David E. Stringer

Inhibition of human ornithine decarboxylase activity by enantiomers of difluoromethylornithine.

Racemic difluoromethylornithine (D/L-DFMO) is an inhibitor of ODC (ornithine decarboxylase), the first enzyme in eukaryotic polyamine biosynthesis. D/L-DFMO is an effective anti-parasitic agent and inhibitor of mammalian cell growth and development. Purified human ODC-catalysed ornithine decarboxylation is highly stereospecific. However, both DFMO enantiomers suppressed ODC activity in a time- and concentration-dependent manner. ODC activity failed to recover after treatment with either L- or D-DFMO and dialysis to remove free inhibitor. The inhibitor dissociation constant (K(D)) values for the formation of enzyme-inhibitor complexes were 28.3+/-3.4, 1.3+/-0.3 and 2.2+/-0.4 microM respectively for D-, L- and D/L-DFMO. The differences in these K(D) values were statistically significant ( P <0.05). The inhibitor inactivation constants (K(inact)) for the irreversible step were 0.25+/-0.03, 0.15+/-0.03 and 0.15+/-0.03 min(-1) respectively for D-, L- and D/L-DFMO. These latter values were not statistically significantly different ( P >0.1). D-DFMO was a more potent inhibitor (IC50 approximately 7.5 microM) when compared with D-ornithine (IC50 approximately 1.5 mM) of ODC-catalysed L-ornithine decarboxylation. Treatment of human colon tumour-derived HCT116 cells with either L- or D-DFMO decreased the cellular polyamine contents in a concentration-dependent manner. These results show that both enantiomers of DFMO irreversibly inactivate ODC and suggest that this inactivation occurs by a common mechanism. Both enantiomers form enzyme-inhibitor complexes with ODC, but the probability of formation of these complexes is 20 times greater for L-DFMO when compared with D-DFMO. The rate of the irreversible reaction in ODC inactivation is similar for the L- and D-enantiomer. This unexpected similarity between DFMO enantiomers, in contrast with the high degree of stereospecificity of the substrate ornithine, appears to be due to the alpha-substituent of the inhibitor. The D-enantiomer may have advantages, such as decreased normal tissue toxicity, over L- or D/L-DFMO in some clinical applications.

Identification and characterization of a diamine exporter in colon epithelial cells.

SLC3A2, a member of the solute carrier family, was identified by proteomics methods as a component of a transporter capable of exporting the diamine putrescine in the Chinese hamster ovary (CHO) cells selected for resistance to growth inhibition by high exogenous concentrations of putrescine. Putrescine transport was increased in inverted plasma membrane vesicles prepared from cells resistant to growth inhibition by putrescine compared with transport in inverted vesicles prepared from non-selected cells. Knockdown of SLC3A2 in human cells, using short hairpin RNA, caused an increase in putrescine uptake and a decrease in arginine uptake activity. SLC3A2 knockdown cells accumulated higher polyamine levels and grew faster than control cells. The growth of SLC3A2 knockdown cells was inhibited by high concentrations of putrescine. Knockdown of SLC3A2 reduced export of polyamines from cells. Expression of SLC3A2 was suppressed in human HCT116 colon cancer cells, which have an activated K-RAS, compared with their isogenic clone, Hkh2 cells, which lack an activated K-RAS allele. Spermidine/spermine N1-acetyltransferase (SAT1) was co-immunoprecipitated by an anti-SLC3A2 antibody as was SLC3A2 with an anti-SAT1 antibody. SLC3A2 and SAT1 colocalized on the plasma membrane. These data provide the first molecular characterization of a polyamine exporter in animal cells and indicate that the diamine putrescine is exported by an arginine transporter containing SLC3A2, whose expression is negatively regulated by K-RAS. The interaction between SLC3A2 and SAT1 suggests that these proteins may facilitate excretion of acetylated polyamines.

The chemopreventive agent α‐difluoromethylornithine blocks Ki‐ras–dependent tumor formation and specific gene expression in Caco‐2 cells

Mutation of the Kirsten‐ras (Ki‐ras) proto‐oncogene occurs frequently in colorectal cancers. α‐Difluoromethylornithine (DFMO), an irreversible inhibitor of the polyamine biosynthetic enzyme, ornithine decarboxylase (ODC), inhibits Ki‐ras transformation and colon tumorigenesis in carcinogen‐treated animal models by mechanisms yet to be elucidated. Caco‐2 cells transfected with an activated Ki‐ras, but not parental cells, formed tumors in severe combined immunodeficient (SCID) mice. DFMO treatment (2% in drinking water) prevented tumor growth. Gene expression profiling was performed to identify Ki‐ras–and DFMO‐dependent patterns of gene expression. Microarray results were validated with real‐time or semi‐quantitative RT‐PCR and/or Western blot analysis. Genes upregulated in Caco‐2 cells expressing an activated Ki‐ras encoded cytoskeletal‐, transport‐, protease‐, and gap junction–associated proteins. These genes are important for normal development and maintenance of colonic epithelial tissue. Caco‐2 cells transfected with an activated Ki‐ras displayed increased expression of the integrin alpha 1 (INGA1) and enhanced cell migration on laminin. These parameters were unaffected by DFMO, but Ki‐ras–dependent migration was inhibited by INGA1 antibodies. Other Ki‐ras–dependent, but DFMO‐independent, genes included transglutaminase (TGase) and kallikrein 6 (KLK6). Ki‐ras–transfected cells also expressed increased levels of connexin43 (Cx43) (RNA and protein), tight junction protein, and endothelin 1. DFMO reversed these increases. The results indicated that the Ki‐ras oncogene caused changes in experimental cell migration and cell‐cell communication genes and that some of these changes could be reversed by DFMO.

Role of polyamines in arginine-dependent colon carcinogenesis in ApcMin/+ mice

We evaluated the role of polyamines in arginine‐dependent intestinal tumorigenesis in ApcMin/+ mice. Arginine is a substrate for ornithine synthesis and thus can influence polyamine production. Supplementing the diet with arginine increased intestinal and colonic polyamine levels and colonic carcinogenesis. Inhibiting polyamine synthesis with D,L‐α‐diflouromethylornithine (DFMO) decreased small intestinal and colonic polyamine pools. In mice provided basal diet, but not when supplemented with arginine, DFMO decreased small intestinal tumor number and burden, and increased intestinal apoptosis. In mice provided supplemental arginine in the diet, DFMO induced late apoptosis and decreased tumorigenesis in the colon. DFMO slightly reduced tumor incidence, number, and size while significantly decreasing tumor burden and grade. These changes in colon tumorigenesis did not occur in mice not provided supplemental arginine. Our study indicates that polyamines play unique roles in intestinal and colonic carcinogenesis in ApcMin/+ mice. Inhibition of polyamine synthesis suppresses the arginine‐dependent risk of colon tumorigenesis, resulting in apoptosis induction and decreased tumorigenesis, in this murine model.

Polyamine transport is mediated by both endocytic and solute carrier transport mechanisms in the gastrointestinal tract

The polyamines spermidine and spermine, and their precursor putrescine, are required for cell growth and cellular functions. The high levels of tissue polyamines are implicated in carcinogenesis. The major sources of exogenous polyamines are diet and intestinal luminal bacteria in gastrointestinal (GI) tissues. Both endocytic and solute carrier-dependent mechanisms have been described for polyamine uptake. Knocking down of caveolin-1 protein increased polyamine uptake in colon cancer-derived HCT116 cells. Dietary supplied putrescine was accumulated in GI tissues and liver in caveolin-1 knockout mice more than wild-type mice. Knocking out of nitric oxide synthase (NOS2), which has been implicated in the release of exogenous polyamines from internalized vesicles, abolished the accumulation of dietary putrescine in GI tissues. Under conditions of reduced endogenous tissue putrescine contents, caused by treatment with the polyamine synthesis inhibitor difluoromethylornithine (DFMO), small intestinal and colonic mucosal polyamine contents increased with dietary putrescine levels, even in mice lacking NOS2. Knocking down the solute carrier transporter SLC3A2 in HCT116-derived Hkh2 cells reduced the accumulation of exogenous putrescine and total polyamine contents in DFMO treated cells, relative to non-DFMO-treated cells. These data demonstrate that exogenous putrescine is transported into GI tissues by caveolin-1- and NOS2-dependent mechanisms, but that the solute carrier transporter SLC3A2 can function bidirectionally to import putrescine under conditions of low tissue polyamines.

Combination Chemoprevention of Intestinal Carcinogenesis in a Murine Model of Familial Adenomatous Polyposis

Familial adenomatous polyposis (FAP) is an autosomal dominantly inherited syndrome in humans. The Apc Min/+ mouse, which expresses a mutant homolog of the adenomatous polyposis coli gene, is a model of FAP in humans. Treatment with the nonsteroidal anti-inflammatory drugs (NSAIDS) sulindac or celecoxib can suppress polyp development in FAP patients, but responses are generally transient and incomplete. Combination chemoprevention with the ornithine decarboxylase inhibitor difluoromethylornithine (DFMO) and either celecoxib or sulindac was evaluated in the Apc Min/+ mouse. Combinations of DFMO and either NSAID reduced intestinal tumor number by more than 80% (P < 0.0001) compared to untreated controls. In addition to the dramatic reduction in tumor number, the combination of DFMO and sulindac reduced the development of high-grade intestinal adenomas compared to sulindac alone (P = 0.003). The fraction of high-grade intestinal adenomas remaining after treatment was similar for the combination of DFMO and celecoxib and celecoxib alone. Only combinations of DFMO plus sulindac reduced total intestinal polyamine contents compared to untreated mice. These data support the rationale for treatment of FAP patients postcolectomy with DFMO combined with either celecoxib or sulindac but indicate that sulindac may be more effective than celecoxib in reducing intestinal polyamine contents and the incidence of high-grade intestinal adenomas when combined with DFMO.

The role of NO synthases in arginine-dependent small intestinal and colonic carcinogenesis

Arginine is catabolized by NOS2 and other nitric oxide synthases to form nitric oxide. We evaluated the roles of dietary arginine and Nos2 in Apc‐dependent intestinal tumorigenesis in Min mice with and without a functional Nos2 gene. NOS2 protein was expressed only in intestinal tissues of ApcMin/+ Nos2+/+ mice. NOS3 expression was higher in intestinal tissues of mice lacking Nos2, mainly in the small intestine. When diet was supplemented with arginine (0.2% and 2% in drinking water), lack of Nos2 results in decreased tumorigenesis in both small intestine and colon. In Nos2 knockout mice, supplemental arginine (up to 2%) caused a decrease in small intestinal tumor number and size. The arginine‐dependent decrease was associated with an increase in nitrotyrosine formation and apoptosis in the region of intestinal stem cells. Mice expressing Nos2 did not show these changes. These mice did, however, show an arginine‐dependent increase in colon tumor number and incidence, while no effect on apoptosis was seen. These changes were associated with increased nitrotyrosine formation in epithelial cells. Mice lacking Nos2 did not show changes in tumorigenesis or nitrotyrosine formation, while demonstrating an arginine‐dependent increase in apoptosis. These data suggest that Nos2 and dietary arginine have significant effects on intestinal and colonic tumorigenesis in Min mice. In both tissues, loss of Nos2 is associated with decreased tumorigenesis when mice are supplemented with dietary arginine. In the small intestine, Nos2 prevents the arginine‐induced decrease in tumor number and size, which is associated with NOS3 expression and increased apoptosis. In the colon, Nos2 is required for the arginine‐induced increase in tumor number and incidence.

Dietary putrescine reduces the intestinal anticarcinogenic activity of sulindac in a murine model of familial adenomatous polyposis.

Abstract: The nonsteroidal antiinflammatory drug sulindac displays chemopreventive activity in patients with familial adenomatous polyposis (FAP). Sulindac metabolites induce apoptosis in colon tumor cells, in part, by a polyamine-dependent mechanism that can be suppressed with exogenous putrescine. To determine the relevance of this mechanism in animals, we treated ApcMin/+ mice, a model of human FAP, with sulindac alone or in combination with dietary putrescine. Sulindac increased steady-state RNA levels and enzymatic activity of the polyamine catabolic enzyme spermidine/spermine N1-acetyltransferase and intestinal levels of monoacetylspermidine, spermidine, and spermine in the small intestine of mice. Sulindac also decreased the activity of the biosynthetic enzyme ornithine decarboxylase but not adenosylmethionine decarboxylase (AMD). Dietary putrescine increased intestinal putrescine contents, whereas the combination of dietary putrescine and sulindac yielded the highest levels of intestinal putrescine and correlated with a statistically significant reduction in AMD enzyme activity. Dietary putrescine did not statistically significantly increase tumorigenesis, although it significantly increased the grade of adenoma dysplasia (P < 0.05). The effectiveness of sulindac to suppress intestinal carcinogenesis was partially abrogated by dietary putrescine. These data suggest that sulindac exerts at least some of its anticarcinogenic effects in mice via a polyamine-dependent mechanism. Because high concentrations of putrescine can be found in certain dietary components, it may be advantageous to restrict dietary putrescine consumption in patients undergoing treatment with sulindac.

Alteration of cellular adhesion by heat shock

Chinese hamster ovary (CHO) cells were analyzed for their ability to reassemble microfilament bundles, to remain attached to a tissue culture surface, or to initiate and complete attachment onto a substrate after heat shock (45 degrees C/10 min). The cells remained attached to the tissue culture surface during and after the heat shock while the actin microfilament bundles were reversibly disrupted. Heat shock inhibited the ability of the cells to initiate and complete attachment onto a new tissue culture surface or onto a plastic surface coated with vitronectin. An inspection of the proteins present in substrate-attached material (SAM) revealed 11 major proteins containing glucosamine whose apparent Mr values were 250,000, 200,000, 150,000, 140,000, 90,000, 86,000, 82,000, 68,000, 54,000, 47,000, and 46,000. Three of the proteins (p200, p150, and p46) bound to wheat germ agglutinin while p150 and p140 bound to concanavalin A. The composition of the 11 proteins of the SAM fraction synthesized previous to the heat shock was not altered during heat shock. However, the appearance of the newly synthesized proteins in the SAM fraction was delayed by heat shock (0.5 h for p150 and 6 h for p82). The ability of heat-shocked cells to reattach onto a vitronectin-coated surface correlated with the appearance of newly synthesized p150 and p82 in the SAM fraction. Our results suggest that in addition to the microfilament bundles, heat shock may reversibly disrupt the cellular adhesion site. Further, p150 and p82, proteins whose appearance in the SAM fraction is delayed by heat shock, may be involved in the cellular attachment onto substrates, including vitronectin.

Pharmacogenomics of the Polyamine Analog 3,8,13,18-tetraaza-10,11-[(E)-1,2-cyclopropyl] eicosane Tetrahydrochloride, CGC-11093, in the Colon Adenocarcinoma Cell Line HCT1161

Polyamine analogs are known to inhibit tumorigenesis at least in part by mimicking some of the regulatory roles of natural polyamines. To begin the identification of those signaling pathways that are involved in differential cellular responses to the synthetic conformationally restricted polyamine analog CGC-11093, we conducted gene expression profiling, proteomic, and genome-wide DNA methylation and histone acetylation analyses of the HCT116 colon adenocarcinoma cell line after treatment with this analog. Gene expression analysis was performed using Affymetrix GeneChip human genome U133 Plus 2.0 arrays. Changes in protein expression were evaluated using 2D polyacrylamide gels followed by LCMS/MS. DNA methylation was measured using 6,800 element CpG island microarrays. Treatment of cells with CGC-11093 at concentrations ranging from 0.1 to 10 μM caused inhibition of cell growth and metabolic activity, but only minimally affected cell viability. Gene expression analysis showed concentration-dependent effects of CGC-11093 on the DNA/RNA binding transcription factor, cell cycle, signaling, transport, cytoskeletal/structural, and serine protease genes. Functional gene analysis revealed distinct expression patterns related to inhibition of cell cycle control, TGF beta signaling, proteasome and RNA polymerase pathways, upregulation of the aminoacyl-tRNA synthesis pathway, and perturbations in the MAPK and Wnt signaling pathways. Microarray results were validated for selected genes with real time RT PCR. Proteomics analysis showed correlative changes in the expression of proteins involved in the regulation of proteasome function (proteasome subunit Y) and tRNA synthesis. CGC-11093 treatment did not produce any detectable changes in DNA methylation or histone acetylation in cells. This study validates specific target pathways for a specific conformationally restricted polyamine analog and suggests the utility of combined gene and DNA methylation microarrays along with proteomic analyses as a useful approach to the evaluation of the mechanisms of action of anticancer drugs.