ZEBULARINE: A UNIQUE MOLECULE FOR AN EPIGENETICALLY BASED STRATEGY IN CANCER CHEMOTHERAPY. THE MAGIC OF ITS CHEMISTRY AND BIOLOGY

1-(β-D-ribofuranosyl)-1,2-dihydropyrimidin-2-one (zebularine) is structurally 4-deamino cytidine. The increased electrophilic character of this simple aglycon endows the molecule with unique chemical and biological properties, making zebularine a versatile starting material for the synthesis of complex nucleosides and an effective inhibitor of cytidine deaminase and DNA cytosine methyltransferase. Zebularine is a stable, antitumor agent that preferentially targets cancer cells and shows activity both in vitro and in experimental animals, even after oral administration.     

Mutagenicity of the cytidine analog zebularine in Escherichia coli

We have examined the mutagenic properties of zebularine, a cytidine analog lacking the amino group at C-4 that has potential use in chemotherapy. Because the hydrate is a strong inhibitor of cytidine deaminase, its use can enhance the potency of other cytosine based compounds such as 5-azacytidine (5AzaC) and cytosine arabinoside (ara-C) that are inactivated by cytidine deaminase. Using the newly developed rpoB/Rifr system in Escherichia coli, we examined base substitution mutations caused by zebularine in the chromosomal rpoB gene. Zebularine is a potent mutagen that causes mainly G : C --> A : T transitions and favors certain hotspots. Mutations are not specific to the rpoB gene, since there is also a strong induction of mutations in the thyA gene. In the absence of mismatch repair, zebularine induces both base substitutions and frame shifts at rates well above those seen in wild-type strains treated with zebularine or in mismatch repair deficient strains without treatment. The nature of these induced mutations indicates that zebularine is stimulating the induction of increased replication errors, in addition to the targeted G : C --> A : T mutations, and that these errors are normally repaired by the mismatch repair system.     

Inhibition of Cytidine Deaminase by Derivatives of 1-(β-D-Ribofuranosyl)-Dihydropyrimidin-2-One (Zebularine)

Abstract

The 2′-deoxy and ara derivatives of 1-β-(D-ribofuranosyl)-1,2-dihydropyrimidin-2-one (zebularine) were synthesized by improved routes and tested for their inhibitory properties against cytidine deaminase. It was shown that the K_i′s of both compounds were comparable to that of the parent zebularine in inhibition studies with purified enzyme. In contrast to zebularine, 2′-deoxy and ara zebularine showed only nominal cytotoxicity against MOLT-4 and L1210 cells in vitro. A model compound for the inhibition of deoxycytidylate deaminase, 2′-deoxyzebularine 5′-monophosphate (6), was also prepared.     

Mechanistic Insights on the Inhibition of C5 DNA Methyltransferases by Zebularine

In mammals DNA methylation occurs at position 5 of cytosine in a CpG context and regulates gene expression. It plays an important role in diseases and inhibitors of DNA methyltransferases (DNMTs)—the enzymes responsible for DNA methylation—are used in clinics for cancer therapy. The most potent inhibitors are 5-azacytidine and 5-azadeoxycytidine. Zebularine (1-(β-D-ribofuranosyl)-2(1H)- pyrimidinone) is another cytidine analog described as a potent inhibitor that acts by forming a covalent complex with DNMT when incorporated into DNA. Here we bring additional experiments to explain its mechanism of action. First, we observe an increase in the DNA binding when zebularine is incorporated into the DNA, compared to deoxycytidine and 5-fluorodeoxycytidine, together with a strong decrease in the dissociation rate. Second, we show by denaturing gel analysis that the intermediate covalent complex between the enzyme and the DNA is reversible, differing thus from 5-fluorodeoxycytidine. Third, no methylation reaction occurs when zebularine is present in the DNA. We confirm that zebularine exerts its demethylation activity by stabilizing the binding of DNMTs to DNA, hindering the methylation and decreasing the dissociation, thereby trapping the enzyme and preventing turnover even at other sites.     

Repair of DNA Damage Induced by the Cytidine Analog Zebularine Requires ATR and ATM in Arabidopsis

DNA damage repair is an essential cellular mechanism that maintains genome stability. Here, we show that the nonmethylable cytidine analog zebularine induces a DNA damage response in Arabidopsis thaliana, independent of changes in DNA methylation. In contrast to genotoxic agents that induce damage in a cell cycle stage-independent manner, zebularine induces damage specifically during strand synthesis in DNA replication. The signaling of this damage is mediated by additive activity of ATAXIA TELANGIECTASIA MUTATED AND RAD3-RELATED and ATAXIA TELANGIECTASIA MUTATED kinases, which cause postreplicative cell cycle arrest and increased endoreplication. The repair requires a functional STRUCTURAL MAINTENANCE OF CHROMOSOMES5 (SMC5)-SMC6 complex and is accomplished predominantly by synthesis-dependent strand-annealing homologous recombination. Here, we provide insight into the response mechanism for coping with the genotoxic effects of zebularine and identify several components of the zebularine-induced DNA damage repair pathway.     

Quantitative determination of zebularine (NSC 309132), a DNA methyltransferase inhibitor, and three metabolites in murine plasma by high-performance liquid chromatography coupled with on-line radioactivity detection

The metabolism of zebularine (NSC 309132), a novel agent that inhibits DNA methyltransferases, is still uncharacterized. To examine the in vivo metabolism of zebularine, an analytical method was developed and validated (based on FDA guidelines) to quantitate 2-[(14)C]-zebularine and its major metabolites in murine plasma. Zebularine and its metabolites uridine, uracil and dihydrouracil were baseline-separated based on hydrophilic interaction chromatography by using an amino column. The assay was accurate and precise in the concentration ranges of 5.0-100 microg/mL for zebularine, 2.5-50 microg/mL for uridine, 1.0-10 microg/mL for uracil and 0.5-5.0 microg/mL for dihydrouracil. This assay is being used to quantitate zebularine and its metabolites in ongoing pharmacokinetic studies of zebularine.     

Zebularine induces enzymatic DNA–protein crosslinks in 45S rDNA heterochromatin of Arabidopsis nuclei

Loss of genome stability leads to reduced fitness, fertility and a high mutation rate. Therefore, the genome is guarded by the pathways monitoring its integrity and neutralizing DNA lesions. To analyze the mechanism of DNA damage induction by cytidine analog zebularine, we performed a forward-directed suppressor genetic screen in the background of Arabidopsis thaliana zebularine-hypersensitive structural maintenance of chromosomes 6b (smc6b) mutant. We show that smc6b hypersensitivity was suppressed by the mutations in EQUILIBRATIVE NUCLEOSIDE TRANSPORTER 3 (ENT3), DNA METHYLTRANSFERASE 1 (MET1) and DECREASE IN DNA METHYLATION 1 (DDM1). Superior resistance of ent3 plants to zebularine indicated that ENT3 is likely necessary for the import of the drug to the cells. Identification of MET1 and DDM1 suggested that zebularine induces DNA damage by interference with the maintenance of CG DNA methylation. The same holds for structurally similar compounds 5-azacytidine and 2-deoxy-5-azacytidine. Based on our genetic and biochemical data, we propose that zebularine induces enzymatic DNA–protein crosslinks (DPCs) of MET1 and zebularine-containing DNA in Arabidopsis, which was confirmed by native chromatin immunoprecipitation experiments. Moreover, zebularine-induced DPCs accumulate preferentially in 45S rDNA chromocenters in a DDM1-dependent manner. These findings open a new avenue for studying genome stability and DPC repair in plants.     

Incorporation of zebularine from its 2'-deoxyribonucleoside triphosphate derivative and activity as a template-coding nucleobase

Zebularine (1-(beta-D-ribofuranosyl)-1,2-dihydropyrimidin-2-one) was studied as both a 2 '-deoxyribosyl 5 '-triphosphate derivative and as a template incorporated into an oligonucleotide. Using a novel pyrosequencing assay, zebularine acted as cytosine analog and was incorporated into DNA with a template pairing profile most similar to cytosine, pairing with greatest efficiency opposite guanine in the template strand. Guanine was incorporated with greater affinity than adenine opposite a zebularine in the template strand. Computer modeling of base-pairing structures supported a better fit of zebularine opposite guanine than adenine. Zebularine acts as a cytosine analog, which supports its activity as an inhibitor of cytosine methyltransferase.     

DNA Methyltransferase Inhibitor Zebularine Inhibits Human Hepatic Carcinoma Cells Proliferation and Induces Apoptosis

Hepatocellular carcinoma is one of the most common cancers worldwide. During tumorigenesis, tumor suppressor and cancer-related genes are commonly silenced by aberrant DNA methylation in their promoter regions. Zebularine (1-(β-_D-ribofuranosyl)-1,2-dihydropyrimidin-2-one) acts as an inhibitor of DNA methylation and exhibits chemical stability and minimal cytotoxicity both in vitro and in vivo. In this study, we explore the effect and possible mechanism of action of zebularine on hepatocellular carcinoma cell line HepG2. We demonstrate that zebularine exhibits antitumor activity on HepG2 cells by inhibiting cell proliferation and inducing apoptosis, however, it has little effect on DNA methylation in HepG2 cells. On the other hand, zebularine treatment downregulated CDK2 and the phosphorylation of retinoblastoma protein (Rb), and upregulated p21^WAF/CIP1 and p53. We also found that zebularine treatment upregulated the phosphorylation of p44/42 mitogen-activated protein kinase (MAPK). These results suggest that the p44/42 MAPK pathway plays a role in zebularine-induced cell-cycle arrest by regulating the activity of p21^WAF/CIP1 and Rb. Furthermore, although the proapoptotic protein Bax levels were not affected, the antiapoptotic protein Bcl-2 level was downregulated with zebularine treatment. In addition, the data in the present study indicate that inhibition of the double-stranded RNA-dependent protein kinase (PKR) is involved in inducing apoptosis with zebularine. These results suggest a novel mechanism of zebularine-induced cell growth arrest and apoptosis via a DNA methylation-independent pathway in hepatocellular carcinoma.     

Incorporation of Zebularine from its 2′-Deoxyribonucleoside Triphosphate Derivative and Activity as a Template-Coding Nucleobase

Zebularine (1-(β-D-ribofuranosyl)-1,2-dihydropyrimidin-2-one) was studied as both a 2 ′-deoxyribosyl 5 ′-triphosphate derivative and as a template incorporated into an oligonucleotide. Using a novel pyrosequencing assay, zebularine acted as cytosine analog and was incorporated into DNA with a template pairing profile most similar to cytosine, pairing with greatest efficiency opposite guanine in the template strand. Guanine was incorporated with greater affinity than adenine opposite a zebularine in the template strand. Computer modeling of base-pairing structures supported a better fit of zebularine opposite guanine than adenine. Zebularine acts as a cytosine analog, which supports its activity as an inhibitor of cytosine methyltransferase.     

Continuous zebularine treatment effectively sustains demethylation in human bladder cancer cells

During tumorigenesis, tumor suppressor and cancer-related genes are commonly silenced by aberrant DNA methylation in their promoter regions. Recently, we reported that zebularine [1-(beta-D-ribofuranosyl)-1,2-dihydropyrimidin-2-one] acts as an inhibitor of DNA methylation and exhibits chemical stability and minimal cytotoxicity both in vitro and in vivo. Here we show that continuous application of zebularine to T24 cells induces and maintains p16 gene expression and sustains demethylation of the 5' region for over 40 days, preventing remethylation. In addition, continuous zebularine treatment effectively and globally demethylated various hypermethylated regions, especially CpG-poor regions. The drug caused a complete depletion of extractable DNA methyltransferase 1 (DNMT1) and partial depletion of DNMT3a and DNMT3b3. Last, sequential treatment with 5-aza-2'-deoxycytidine followed by zebularine hindered the remethylation of the p16 5' region and gene resilencing, suggesting the possible combination use of both drugs as a potential anticancer regimen.     

Zebularine exerts its antiproliferative activity through S phase delay and cell death in human malignant mesothelioma cells

Malignant mesothelioma is an asbestos-related aggressive tumor and current therapy remains ineffective. Zebularine as a DNA methyltransferase (DNMT) inhibitor has an anti-tumor effect in several human cancer cells. The aim of the present study was to investigate whether zebularine could induce antiproliferative effect in human malignant mesothelioma cells. Zebularine induced cell growth inhibition in a dose-dependent manner. In addition, zebularine dose-dependently decreased expression of DNMT1 in all malignant mesothelioma cells tested. Cell cycle analysis indicated that zebularine induced S phase delay. Zebularine also induced cell death in malignant mesothelioma cells. In contrast, zebularine did not induce cell growth inhibition and cell death in human normal fibroblast cells. These results suggest that zebularine has a potential for the treatment of malignant mesothelioma by inhibiting cell growth and inducing cell death.     

The DNA methyltransferase inhibitor zebularine induces mitochondria-mediated apoptosis in gastric cancer cells in vitro and in vivo

DNA methyltransferase (DNMT) inhibitor zebularine has been reported to potentiate the anti-tumor effect by reactivating the expression of tumor suppressor genes and apoptosis-related genes in various malignant cells. However, the apoptotic signaling pathway in gastric cancer cells induced by zebularine is not well understood. In the study, the effects of zebularine on the growth and apoptosis of gastric cancer cells were investigated by MTT assay, Hoechst assay, Western blot analysis, flow cytometric analysis of annexin V-FITC/PI staining, and TUNEL assay. Zebularine was an effective inhibitor of human gastric cancer cells proliferation in vitro and in vivo. The effects were dose dependent. A zebularine concentration of 50 μM accounted for the inhibition of cell proliferation of 67% at 48 h. The treatment with zebularine upregulated Bax, and decreased Bcl-2 protein. Caspase-3 was activated, suggesting that the apoptosis is mediated by mitochondrial pathways. Moreover, zebularine injection successfully inhibited the tumor growth via apoptosis induction which was demonstrated by TUNEL assay in xenograft tumor mouse model. These results demonstrated that zebularine induced apoptosis in gastric cancer cells via mitochondrial pathways, and zebularine might become a therapeutic approach for the treatment of gastric cancer.     

Zebularine induces replication-dependent double-strand breaks which are preferentially repaired by homologous recombination

Zebularine is a second-generation, highly stable hydrophilic inhibitor of DNA methylation with oral bioavailability that preferentially target cancer cells. It acts primarily as a trap for DNA methyl transferases (DNMTs) protein by forming covalent complexes between DNMT protein and zebularine-substrate DNA. It’s well documented that replication-blocking DNA lesions can cause replication fork collapse and thereby to the formation of DNA double-strand breaks (DSB). DSB are dangerous lesions that can lead to potentially oncogenic genomic rearrangements or cell death. The two major pathways for repair of DSB are non-homologous end joining (NHEJ) and homologous recombination (HR). Recently, multiple functions for the HR machinery have been identified at arrested forks. Here we investigate in more detail the importance of the lesions induced by zebularine in terms of DNA damage and cytotoxicity as well as the role of HR in the repair of these lesions. When we examined the contribution of NHEJ and HR in the repair of DSB induced by zebularine we found that these breaks were preferentially repaired by HR. Also we show that the production of DSB is dependent on active replication. To test this, we determined chromosome damage by zebularine while transiently inhibiting DNA synthesis. Here we report that cells deficient in single-strand break (SSB) repair are hypersensitive to zebularine. We have observed more DSB induced by zebularine in XRCC1 deficient cells, likely to be the result of conversion of SSB into toxic DSB when encountered by a replication fork. Furthermore we demonstrate that HR is required for the repair of these breaks. Overall, our data suggest that zebularine induces replication-dependent DSB which are preferentially repaired by HR.     

The effect of epigenetic regulation of fucosylation on TRAIL-induced apoptosis

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis in many cancer cells but not in normal ones. Recombinant TRAIL and agonistic antibodies to its cognate receptors are currently being studied as promising anticancer drugs. However, preclinical and clinical studies have shown that many types of human cancers are resistant to TRAIL agonists. We previously reported that a deficiency of fucosylation, which is one of the most common oligosaccharide modifications, leads to resistance to TRAIL-induced apoptosis. In contrast, DNA methylation is associated with silencing of various tumor suppressor genes and resistance of cancer cells to anticancer drugs. The aim of this study is to clarify the involvement of DNA methylation in the regulation of cellular fucosylation and the susceptibility to TRAIL-induced apoptosis. When nineteen cancer cell lines with relatively low fucosylation levels were treated with a novel methyltransferase inhibitor, zebularine, an increase in the fucosylation level was observed in many cancer cell lines. The expression of fucosylation-related genes, such as the FX, GDP-fucose transporter, and Fut4 genes, was significantly increased after the treatment with zebularine. Moreover, a synergistic effect of zebularine on TRAIL-induced apoptosis was observed in several cancer cell lines, in which fucosylation was increased by treatment with zebularine. This synergistic effect was independent of the expression of TRAIL receptors and caspase-8. These results indicate that cellular fucosylation is regulated through DNA methylation in many cancer cells. Moreover, zebularine might be useful as a combination drug with TRAIL-based therapies in patients with TRAIL-resistant cancer.