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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.

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 cm3) than those on placebo (2.95 cm3; 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. (Cancer Epidemiol Biomarkers Prev 2008;17(2):292–9)

The conformationally restricted polyamine analog PG-11047 has significant growth inhibitory activity against prostate and lung cancer cell lines and is currently under evaluation in several clinical trials, both alone and in combination with other drugs, for the treatment of relapsed or refractory cancer. The objective of this study was to identify the molecular signature of genes responsive to PG-11047 treatment and the biochemical effects of this drug in the HCT116 colon cancer cell line.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.Treatment of cells with PG-11047 at concentrations ranging from 0.1 to 10 microM caused inhibition of cell growth. The activity of PG-11047 was found to correlate with its transcriptional effects on cell cycle control, focal adhesion, adherent and gap junction genes, MAPK-, Wnt- and, TGF-beta signaling pathways, transport and DNA/RNA transcription factor genes. PG-11047 caused depletion of polyamine pools. Proteomics analysis showed that PG-11047 restricts the modification of eukaryotic translation initiation factor 5A (eIF5A), resulting in suppression of general protein synthesis in PG-11047-treated cells.These data show that PG-11047 has a broad spectrum of anticancer activity in colon cancer cells.

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 Apc(Min/+) 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 Apc(Min/+) Nos2(+/+) mice, ut not in Nos2 knockout mice. Additionally, celecoxib suppressed intestinal steady state ornithine decarboxylase RNA levels (p < 0.001), induced steady state spermidine/spermine N-1-acetyltransferase RNA levels (p = 0.002), decreased putrescine levels (P = 0.04) and decreased tumor number in the small intestines of Apc(Min/+) 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. (c) 2006 Wiley-Liss, Inc.

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.

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. © 2006 Wiley-Liss, Inc.

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. © 2005 Wiley-Liss, Inc.

Most sporadic colon adenomas acquire mutations in the adenomatous polyposis coli gene ( APC ) and show defects in APC -dependent signaling. APC influences the expression of several genes, including the c- myc oncogene and its antagonist Mad1 . Ornithine decarboxylase (ODC), the first enzyme in polyamine synthesis, is a transcriptional target of c- myc and a modifier of APC -dependent tumorigenesis. A single-nucleotide polymorphism exists in intron 1 of the human ODC gene, which lies between two myc -binding domains. This region is known to affect ODC transcription, but no data exist on the relationship of this polymorphism to risk of colorectal neoplasia in humans. We show that individuals homozygous for the minor ODC A-allele who reported using aspirin are ≈0.10 times as likely to have an adenoma recurrence as non-aspirin users homozygous for the major G-allele. Mad1 selectively suppressed the activity of the ODC promoter containing the A-allele, but not the G-allele, in a human colon cancer-derived cell line (HT29). Aspirin (≥10 μM) did not affect ODC allele-specific promoter activity but did activate polyamine catabolism and lower polyamine content in HT29 cells. We propose that the ODC polymorphism and aspirin act independently to reduce the risk of adenoma recurrence by suppressing synthesis and activating catabolism, respectively, of colonic mucosal polyamines. These findings confirm the hypothesis that the ODC polymorphism is a genetic marker for colon cancer risk, and support the use of ODC inhibitors and aspirin, or other nonsteroidal antiinflammatory drugs (NSAIDs), in combination as a strategy for colon cancer prevention.