and was not inhibited by valinomycin in the presence of potassium ions. Reserpine and verapamil blocked [3H]putrescine uptake into inverted vesicles. Verapamil treatment caused an increase in intracellular contents of putrescine, cadaverine, and N8-acetylspermidine, in unstressed proliferating cells, or of N1-acetylspermidine, in cells subjected to heat shock to induce acetylation of spermidine at N1. These data indicate that putrescine export in Chinese hamster cells is mediated by a non-electrogenic antiporter capable of using protons as the counter ion. Physiological substrates for this exporter include putrescine, cadaverine, and monoacetyl spermidine and have the general structure NH3+-(CH2)n-NH2 + R at acidic or neutral pH.