Autophagy induction by exogenous polyamines is an artifact of bovine serum amine oxidase activity in culture serum.

Polyamines are small polycationic alkylamines involved in many fundamental cellular processes, including proliferation, nucleic acid synthesis, apoptosis, and protection from oxidative damage. It has been proposed that in addition to these functions, elevated levels of polyamines promote longevity in various biological systems, including yeast, Drosophila, and murine models. A series of in vitro mechanistic studies by multiple investigators has led to the conclusion that addition of exogenous spermidine promotes longevity through autophagy induction; however, these experiments were confounded by the use of mammalian cell culture systems supplemented with fetal bovine serum. Using cell viability assays, LC3B immunoblots, and live-cell fluorescence microscopy, we report here that in the presence of ruminant serum, exogenously added polyamines are quickly oxidized by the copper-containing bovine serum amine oxidase. This polyamine oxidation resulted in the production of harmful byproducts including hydrogen peroxide, ammonia, and reactive aldehydes. Our data demonstrate that it is critically important to prevent confounding bovine serum amine oxidase-induced cytotoxicity in mechanistic studies of the roles of polyamines in autophagy.

Elevation of cellular Mg2+ levels by the Mg2+ transporter, Alr1, supports growth of polyamine-deficient Saccharomyces cerevisiae cells.

The polyamines putrescine, spermidine, and spermine are required for normal eukaryotic cellular functions. However, the minimum requirement for polyamines varies widely, ranging from very high concentrations (mm) in mammalian cells to extremely low in the yeast Saccharomyces cerevisiae Yeast strains deficient in polyamine biosynthesis (spe1Δ, lacking ornithine decarboxylase, and spe2Δ, lacking SAM decarboxylase) require externally supplied polyamines, but supplementation with as little as 10-8 m spermidine restores their growth. Here, we report that culturing a spe1Δ mutant or a spe2Δ mutant in a standard polyamine-free minimal medium (SDC) leads to marked increases in cellular Mg2+ content. To determine which yeast Mg2+ transporter mediated this increase, we generated mutant strains with a deletion of SPE1 or SPE2 combined with a deletion of one of the three Mg2+ transporter genes, ALR1, ALR2, and MNR2, known to maintain cytosolic Mg2+ concentration. Neither Alr2 nor Mnr2 was required for increased Mg2+ accumulation, as all four double mutants (spe1Δ alr2Δ, spe2Δ alr2Δ, spe1Δ mnr2Δ, and spe2Δ mnr2Δ) exhibited significant Mg2+ accumulation upon polyamine depletion. In contrast, a spe2Δ alr1Δ double mutant cultured in SDC exhibited little increase in Mg2+ content and displayed severe growth defects compared with single mutants alr1Δ and spe2Δ under polyamine-deficient conditions. These findings indicate that Alr1 is required for the up-regulation of the Mg2+ content in polyamine-depleted cells and suggest that elevated Mg2+ can support growth of polyamine-deficient S. cerevisiae mutants. Up-regulation of cellular polyamine content in a Mg2+-deficient alr1Δ mutant provided further evidence for a cross-talk between Mg2+ and polyamine metabolism.

Curcumin mediates polyamine metabolism and sensitizes gastrointestinal cancer cells to antitumor polyamine-targeted therapies.

Curcumin, a natural polyphenol that contributes to the flavor and yellow pigment of the spice turmeric, is known for its antioxidant, anti-inflammatory, and anticarcinogenic properties. Capable of affecting the initiation, promotion, and progression of carcinogenesis through multiple mechanisms, curcumin has potential utility for both chemoprevention and chemotherapy. Previous studies demonstrated that curcumin can inhibit ornithine decarboxylase (ODC) activity in human leukemia and breast cancer cells, and pretreatment with dietary curcumin blocks carcinogen-induced ODC activity in rodent models of skin, colon, and renal cancer. The current study investigated the regulation of polyamine metabolism in human gastric and colon carcinoma cell lines in response to curcumin. Curcumin treatment significantly induced spermine oxidase (SMOX) mRNA and activity, which results in the generation of hydrogen peroxide, a source of ROS. Simultaneously, curcumin down regulated spermidine/spermine N1-acetyltransferase (SSAT) activity and the biosynthetic enzymes ODC and S-adenosylmethionine decarboxylase (SAMDC), thereby diminishing intracellular polyamine pools. Combination treatments using curcumin with the ODC inhibitor 2-difluoromethylornithine (DFMO), an agent currently in clinical chemoprevention trials, significantly enhanced inhibition of ODC activity and decreased growth of GI cancer cell lines beyond that observed with either agent alone. Similarly, combining curcumin with the polyamine analogue bis(ethyl)norspermine enhanced growth inhibition that was accompanied by enhanced accumulation of the analogue and decreased intracellular polyamine levels beyond those observed with either agent alone. Importantly, cotreatment with curcumin permitted the lowering of the effective dose of ODC inhibitor or polyamine analogue. These studies provide insight into the polyamine-related mechanisms involved in the cancer cell response to curcumin and its potential as a chemopreventive or chemotherapeutic agent in the GI tract.

Efficacy and Safety of Curcumin in Treatment of Intestinal Adenomas in Patients With Familial Adenomatous Polyposis.

BACKGROUND & AIMS: Familial adenomatous polyposis is an autosomal dominant disorder characterized by the development of hundreds of colorectal adenomas and eventually colorectal cancer. Oral administration of the spice curcumin has been followed by regression of polyps in patients with this disorder. We performed a double-blinded randomized trial to determine the safety and efficacy of curcumin in patients with familial adenomatous polyposis. METHODS: This study included 44 patients with familial adenomatous polyposis (18-85 years old) who had not undergone colectomy or had undergone colectomy with ileorectal anastomosis or ileal anal pouches, had at least 5 intestinal adenomatous polyps, and had enrolled in Puerto Rico or the United States from September 2011 through November 2016. Patients were randomly assigned (1:1) to groups given 100% pure curcumin (1,500 mg orally, twice per day) or identical-appearing placebo capsules for 12 months. The number and size of lower gastrointestinal tract polyps were evaluated every 4 months for 1 year. The primary outcome was the number of polyps in the curcumin and placebo groups at 12 months or at the time of withdrawal from the study according to the intention-to-treat principle. RESULTS: After 1 year of treatment, the average rate of compliance was 83% in the curcumin group and 91% in the placebo group. After 12 weeks, there was no significant difference in the mean number of polyps between the placebo group (18.6; 95% CI, 9.3-27.8) and the curcumin group (22.6; 95% CI, 12.1-33.1; P = .58). We found no significant difference in mean polyp size between the curcumin group (2.3 mm; 95% CI, 1.8-2.8) and the placebo group (2.1 mm; 95% CI, 1.5-2.7; P = .76). Adverse events were few, with no significant differences between groups. CONCLUSIONS: In a double-blinded randomized trial of patients with familial adenomatous polyposis, we found no difference in the mean number or size of lower intestinal tract adenomas between patients given curcumin 3,000 mg/day and those given placebo for 12 weeks. Clinicaltrials.gov ID NCT00641147.

Regulation of Polyamine Metabolism by Curcumin for Cancer Prevention and Therapy.

Curcumin (diferuloylmethane), the natural polyphenol responsible for the characteristic yellow pigment of the spice turmeric (Curcuma longa), is traditionally known for its antioxidant, anti-inflammatory, and anticarcinogenic properties. Capable of affecting the initiation, promotion, and progression of carcinogenesis through multiple mechanisms, curcumin has potential utility for both chemoprevention and chemotherapy. In human cancer cell lines, curcumin has been shown to decrease ornithine decarboxylase (ODC) activity, a rate-limiting enzyme in polyamine biosynthesis that is frequently upregulated in cancer and other rapidly proliferating tissues. Numerous studies have demonstrated that pretreatment with curcumin can abrogate carcinogen-induced ODC activity and tumor development in rodent tumorigenesis models targeting various organs. This review summarizes the results of curcumin exposure with regard to the modulation of polyamine metabolism and discusses the potential utility of this natural compound in conjunction with the exploitation of dysregulated polyamine metabolism in chemopreventive and chemotherapeutic settings.

L-arginine supplementation improves responses to injury and inflammation in dextran sulfate sodium colitis.

Inflammatory bowel disease (IBD), consisting of Crohn's disease and ulcerative colitis (UC), results in substantial morbidity and is difficult to treat. New strategies for adjunct therapies are needed. One candidate is the semi-essential amino acid, L-arginine (L-Arg), a complementary medicine purported to be an enhancer of immunity and vitality in the lay media. Using dextran sulfate sodium (DSS) as a murine colonic injury and repair model with similarities to human UC, we assessed the effect of L-Arg, as DSS induced increases in colonic expression of the y(+) cationic amino acid transporter 2 (CAT2) and L-Arg uptake. L-Arg supplementation improved the clinical parameters of survival, body weight loss, and colon weight, and reduced colonic permeability and the number of myeloperoxidase-positive neutrophils in DSS colitis. Luminex-based multi-analyte profiling demonstrated that there was a marked reduction in proinflammatory cytokine and chemokine expression with L-Arg treatment. Genomic analysis by microarray demonstrated that DSS-treated mice supplemented with L-Arg clustered more closely with mice not exposed to DSS than to those receiving DSS alone, and revealed that multiple genes that were upregulated or downregulated with DSS alone exhibited normalization of expression with L-Arg supplementation. Additionally, L-Arg treatment of mice with DSS colitis resulted in increased ex vivo migration of colonic epithelial cells, suggestive of increased capacity for wound repair. Because CAT2 induction was sustained during L-Arg treatment and inducible nitric oxide (NO) synthase (iNOS) requires uptake of L-Arg for generation of NO, we tested the effect of L-Arg in iNOS(-/-) mice and found that its benefits in DSS colitis were eliminated. These preclinical studies indicate that L-Arg supplementation could be a potential therapy for IBD, and that one mechanism of action may be functional enhancement of iNOS activity.

Polyaminohydroxamic acids and polyaminobenzamides as isoform selective histone deacetylase inhibitors.

A series of polyaminohydroxamic acids (PAHAs) and polyaminobenzamides (PABAs) were synthesized and evaluated as isoform-selective histone deacetylase (HDAC) inhibitors. These analogues contain a polyamine chain to increase affinity for chromatin and facilitate cellular import. Seven PAHAs inhibited HDAC >50% (1 microM), and two PABAs inhibited HDAC >50% (5 microM). Compound 17 increased acetylated alpha-tubulin in HCT116 colon tumor cells 253-fold but only modestly increased p21 (waf1) and acetylated histones 3 and 4, suggesting that 17 selectively inhibits HDAC 6. PABA 22 alone minimally increased p21 (waf1) and acetylated histones 3 and 4 but caused dose-dependent increases in p21 (waf1) in combination with 0.1 microM 5-azadeoxycytidine. Finally, 22 appeared to be a substrate for the polyamine transport system. None of these compounds were cytotoxic at 100 microM. PAHAs and PABAs exhibit strikingly different cellular effects from SAHA and have the potential for use in combination antitumor therapies with reduced toxicity.

Protective role of arginase in a mouse model of colitis.

Arginase is the endogenous inhibitor of inducible NO synthase (iNOS), because both enzymes use the same substrate, l-arginine (Arg). Importantly, arginase synthesizes ornithine, which is metabolized by the enzyme ornithine decarboxylase (ODC) to produce polyamines. We investigated the role of these enzymes in the Citrobacter rodentium model of colitis. Arginase I, iNOS, and ODC were induced in the colon during the infection, while arginase II was not up-regulated. l-Arg supplementation of wild-type mice or iNOS deletion significantly improved colitis, and l-Arg treatment of iNOS(-/-) mice led to an additive improvement. There was a significant induction of IFN-gamma, IL-1, and TNF-alpha mRNA expression in colitis tissues that was markedly attenuated with l-Arg treatment or iNOS deletion. Treatment with the arginase inhibitor S-(2-boronoethyl)-l-cysteine worsened colitis in both wild-type and iNOS(-/-) mice. Polyamine levels were increased in colitis tissues, and were further increased by l-Arg. In addition, in vivo inhibition of ODC with alpha-difluoromethylornithine also exacerbated the colitis. Taken together, these data indicate that arginase is protective in C. rodentium colitis by enhancing the generation of polyamines in addition to competitive inhibition of iNOS. Modulation of the balance of iNOS and arginase, and of the arginase-ODC metabolic pathway may represent a new strategy for regulating intestinal inflammation.

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 PPAR gamma 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 PPAR delta and PPAR gamma. 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.