Natalia A. Ignatenko

Cyclooxygenase-independent Induction of Apoptosis by Sulindac Sulfone Is Mediated by Polyamines in Colon Cancer

Sulindac, a non-steroidal anti-inflammatory prodrug, is metabolized into pharmacologically active sulfide and sulfone derivatives. Sulindac sulfide, but not sulindac sulfone, inhibits cyclooxygenase (COX) enzyme activities, yet both derivatives have growth inhibitory effects on colon cancer cells. Microarray analysis was used to detect COX-independent effects of sulindac on gene expression in human colorectal cells. Spermidine/sperm-ine N1-acetyltransferase (SSAT) gene, which encodes a polyamine catabolic enzyme, was induced by clinically relevant sulindac sulfone concentrations. Northern blots confirmed increased SSAT RNA levels in these colon cancer cells. Deletion analysis and mutational studies were done to map the sulindac sulfone-dependent response sequences in the SSAT 5′-flanking sequences. This led us to the identification of two peroxisome proliferator-activated receptor (PPAR) response elements (PPREs) in the SSAT gene. PPRE-2, at +48 bases relative to the transcription start site, is required for the induction of SSAT by sulindac sulfone and is specifically bound by PPARγ in the Caco-2 cells as shown by transfection and gel shift experiments. PPRE-1, at–323 bases relative to the start site, is not required for the induction of SSAT by sulindac sulfone but can be bound by both PPARδ and PPARγ. Sulindac sulfone reduced cellular polyamine contents in the absence but not in the presence of verapamil, an inhibitor of the export of monoacetyl diamines, inhibited cell proliferation and induced apoptosis. The induced apoptosis could be partially rescued by exogenous putrescine. These data suggest that apoptosis induced by sulindac sulfone is mediated, in part, by the COX-independent, PPAR-dependent transcriptional activation of SSAT, leading to reduced tissue polyamine contents in human colon cancer cells.

Suppression of polyamine catabolism by activated Ki‐ras in human colon cancer cells

An activated Ki‐ras was expressed in the human colon adenocarcinoma cell line Caco‐2 to study the effects of Ki‐ras oncogene on polyamine metabolism during gastrointestinal tumorigenesis. Multiple clones selected for expression of the mutant Ki‐ras transgene displayed a suppression of transcription of a key catabolic enzyme in polyamine catabolism spermidine/spermine N1‐acetyltransferase (SSAT). Gene expression analysis, with cDNA microarrays, showed that Ki‐ras transfected clones had decreased levels of expression, compared to mock transfected cells, of peroxisome proliferator‐activated receptor gamma (PPARγ), a member of the nuclear hormone receptor family and an important regulator of cell proliferation and differentiation. The activated Ki‐ras suppressed SSAT expression by a mechanism involving the PPARγ response element (PPRE) located at +48 bp relative to the transcription start site of the SSAT gene. Transient expression of the PPARγ protein in Ki‐ras expressing Caco‐2 clones, or treatment with the PPARγ ligand ciglitazone, led to an increase in the SSAT promoter activity. A MEK1/2 inhibitor PD98059 induced transcription of both PPARγ and SSAT genes in the activated Ki‐ras clones, suggesting that the mitogen‐activated protein kinases (MAPKs) were involved in the regulation of SSAT expression by PPARγ. We concluded that mutated Ki‐ras suppressed SSAT via a transcriptional mechanism involving the PPARγ signaling pathway.

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.

Risk and risk reduction involving arginine intake and meat consumption in colorectal tumorigenesis and survival

Elevated polyamine and nitric oxide levels (both derived from arginine) promote tumorigenesis, whereas non‐steroidal anti‐inflammatory drugs (NSAIDs) inhibit colorectal cancer (CRC) incidence in experimental and epidemiologic studies. We investigated dietary arginine‐induced intestinal tumorigenesis and NSAID‐inhibitory effects in ApcMin/+ mice differentially expressing nitric oxide synthase‐2 (Nos2). We also studied effects of estimated arginine exposures through meat consumption on tumor characteristics and survival in human CRC cases. Dietary arginine increased high‐grade colon adenoma incidence in ApcMin/+Nos2+/+ mice, but not in Nos2 knockout mice. Additionally, celecoxib suppressed intestinal steady state ornithine decarboxylase RNA levels (p < 0.001), induced steady state spermidine/spermine N1‐acetyltransferase RNA levels (p = 0.002), decreased putrescine levels (p = 0.04) and decreased tumor number in the small intestines of ApcMin/+Nos2+/+ mice (p = 0.0003). Five hundred and eleven cases from our NCI‐supported CRC gene‐environment study were analyzed based on self‐reported meat (as a surrogate for arginine) consumption. Familial CRC cases (n = 144) in the highest meat consumption quartile (Q4) had no statistically significant differences in tumor grade compared to cases in Q1–Q3 (p = 0.32); however, they were observed to have decreased overall survival (OS) (10‐year OS = 42% vs. 65%; p = 0.017), and increased risk of death in an adjusted analysis (hazards ratio [HR] = 2.24; p = 0.007). No differences in tumor grade, OS or adjusted HR were observed for sporadic CRC cases (n = 367) based on meat consumption. Our results suggest important roles for arginine and meat consumption in CRC pathogenesis, and have implications for CRC prevention.

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.

Role of c-Myc in intestinal tumorigenesis of the ApcMin/+ mouse

The c-MYC oncogene plays an important role in tumorigenesis and is commonly highly expressed in gastrointestinal cancers. In colon cells, c-MYC is regulated by the adenomatous polyposis coli (Apc) tumor suppressor gene. Multiple intestinal neoplasia (ApcMin/+ or Min) mice are heterozygous for a truncating Apc mutation and serve as a model of familial adenomatous polyposis (FAP) disease. To study the role of c-Myc in the mutant Apc-mediated colon tumorigenesis, we have developed a transgenic mouse with the conditional deletion of the floxed c-Myc alleles in the intestinal crypts of ApcMin/+ mice (ApcMin/+; c-Mycfl/fl). The floxed c-Myc deletion was initiated via a Cre recombinase controlled by the intestine-specific transcriptional regulatory elements of the liver fatty acid-binding protein gene (Fabpl4×at-132). Fabpl4×at-132-mediated Cre expression and recombination resulted in a two-fold decrease in c-MYC protein expression with no effect on intestinal tract morphology. Small intestinal tumorigenesis was significantly suppressed throughout the small intestinal tract of ApcMin/+; c-Mycfl/fl mice compared to c-Myc wild type littermates. In ApcMin/+; c-Mycfl/fl mice, the intestinal apoptosis was higher in the areas of the small intestine with the decreased c-Myc protein expression (P= 0.0016, compared to their littermates with the wild type c-Myc). Thus, conditional inactivation of c-Myc, mediated by Fabpl4×at-132-driven Cre-recombinase, suppresses Apc-dependent intestinal tumorigenesis in adult ApcMin/+) mice, without apparent effect on normal intestinal mucosa.

Unique dietary‐related mouse model of colitis

Background: A high‐fat diet is a risk factor for the development of inflammatory bowel disease (IBD) in humans. Deoxycholate (DOC) is increased in the colonic contents in response to a high‐fat diet. Thus, an elevated level of DOC in the colonic lumen may play a role in the natural course of development of IBD. Methods: Wild‐type B6.129 mice were fed an AIN‐93G diet, either supplemented with 0.2% DOC or unsupplemented and sacrificed at 1 week, 1 month, 3 months, 4 months, and 8 months. Colon samples were assessed by histopathological, immunohistochemical, and cDNA microarray analyses. Results: Mice fed the DOC‐supplemented diet developed focal areas of colonic inflammation associated with increases in angiogenesis, nitrosative stress, DNA/RNA damage, and proliferation. Genes that play a central role in inflammation and angiogenesis and other related processes such as epithelial barrier function, oxidative stress, apoptosis, cell proliferation/cell cycle/DNA repair, membrane transport, and the ubiquitin‐proteasome pathway showed altered expression in the DOC‐fed mice compared with the control mice. Changes in expression of individual genes (increases or reductions) correlated over time. These changes were greatest 1 month after the start of DOC feeding. Conclusions: The results suggest that exposure of the colonic mucosa to DOC may be a key etiologic factor in IBD. The DOC‐fed mouse model may reflect the natural course of development of colitis/IBD in humans, and thus may be useful for determining new preventive strategies and lifestyle changes in affected individuals.

Kallikrein 6 is a mediator of K-RAS-dependent migration of colon carcinoma cells.

Abstract Kallikrein 6 (KLK6) is a trypsin-like serine peptidase whose relevance in various types of cancers is currently being explored. Previous studies have shown that KLK6 mRNA is upregulated in colon and gastric cancers; however, the regulatory mechanisms and phenotypic consequences of this upregulation are largely unknown. Activating K-RAS mutations are common in colon cancer, occurring in approximately 50% of cases. We have recently reported the upregulation of KLK6 mRNA in Caco2 human colon cancer cells stably transfected with a mutant K-RAS allele (K-RASG12V). In this study we examined the pattern of K-RAS-dependent KLK6 expression and secretion in colon cancer cells. Using pharmacological inhibitors of pathways downstream of K-RAS, we could show that the PI3K and p42/44 MAPK pathways play an important role in the induction of KLK6 in mutant K-RAS-expressing colon cancer cells. Increased KLK6 expression enhanced colon cancer cell migration through laminin and Matrigel. Inhibition of KLK6 using small interference RNA treatment or a specific KLK6 antibody in Caco2 cells stably expressing the mutant K-RAS and in SW480 cells carrying a mutation in the K-RAS oncogene resulted in a reduction in invasiveness through cell culture inserts. These data support the oncogenic role of KLK6 in colorectal cancer.

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.