The recently discovered enzyme lysine-specific demethylase 1 (LSD1) plays an important role in the epigenetic control of gene expression, and aberrant gene silencing secondary to LSD1 dysregulation is thought to contribute to the development of cancer. We reported that (bis)guanidines, (bis)biguanides and their urea- and thiourea isosteres are potent inhibitors of LSD1, and induce the re-expression of aberrantly silenced tumor suppressor genes in tumor cells in vitro. We now report a series of small molecule amidoximes that are moderate inhibitors of recombinant LSD1, but that produce dramatic changes in methylation at the histone 3 lysine 4 (H3K4) chromatin mark, a specific target of LSD1, in Calu-6 lung carcinoma cells. In addition, these analogues increase cellular levels of secreted frizzle-related protein (SFRP) 2, H-cadherin (HCAD) and transcription factor GATA4. These compounds represent leads for an important new series of drug-like epigenetic modulators with the potential for use as antitumor agents.
Epigenetics; lysine-specific demethylase 1; histones; histone demethylase; amidoxime; gene expression; enzyme inhibitor; secreted frizzle-related protein; H-cadherin; GATA4; Calu-6 human anaplastic non-small cell lung carcinoma
Our previous studies demonstrated that specific polyamine analogues, oligoamines, down-regulated the activity of a key polyamine biosynthesis enzyme, ornithine decarboxylase (ODC), and suppressed expression of estrogen receptor alpha (ERα) in human breast cancer cells. However, the mechanism underlying the potential regulation of ERα expression by polyamine metabolism has not been explored. Here, we demonstrated that RNAi-mediated knockdown of ODC (ODC KD) down-regulated the polyamine pool, and hindered growth in ERα-positive MCF7 and T47D and ERα-negative MDA-MB-231 breast cancer cells. ODC KD significantly induced the expression and activity of the key polyamine catabolism enzymes, spermine oxidase (SMO) and spermidine/spermine N1-acetyltransferase (SSAT). However, ODC KD-induced growth inhibition could not be reversed by exogenous spermidine or overexpression of antizyme inhibitor (AZI), suggesting that regulation of ODC on cell proliferation may involve the signaling pathways independent of polyamine metabolism. In MCF7 and T47D cells, ODC KD, but not DFMO treatment, diminished the mRNA and protein expression of ERα. Overexpression of antizyme (AZ), an ODC inhibitory protein, suppressed ERα expression, suggesting that ODC plays an important role in regulation of ERα expression. Decrease of ERα expression by ODC siRNA altered the mRNA expression of a subset of ERα response genes. Our previous analysis showed that oligoamines disrupt the binding of Sp1 family members to an ERα minimal promoter element containing GC/CA-rich boxes. By using DNA affinity precipitation and mass spectrometry analysis, we identified ZBTB7A, MeCP2, PARP-1, AP2, and MAZ as co-factors of Sp1 family members that are associated with the ERα minimal promoter element. Taken together, these data provide insight into a novel antiestrogenic mechanism for polyamine biosynthesis enzymes in breast cancer.
Polyamines; Ornithine decarboxylase; Estrogen receptor α; Antizyme; Breast cancer
The full molecular consequences of oncogene activation during tumorigenesis are not well understood, but several studies have recently linked oncogene activation to epigenetic silencing of specific genes.1,2 Transcriptional repressor Id1 is overexpressed in many malignancies including melanoma, and Id1 targets include tumor suppressor genes TSP1, CDKN2A (p16) and CDKN1A (p21), which are frequently epigenetically silenced in cancer. We confirmed that both TSP1 and CDKN2A have abnormal promoter region DNa methylation in primary melanoma, but the mechanism by which this silencing occurs remains unknown. Here we explore the effects of stable lentiviral Id1 overexpression on the expression of these Id1 target genes in human melanoma cell lines. Overexpressed Id1 was functional and bound transcriptional activator E2A, but did not sequester E2A from gene promoters and repress gene expression. Therefore, these Id1 target genes were resistant to Id1-mediated gene silencing. Our results suggest that Id1 activation may need to occur at discrete stages in cooperation with additional gene dysregulation to repress and induce epigenetic silencing of tumor suppressor genes during melanoma progression.
Id1; thrombospondin; melanoma; DNA methylation; oncogene
This chapter provides an overview of the polyamine field and introduces the 32 other chapters that make up this volume. These chapters provide a wide range of methods, advice, and background relevant to studies of the function of polyamines, the regulation of their content, their role in disease, and the therapeutic potential of drugs targeting polyamine content and function. The methodology provided in this new volume will enable laboratories already working in this area to expand their experimental techniques and facilitate the entry of additional workers into this rapidly expanding field.
Putrescine; Spermidine; Spermine; Hypusine; Antizyme; Polyamine transport
Spermine oxidase (SMO), the most recently characterized polyamine metabolic enzyme, catalyzes the direct back-conversion of spermine to spermidine in an FAD-dependent reaction that also yields the byproducts hydrogen peroxide (H2O2) and 3-aminopropanal. These metabolites, particularly H2O2, have been implicated in cytotoxic cellular responses to specific antitumor polyamine analogs, as well as in the inflammation-associated generation of DNA damage. This chapter describes a rapid, sensitive, and inexpensive method for the chemiluminescent measurement of SMO (or alternatively, N1-acetyl polyamine oxidase, APAO) enzyme activity in cultured cell lysates, without the need for radioactive reagents or the use of high performance liquid chromatography (HPLC). Specifically, H2O2 production by SMO is coupled to chemiluminescence generated by the horseradish peroxidase-catalyzed oxidation of luminol. Detailed protocols for preparation of reagents, harvesting cell lysates, generation of a standard curve, assaying of samples, and calculation of SMO enzyme activity are presented.
Spermine oxidase; Polyamine catabolism; Hydrogen peroxide; Chemiluminescence
Polyamines are essential for normal growth; however, the requirement for, and the metabolism of, polyamines are frequently dysregulated in cancer. Polyamine analogues have demonstrated promising preclinical results in multiple model systems of cancer, but their clinical utility has been limited by apparent toxicity. A representative compound of a new generation of short chain, conformationally restricted polyamine analogues, CGC-11047 has been synthesized and ongoing phase I clinical trials indicate it to be well tolerated at weekly doses of 610 mg (dose escalation is still in progress). Therefore, studies were designed to gain a better understanding of its effects on cellular polyamine biochemistry and efficacy in the treatment of human lung cancer models in vitro and in vivo.
Human lung cancers cell lines representing non-small cell and small cell lung cancers were investigated for their growth and biochemical response to CGC-11047. Effects of in vitro treatment with CGC-11047 on cell growth, the activity of the polyamine biosynthetic enzyme ornithine decarboxylase (ODC), and the expression and activity of the polyamine catabolic enzymes spermidine/spermine N1-acetyltransferase (SSAT) and spermine oxides (SMO) were measured. Additionally, the overall effects on intracellular polyamine pools were monitored. Finally, the in vivo efficacy of CGC-11047 in the treatment of a nude mouse model of human non-small cell lung cancer was evaluated.
CGC-11047 effectively inhibited the growth of both small cell and non-small cell lung cancer cells in vitro. The greatest biochemical effects were observed in the non-small cell lung cancer cells where in addition to a profound down regulation of ODC activity, there was a significant increase in polyamine catabolism leading to a greater degree of polyamine pool depletion and greater accumulation of CGC-11047 when compared with the changes observed for the small cell lines. Importantly, CGC-11047 was found to be highly significant (P < 0.0001) in delaying the progression of established tumors in an in vivo model of human non-small cell lung cancer.
CGC-11047 represents a promising new polyamine analogue that warrants further preclinical and, potentially, clinical evaluation in lung cancer.
Polyamines; Analogues; Lung cancer; SSAT; SMO; ODC
The recent discovery of the direct oxidation of spermine via spermine oxidase (SMO) as a mechanism through which specific antitumor polyamine analogues exert their cytotoxic effects has fueled interest in the study of the polyamine catabolic pathway. A major byproduct of spermine oxidation is H2O2, a source of toxic reactive oxygen species. Recent targeted small interfering RNA studies have confirmed that SMO-produced reactive oxygen species are directly responsible for oxidative stress capable of inducing apoptosis and potentially mutagenic DNA damage. In the present study, we describe a second catalytically active splice variant protein of the human spermine oxidase gene, designated SMO5, which exhibits substrate specificities and affinities comparable to those of the originally identified human spermine oxidase-1, SMO/PAOh1, and, as such, is an additional source of H2O2. Importantly, overexpression of either of these SMO isoforms in NCI-H157 human non-small cell lung carcinoma cells resulted in significant localization of SMO protein in the nucleus, as determined by confocal microscopy. Furthermore, cell lines overexpressing either SMO/PAOh1 or SMO5 demonstrated increased spermine oxidation in the nucleus, with accompanying alterations in individual nuclear polyamine concentrations. This increased oxidation of spermine in the nucleus therefore increases the production of highly reactive H2O2 in close proximity to DNA, as well as decreases nuclear spermine levels, thus altering the protective roles of spermine in free radical scavenging and DNA shielding, and resulting in an overall increased potential for oxidative DNA damage in these cells. The results of these studies therefore have considerable significance both with respect to targeting polyamine oxidation as an antineoplastic strategy, and in regard to the potential role of spermine oxidase in inflammation-induced carcinogenesis.
carcinogenesis; H2O2; oxidation; polyamine; SMO
Polyamine analogs have demonstrated considerable activity against many important solid tumor models including breast cancer. However, the precise mechanisms of antitumor activities of polyamine analogs are not entirely understood. The cytotoxicity of a newly developed polyamine analog compound, SL11144, against human breast cancer was assessed. Treatment of human breast cancer cell lines in culture with SL11144 decreased cell proliferation and induced programmed cell death in a time- and dose-dependent manner. SL11144 also profoundly inhibited the growth of MDA-MB-231 xenografts in host nude mice without overt toxic effects. Treatment of MDA-MB-435 cells with SL11144 led to the release of cytochrome c from mitochondria into cytosol, activation of caspase-3, and poly(ADP-ribose) polymerase cleavage. SL11144 decreased Bcl-2 and increased Bax protein levels in MDA-MB-231 cells. Furthermore, activator protein 1 transcriptional factor family member c-Jun was up-regulated by SL11144 in MDA-MB-435 and MDA-MB-231 cells, but not in MCF7 cells. In addition, significant inhibition of ornithine decarboxylase activity and a decrease in polyamine pools were demonstrated. These results demonstrate that the novel polyamine analog SL11144 has effective antineoplastic action against human breast cancer cells in vitro and in vivo and that multiple apoptotic mechanisms are associated with its cytotoxic effect in specific human breast cancer cell lines.
The critical role of polyamines in cell growth has led to the development of a number of agents that interfere with polyamine metabolism including a novel class of polyamine analogues, oligoamines. Here we demonstrate that oligoamines specifically suppress the mRNA and protein expression of estrogen receptor α (ERα) and ERα target genes in ER-positive human breast cancer cell lines, whereas neither ERβ nor other steroid hormonal receptors are affected by oligoamines. The constitutive expression of a cytomegalovirus promoter-driven exogenous ERα in ER-negative MDA-MB-231 human breast cancer cells was not altered by oligoamines, suggesting that oligoamines specifically suppress ERα transcription rather than affect mRNA or protein stability. Further analysis demonstrated that oligoamines disrupted the DNA binding activity of Sp1 transcription factor family members to an ERα minimal promoter element containing GC/CA-rich boxes. Treatment of MDA-MB-231 cells with the JNK-specific inhibitor SP600125 or expression of the c-Jun dominant negative inhibitor TAM67 blocked the oligoamine-activated JNK/c-Jun pathway and enhanced oligoamine-inhibited ERα expression, suggesting that AP-1 is a positive regulator of ERα expression and that oligoamine-activated JNK/AP-1 activity may antagonize the down-regulation of ERα induced by oligoamines. Taken together, these results suggest a novel antiestrogenic mechanism for specific polyamine analogues in human breast cancer cells.
Substrate activities of various linear polyamines to human spermine oxidase (hSMO) were investigated. The activities were evaluated by monitoring the amount of H2O2 released from sample polyamines by hSMO. H2O2 was measured by a HPLC method that analyzed fluorescent dimers derived from the oxidation of homovanillic acid in the presence of horseradish peroxidase. Six triamines were tested and were found not to be hSMO substrates. Of sixteen tetramines tested, spermine (Spm) was the most active substrate, followed by homospermine and N-butylated Spm. Pentamines showed a characteristic pattern of substrate activity. Of thirteen pentamines tested, 3343 showed higher substrate activity than Spm, and 4343 showed similar activity to Spm. The activities of the other pentamines were as follows: 3443, 4443, 4344, 3344, 4334, 4444, and 3334 (in decreasing order). Product amines released from these pentamines by hSMO were then analyzed by HPLC. Triamine was the only observed product, and the amount of triamine was nearly equivalent to that of released H2O2. A marked difference in the pH dependency curves between tetramines and pentamines suggested that hSMO favored reactions with a non-protonated secondary nitrogen at the cleavage site. The Km and Vmax values for Spm and 3343 at pH 7.0 and 9.0 were consistent with the higher substrate activity of 3343 compared to Spm, as well as with the concept of a non-protonated secondary nitrogen at the cleavage site being preferred, and 3343 was well degraded at a physiological pH by hSMO.
Spermine oxidase; Polyamine; Pentamine; Hydrogen peroxide; Homovanillic acid
Over the past three decades the metabolism and functions of the polyamines have been actively pursued as targets for antineoplastic therapy. Interactions between cationic polyamines and negatively charged nucleic acids play a pivotal role in DNA stabilization and RNA processing that may affect gene expression, translation and protein activity. Our growing understanding of the unique roles that the polyamines play in chromatin regulation, and the discovery of novel proteins homologous with specific regulatory enzymes in polyamine metabolism, have led to our interest in exploring chromatin remodelling enzymes as potential therapeutic targets for specific polyamine analogues. One of our initial efforts focused on utilizing the strong affinity that the polyamines have for chromatin to create a backbone structure, which could be combined with active-site-directed inhibitor moieties of HDACs (histone deacetylases). Specific PAHAs (polyaminohydroxamic acids) and PABAs (polyaminobenzamides) polyamine analogues have demonstrated potent inhibition of the HDACs, re-expression of p21 and significant inhibition of tumour growth. A second means of targeting the chromatin-remodelling enzymes with polyamine analogues was facilitated by the recent identification of flavin-dependent LSD1 (lysine-specific demethylase 1). The existence of this enzyme demonstrated that histone lysine methylation is a dynamic process similar to other histone post-translational modifications. LSD1 specifically catalyses demethylation of mono- and di-methyl Lys4 of histone 3, key positive chromatin marks associated with transcriptional activation. Structural and catalytic similarities between LSD1 and polyamine oxidases facilitated the identification of biguanide, bisguanidine and oligoamine polyamine analogues that are potent inhibitors of LSD1. Cellular inhibition of LSD1 by these unique compounds led to the re-activation of multiple epigenetically silenced genes important in tumorigenesis. The use of these novel polyamine-based HDAC or LSD1 inhibitors represents a highly promising and novel approach to cancer prevention and therapy.
Enzymes in the biosynthetic and catabolic polyamine pathway have long been considered targets for drug development, and early drug discovery efforts in the polyamine area focused on the design and development of specific inhibitors of the biosynthetic pathway, or polyamine analogues that specifically bind DNA. More recently, it has become clear that the natural polyamines are involved in numerous known and unknown cellular processes, and disruption of polyamine functions at their effector sites can potentially produce beneficial therapeutic effects. As new targets for polyamine drug discovery continue to evolve, the rational design of polyamine analogues will result in more structurally diverse agents. In addition, the physical linkage of polyamine-like structures to putative drug molecules can have beneficial effects resulting from increases in DNA affinity and selective cellular uptake. The present chapter will summarize recent advances in the development of alkylpolyamine analogues as antitumour agents, and describe subsequent advances that have resulted from incorporating polyamine character into more diverse drug molecules. Specifically, new polyamine analogues, and the role of polyamine fragments in the design of antiparasitic agents, antitumour metal complexes, histone deacetylase inhibitors and lysine-specific demethylase 1 inhibitors, will be described.
Aberrant epigenetic repression of gene expression has been implicated in most cancers, including breast cancer. The nuclear amine oxidase, lysine-specific demethylase 1 (LSD1) has the ability to broadly repress gene expression by removing the activating mono- and di-methylation marks at the lysine 4 residue of histone 3 (H3K4me1 & me2). Additionally, LSD1 is highly expressed in estrogen receptor α negative (ER−) breast cancer cells. Since epigenetic marks are reversible, they make attractive therapeutic targets. Here we examine the effects of polyamine analogue inhibitors of LSD1 on gene expression, with the goal of targeting LSD1 as a therapeutic modality in the treatment of breast cancer. Exposure of the ER-negative human breast cancer cells, MDA-MB-231, to the LSD1 inhibitors, 2d or PG11144, significantly increases global H3K4me1 and H3K4me2, and alters gene expression. Array analysis indicated that 98 (75 up and 23 down) and 477 (237 up and 240 down) genes changed expression by at least 1.5-fold or greater after treatment with 2d and PG11144, respectively. The expression of twelve up-regulated genes by 2d and fourteen up-regulated genes by PG11144 was validated by quantitative RT-PCR. Quantitative chromatin immunoprecipitation (ChIP) analysis demonstrated that up-regulated gene expression by polyamine analogues is associated with increase of the active histone marks H3K4me1, H3K4me2 and H3K9ac, and decrease of the repressive histone marks H3K9me2 and H3K27me3, in the promoter regions of the relevant target genes. These data indicate that the pharmacologic inhibition of LSD1 can effectively alter gene expression and that this therapeutic strategy has potential.
epigenetics; chromatin; histone methylation; acetylation; gene silencing
Ornithine decarboxylase antizyme 1 (AZ1) is a major regulatory protein responsible for the regulation and degradation of ornithine decarboxylase (ODC). To better understand the role of AZ1 in polyamine metabolism and in modulating the response to anticancer polyamine analogues, a small interfering RNA strategy was used to create a series of stable clones in human H157 non-small cell lung cancer cells (NSCLC) that expressed less than 5–10 % of basal AZ1 levels. Antizyme 1 knockdown clones accumulated greater amounts of the polyamine analogue N1,N 11 bis(ethyl)norspermine (BENSpm) and were more sensitive to analogue treatment. The possibility of a loss of polyamine uptake regulation in the knockdown clones was confirmed by polyamine uptake analysis. These results are consistent with the hypothesis that AZ1 knockdown leads to dysregulation of polyamine uptake, resulting in increased analogue accumulation and toxicity. Importantly, there appears to be little difference between AZ1 knockdown cells and cells with normal levels of AZ1 with respect to ODC regulation, suggesting that another regulatory protein, potentially AZ2, compensates for the loss of AZ1. The results of these studies are important for the understanding of both the regulation of polyamine homeostasis and in understanding the factors that regulate tumor cell sensitivity to the anti-tumor polyamine analogues.
antizyme; ornithine decarboxylase; polyamines; spermine; BENSpm
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 µM), and two PABAs inhibited HDAC >50% (5 µM). Compound 17 increased acetylated α-tubulin in HCT116 colon tumor cells 253-fold but only modestly increased p21waf1 and acetylated histones 3 and 4, suggesting that 17 selectively inhibits HDAC 6. PABA 22 alone minimally increased p21waf1 and acetylated histones 3 and 4 but caused dose-dependent increases in p21waf1 in combination with 0.1 µM 5-azadeoxycytidine. Finally, 22 appeared to be a substrate for the polyamine transport system. None of these compounds were cytotoxic at 100 µM. PAHAs and PABAs exhibit strikingly different cellular effects from SAHA and have the potential for use in combination antitumor therapies with reduced toxicity.
The reversible acetylation of histones is critical for regulation of eukaryotic gene expression. The histone deacetylase inhibitors trichostatin (TSA, 1), MS-275 (2) and suberoylanilide hydroxamic acid (SAHA, 3) arrest growth in transformed cells and in human tumor xenografts. However, 1–3 suffer from lack of specificity among the various HDAC isoforms, prompting us to design and synthesize polyaminohydroxamic acid (PAHA) derivatives 6–21. We felt that PAHAs would be selectively directed to chromatin and associated histones by the positively charged polyamine side chain. At 1 μM, compounds 12, 15 and 20 inhibited HDAC by 74.86, 59.99 and 73.85%, respectively. Although 20 was a less potent HDAC inhibitor than 1, it was more potent than 2, more effective as an initiator of histone hyperacetylation, and significantly more effective than 2 at re-expressing p21Waf1 in ML-1 leukemia cells. On the basis of these results, PAHAs 6–21 represent an important new chemical class of HDAC inhibitors.
Acute promyelocytic leukemia (APL), a cytogenetically distinct subtype of acute myeloid leukemia (AML), characterized by the t(15;17)-associated PML-RARA fusion, has been successfully treated with therapy utilizing all-trans-retinoic acid (ATRA) to differentiate leukemic blasts. However, among patients with non- APL AML, ATRA-based treatment has not been effective. Here we show that, through epigenetic reprogramming, inhibitors of lysine- specific demethylase 1 (LSD1, also called KDM1A), including tranylcypromine (TCP), unlocked the ATRA-driven therapeutic response in non-APL AML. LSD1 inhibition did not lead to a large-scale increase in histone 3 Lys4 dimethylation (H3K4me2) across the genome, but it did increase H3K4me2 and expression of myeloid-differentiation–associated genes. Notably, treatment with ATRA plus TCP markedly diminished the engraftment of primary human AML cells in vivo in nonobese diabetic (NOD)- severe combined immunodeficient (SCID) mice, suggesting that ATRA in combination with TCP may target leukemia-initiating cells. Furthermore, initiation of ATRA plus TCP treatment 15 d after engraftment of human AML cells in NOD-SCID γ (with interleukin-2 (IL-2) receptor γ chain deficiency) mice also revealed the ATRA plus TCP drug combination to have a potent anti-leukemic effect that was superior to treatment with either drug alone. These data identify LSD1 as a therapeutic target and strongly suggest that it may contribute to AML pathogenesis by inhibiting the normal pro-differentiative function of ATRA, paving the way for new combinatorial therapies for AML.
Chromatin remodelling enzymes such as the histone deacetylases (HDACs) and histone demethylases such as lysine-specific demethylase 1 (LSD1) have been validated as targets for cancer drug discovery. Although a number of HDAC inhibitors have been marketed or are in human clinical trials, the search for isoform-specific HDAC inhibitors is an ongoing effort. In addition, the discovery and development of compounds targeting histone demethylases are in their early stages. Epigenetic modulators used in combination with traditional antitumor agents such as 5-azacytidine represent an exciting new approach to cancer chemotherapy. We have developed multiple series of HDAC inhibitors and LSD1 inhibitors that promote the re-expression of aberrantly silenced genes that are important in human cancer. The design, synthesis and biological activity of these analogues is described herein.
Epigenetic silencing of gene expression is important in cancer. Aberrant DNA CpG island hypermethylation and histone modifications are involved in the aberrant silencing of tumour-suppressor genes. LSD1 (lysine-specific demethylase 1) is a H3K4 (histone H3 Lys4) demethylase associated with gene repression and is overexpressed in multiple cancer types. LSD1 has also been implicated in targeting p53 and DNMT1 (DNA methyltransferase 1), with data suggesting that the demethylating activity of LSD1 on these proteins is necessary for their stabilization. To examine the role of LSD1 we generated LSD1 heterozygous (LSD1+/−) and homozygous (LSD1−/−) knockouts in the human colorectal cancer cell line HCT116. The deletion of LSD1 led to a reduced cell proliferation both in vitro and in vivo. Surprisingly, the knockout of LSD1 in HCT116 cells did not result in global increases in its histone substrate H3K4me2 (dimethyl-H3K4) or changes in the stability or function of p53 or DNMT1. However, there was a significant difference in gene expression between cells containing LSD1 and those null for LSD1. The results of the present study suggested that LSD1 is critical in the regulation of cell proliferation, but also indicated that LSD1 is not an absolute requirement for the stabilization of either p53 or DNMT1.
chromatin; epigenetics; FAD-dependent oxidase; histone modification; transcriptional repression; AAV, adeno-associated viral; AOL, amine oxidase-like; ATRA, all-trans retinoic acid; ChIP, chromatin immunoprecipitation; COBRA, combined bisulfite restriction analysis; CoREST, RE1-silencing transcription factor corepressor 1; DNMT1, DNA methyltransferase 1; ES, embryonic stem; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HA, homology arm; HDAC, histone deacetylase; HEK, human embryonic kidney; H3K4, histone H3 Lys4; H3K9, histone H3 Lys9; H3K9ac, H3K9 acetylation; JARID1, Jumonji, AT rich interactive domain 1; LINE-1, long interspersed nucleotide element 1; LSD, lysine-specific demethylase; me1, monomethyl; me2, dimethyl; me3, trimethyl; pAAV, AAV plasmid; PCNA, proliferating cell nuclear antigen; qPCR, quantitative PCR; SEPT, synthetic exon promoter trap; SET7/9, SET domain-containing histone methyltransferase 7/9; SWIRM, Swi3p/Rsc8p/Moira; TSS, transcriptional start site; VAT1L, vesicle amine transport protein 1 homologue-like; VIM, vimentin
Cancer cells simultaneously harbor global losses and gains in DNA methylation. We demonstrate that inducing cellular oxidative stress by treatment with hydrogen peroxide, recruits DNA methyltransferase 1 (DNMT1) to damaged chromatin. DNMT1 becomes part of a complex(es) containing DNMT3B and members of Polycomb Repressive Complex 4. Hydrogen peroxide treatment causes translocalization of these proteins from non-GC-rich to GC-rich areas. Key components are similarly enriched at gene promoters in an in vivo colitis model. While high expression genes enriched for members of the complex have histone mark and nascent transcription changes, CpG island-containing low expression genes gain promoter DNA methylation. Thus, oxidative damage induces formation and localization of a silencing complex that may explain cancer-specific aberrant DNA methylation and transcriptional silencing.
The histone deacetylase inhibitor, largazole 1 was synthesized by a convergent approach which involved several efficient and high yielding single pot multistep protocols. Initial attempts using t-butyl as thiol protecting group proved problematic and synthesis was accomplished by switching to trityl protecting group. This synthetic protocol provides a convenient approach to many new largazole analogues. Three side chain analogues with multiple heteroatoms for chelation with Zn2+ were synthesized and their biological activities were evaluated. They were less potent than largazole 1 in growth inhibition of HCT116 colon carcinoma cell line and in inducing increases in global H3 acetylation. Largazole 1 and the three side chain analogues had no effect on HDAC6 as indicated by the lack of increased acetylation of α-tubulin.
BACKGROUND & AIMS
Helicobacter pylori-induced gastric carcinogenesis has been linked to the microbial oncoprotein CagA. Spermine oxidase (SMO) metabolizes the polyamine spermine into spermidine and generates H2O2 that causes apoptosis and DNA damage. We determined if pathogenic effects of CagA are attributable to SMO.
Levels of SMO, apoptosis, and DNA damage (8-oxoguanosine) were measured in gastric epithelial cell lines infected with cagA+ or cagA− H. pylori strains, or transfected with a CagA expression plasmid, in the absence or presence of SMO small interfering RNA, or an SMO inhibitor. The role of CagA in induction of SMO and DNA damage was assessed in H. pylori-infected gastritis tissues from humans, gerbils, and both wild-type and hypergastrinemic INS-GAS mice, using immunohistochemistry and flow cytometry.
cagA+ strains or ectopic expression of CagA, but not cagA− strains, led to increased levels of SMO, apoptosis, and DNA damage in gastric epithelial cells, and knockdown or inhibition of SMO blocked apoptosis and DNA damage. There was increased SMO expression, apoptosis, and DNA damage in gastric tissues from humans infected with cagA+, but not cagA− strains. In gerbils and mice, DNA damage was CagA-dependent and present in cells that expressed SMO. Gastric epithelial cells with DNA damage that were negative for markers of apoptosis accounted for 42–69% of cells in gerbils and INS-GAS mice with dysplasia and carcinoma.
By inducing SMO, H. pylori CagA generates cells with oxidative DNA damage, and a subpopulation of these cells are resistant to apoptosis and thus at high risk for malignant transformation.
polyamines; epithelial cells; DNA damage
Rapid synthesis of the polyamine catabolic enzyme spermidine/spermine-N1-acetyltransferase (SSAT) in response to increased polyamines is an important polyamine homeostatic mechanism. Indirect evidence has suggested that there is an important control mechanism involving the release of a translational repressor protein that allows the immediate initiation of SSAT protein synthesis without RNA transcription, maturation, or translocation. To identify a repressor protein, we used a mass spectroscopy-based RNA-protein interaction system and found six proteins that bind to the coding region of SSAT mRNA. Individual small interfering RNA (siRNA) experiments showed that nucleolin knockdown enhances SSAT translation. Nucleolin exists in several isoforms, and we report that the isoform that binds to SSAT mRNA undergoes autocatalysis in the presence of polyamines, a result suggesting that there is a negative feedback system that helps control the cellular content of polyamines. Preliminary molecular interaction data show that a nucleolin isoform binds to a 5′ stem-loop of the coding region of SSAT mRNA. The glycine/arginine-rich C terminus of nucleolin is required for binding, and the four RNA recognition motif domains are included in the isoform that blocks SSAT translation. Understanding SSAT translational control mechanisms has the potential for the development of therapeutic strategies against cancer and obesity.
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.
Epigenetic gene silencing is an important mechanism in the initiation and progression of cancer. Abnormal DNA CpG island hypermethylation and histone modifications are involved in aberrant silencing of tumour-suppressor genes. LSD1 (lysine-specific demethylase 1) was the first enzyme identified to specifically demethylate H3K4 (Lys4 of histone H3). Methylated H3K4 is an important mark associated with transcriptional activation. The flavin adenine dinucleotide-binding amine oxidase domain of LSD1 is homologous with two polyamine oxidases, SMO (spermine oxidase) and APAO (N1-acetylpolyamine oxidase). We have demonstrated previously that long-chain polyamine analogues, the oligoamines, are inhibitors of LSD1. In the present paper we report the synergistic effects of specific oligoamines in combination with DFMO (2-difluoromethylornithine), an inhibitor of ornithine decarboxylase, in human colorectal cancer cells. DFMO treatment depletes natural polyamines and increases the uptake of exogenous polyamines. The combination of oligoamines and DFMO results in a synergistic re-expression of aberrantly silenced tumour-suppressor genes, including SFRP2 (secreted frizzled-related protein 2), which encodes a Wnt signalling pathway antagonist and plays an anti-tumorigenic role in colorectal cancer. The treatment-induced re-expression of SFRP2 is associated with increased H3K4me2 (di-methyl H3K4) in the gene promoter. The combination of LSD1-inhibiting oligoamines and DFMO represents a novel approach to epigenetic therapy of cancer.
epigenetic; histone; lysine-specific demethylase 1 (LSD1); ornithine decarboxylase; polyamine; CDKN2A, cyclin-dependent kinase inhibitor 2A; ChIP, chromatin immunoprecipitation; dcSAM, decarboxylated S-adenosylmethionine; DFMO, 2-difluoromethylornithine; DNMT, DNA methyltransferase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GATA, GATA-binding protein; HDAC, histone deacetylase; H3K4, Lys4 of histone H3; H3K4me1, mono-methyl H3K4; H3K4me2, di-methyl H3K4; H3K9, Lys9 of histone H3; H3K9me2, di-methyl H3K9; LSD1, lysine-specific demethylase 1; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; ODC, ornithine decarboxylase; PARP, poly(ADP-ribose) polymerase; qPCR, quantitative PCR; SFRP, secreted frizzled-related protein; TSS, transcriptional start site